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Studies on the thymus and ontogeny of lymphocyteheterogeneity in the clawed toad Xenopus laevis

(Daudin)

Williams, Nigel Howard

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Williams, Nigel Howard (1981) Studies on the thymus and ontogeny of lymphocyte heterogeneity in theclawed toad Xenopus laevis (Daudin), Durham theses, Durham University. Available at Durham E-ThesesOnline: http://etheses.dur.ac.uk/7834/

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2

r-

Studies on the thymus and ontogeny of lymphocyte

heterogeneity i n the clawed toad Xenopus laevis (Daudin)

by

Nigel Howcurd Williams B.Sc. (Dunelm)

The copyright of this thesis rests with the author.

No quotation from it should be published without

his prior written consent and information derived

from it should be acl nowledged.

,.. being a thesis presented i n candidatvire

for the degree of Doctor of Philosophy

i n the University of Durham.

November 1981

( i )

Abstract

CONTENTS Page

( i i i )

CHAPTER 1: General Introduction

CHAPTER 2: Lymphocyte r e a c t i v i t y to 'T' and 'B' c e l l mitogens: Studies on thymus and spleen of the adult. Introduction Materials and Methods Results (a) - HTdR incorporation and v i a b i l i t y of

lymphocytes i n mitogen-free cultures (b) P r o l i f e r a t i v e responses of thymocytes

and splenocytes to T and B c e l l mitogens Discussion Tables Figures

CHAPTER 3: Ontogeny of mitogen responsiveness i n thymus and spleen Introduction Materials and Methods Results (a) Preliminary studies comparing miniaturised

and standard culture techniques (b) Ontogeny of mitogen responsiveness (c) Characterisation of thymocyte mitogen

responses ( i ) Autoradiographic evidence of T and

B mitogen stimulation ( i i ) The e f f e c t of nylon wool treatment

on T Eind B mitogen stimulation Discussion Tables Figures

CHAPTER 4: Mitogen and mixed lymphocyte culture responses following thymectomy: studies investigating putative T lymphocyte heterogeneity

Introduction Materials and Methods Results (a) Preliminary studies on mitogen

stimulation of PBL (b) Mitogen studies on lymphocytes from a l l o -

immune toadlets (c) MLC and mitogen studies on lymphocytes from

toadlets thymectomixed a t 3-4 weeks of age

Discussion Tables Figures

12 12 14 17

20 26 31

32 32 33 37

37 38

40

40

41 42 50 61

71

71 73 76

76

77

78 78 83 87

( i i ) Page

CHAPTER 5: Ontogeny of in^ vivo lymphocyte r e a c t i v i t y to the hapten trinitrophenyl conjugated to a thymus-dependent and thymus-independent c a r r i e r Introduction Materials and Methods Results (a) Ontogeny of induced splenocyte PFC

r e a c t i v i t y (b) Ontogeny of induced antigen-binding

c e l l s i n the spleen (c) Antibody forming c e l l s within the

thymus Discussion Tables Figures

CHAPTER 6: Thymus histogenesis over metamorphosis and i n organ culture Introduction Materials and Methods Results (a) Thymus morphology (b) Organ culture experiments Discussion Tables Figures

CHAPTER 7: Concluding remarks

Acknowledgements

Bibliography

89 89 91 95

95

96

98 98

104 110

113 113 114 117 117 119 120 123 124

132

138

139

( i i i )

DECLARATIC»}

Some of the work presented i n Chapters 2,3 and 4 form the

b a s i s of four publications*. Assistance of collaborators i n Chapters

2 and 4 i s g r a t e f u l l y acknowledged.

* ( i ) Horton, J.D., Smith, A.R., Williams, N.H., Smith, A., and

Sherif, N.E.H.S. (1980). "Lymphocyte r e a c t i v i t y to 'T' and

'B' c e l l mitogens i n Xenopus l a e v i s ; studies on thymus and

spleen". Devel. Comp. Immunol. £: 75-86.

Cii ] Horton, J.D., Smith, A.R., Williams, N.H., Edwards, B.F. and

Ruben, L.N. (1980). "B-equivalent lymphocyte developnent i n

the amphibian thymus?". I n : Development and Differentiation

of Vertebrate Lymphocytes, Ed. J.D. Horton. Elsevier/Amsterdam,

pp. 173-182.

( i i i ) Smith, A.R., Williams, N.H. and Horton, J,D. (1980).

" I n v i t r o r e a c t i v i t y of Xenopus lymphocytes to T c e l l mitogens

following a l l o g r a f t r e j e c t i o n emd to sheep erythrocytes

a f t e r i n vivo priming". I n : Phylogeny of Immunological Memory,

Ed. M.J. Manning. Elsevier/Amsterdam, pp. 197-205.

Civ) Williams, N.H. and Horton, J.D. (1981). "Ontogeny of

mitogen responsiveness i n thymus and spleen of Xenopus l a e v i s " .

I n : "Aspects of Developnental and Comparative Immunology I "

Ed. J.B. Solomon. Pergamon: Oxford, pp.493-494.

(iv)

STATEMENT OF COPYRIGHT

"The copyright of t h i s thesis r e s t s with the author. No

quotation from i t should be published without the prior

written consent and information derived from i t shoxild be

acknowledged".

(V)

ABSTRACT

(1) Functionally mature lymphocytes can be demonstrated i n v i t r o

by t h e i r p r o l i f e r a t i o n (measured by s c i n t i l l a t i o n counting) i n response

to mitogens. Studies i n Chapter 2 examine the responsiveness of adult

Xenopus lymphocytes from both the thymus and the spleen to B and T c e l l

mitogens. Optimum mitogen doses and l e v e l s of foetal c a l f servmi

supplementation are established. Good stimulation indices are found when

splenocytes are stimulated with both PHA and Con A. Thymocytes also

respond well to these T c e l l mitogens, but a small response to B c e l l

mitogens (E^. c o l i LPS and PPD) i s also found.

(2) The ontogeny of lymphocyte responsiveness to T and B c e l l mitogens

i s examined i n Chapter 3. Very l i t t l e stimulation i s found i n the l a r v a l

thymus, but shortly a f t e r the end of metamorphosis a thymocyte response

begins to emerge and by 6 months of age good stimulation i s found with

both T and B c e l l mitogens. While good l e v e l s of thymocyte stimulation

with the T c e l l mitogens are maintained i n l a t e r l i f e , the response to the

B c e l l mitogen LPS declines to only a low l e v e l by 12 months of age.

Mitogenic responses emerge i n the spleen af t e r the end of metamorphosis

i n a s i m i l a r manner to thymocytes and good responses were found i n a l l

the subsequent ages tested. The transient response of thymic c e l l s

to LPS, which i s maximal at 6 months of age, was confirmed by auto­

radiography and was shown to be dependent on a population of nylon-wool

adherent lymphocytes. ^

(3) The l a r v a l thymus plays a c r i t i c a l role i n the establishment of

T c e l l functions, and Chapter 4 looks at the effect of thymectomy at

d i f f e r e n t stages of l a r v a l development on the subsequent alloimmune

response, mixed lymphocyte r e a c t i v i t y and T c e l l mitogen responsiveness.

The r e s u l t s reveal that (a) skin a l l o g r a f t rejection displayed by

7-day thymectomized animals i s effected i n the absence of spleen and

(vi)

blood lymphocytes reactive to T c e l l mitogens, (b) T-mitogen responsive

c e l l s and MLR T c e l l s ceinnot e a s i l y be separated into d i s t i n c t

populations i n terms of t h e i r degree of thymus dependency.

(4) Chapter 5 looks at the ontogeny of both T helper c e l l and B

c e l l function by examining the i n vivo antigen-binding r e a c t i v i t y

of splenocytes to a T-dependent (TNP-SRBC) and a T-independent (TNP-

LPS) antigen. Good l e v e l s of TtfP binding c e l l s can be induced i n

the spleen when larvae are only 3 weeks old following injection of

TNP-SRBC (after low dose priming with SRBC) or TNP-LPS. The l e v e l

of cuitigen binding i n both cases appears to peak at metamorphosis.

However, when c e l l u l a r antibody production was measured, no plaque

forming c e l l s could be induced i n the spleen u n t i l after the end

of metamorphosis.

(5) Chapter 6 presents (a) a b r i e f h i s t o l o g i c a l study of the thymus

i n l a t e l a r v a l l i f e and at metamorphosis. This work characterises

the dramatic depletion of thymocyte numbers that occurs at the end

of metamorphosis and the renewed lymphopoiesis immediately afterwards,

and (b) preliminary r e s u l t s on the e f f e c t of 7-day organ culture on

thymus histogenesis.

1

CHAPTER 1

General Introduction

Thymus function and lymphocyte heterogeneity i n birds and mammals

I t i s now beyond question that the thymus i s of central immunological

importance (Miller, 1979). The f i r s t major breakthrough that c l e a r l y

linked the thymus with the development of the immvme system came i n

1961, when Mill e r published h i s findings on the effects of thymectomy

i n the newborn mouse. He revealed that neonatal thymectomy (but not

ad<iLt thymectomy) was associated with reduced lymphocyte numbers i n

blood and lymphoid t i s s u e s and abrogated the a b i l i t y of the mouse to

r e j e c t skin a l l o g r a f t s . In the next few years i t became apparent

that neonatal thymectomy i n mammals not only affected c e l l u l a r immunity,

but also impaired antibody responses to some antigens (Miller, 1962,

1963) . I t was r e a l i s e d l a t e r that i t was only the performance,

not the l e v e l of antibody-forming c e l l s which was altered by neonatal

thymectomy (see discussion i n Miller, 1979).

In birds i t became cl e a r early on that two separate 'central'

lymphoid organs were responsible for estcQjlishing humoral and c e l l u l a r

immunity. Thus neonatal removal of the bursa of Fabricius (a

cloacal lymphoid organ found only i n birds) impeiired antibody

production but not a l l o g r a f t rejection i n chickens, whereas early

thymectomy isopaired alloimmune r e a c t i v i t y but had only variable

e f f e c t s on antibody production (Glick, 1956; Szenberg and Warner,

1962; Cooper, Peterson, South and Good, 1966). The search for a

mammalian equivalent of the avian bursa involved protracted

investigations (see Greaves, Owen and Raff, 1973, for discussion).

I t now appears that the foe t a l l i v e r and spleen are centrally

involved i n production of antibody-forming c e l l precursors i n the

mammaliem embryo (Owen, Raff and Cooper, 1975), the bone marrow

assuming t h i s role i n l a t e r l i f e .

These extirpation experiments, backed up with observations

on maumnals with congenital immunodeficient states (such as those on

the nude mouse, with i t s hypoplastic, non-lyii5>hoid thymus - see

Pantelovuris, 1971, 1973; Rygaard, 1973) gave r i s e to the concept

of thymus-dependent (T) and bursa-(or mammalian bursal-equivalent)-

dependent (B) lymphocytes for the 2 ontogenically-distinct c e l l u l a r

popvilations found i n the 'peripheral' lymphoid ti s s u e s (e.g. Iyn5)h

nodes). T and B lymphocytes are c l e a r l y also functionally d i s t i n c t

populations; thus T c e l l s , were shown to be ceiitr a l l y involved i n

c e l l u l a r immunity, while B lymphocytes gave r i s e to antibody-producing

c e l l precursors.

Why the thymus should play a role i n antibody production to

certain antigens ( c a l l e d 'thymus-dependent') remained a inystery u n t i l

the l a t e •60s. Then Claman, Chaperon and T r i p l e t t (1966) revealed

that c e l l s from mouse bone marrow and thymus (each independently capable

of very l i t t l e cintlbody production) could, vrtien injected together into

i r r a d i a t e d r e c i p i e n t s , a ct s y n e r g i s t i c a l l y to give a good antibody

response when challenged with foreign erythrocytes. Mareover, a l l the

antibody i n t h i s type of experiment was shown to be produced by

i n j e c t e d bone marrow c e l l s (Davies, Leuchars, Walls, Marchant and

E l l i o t t , 1967; Mitchell and M i l l e r , 1968). I t appeared that the

T lymphocytes somehow 'helped' bone marrow derived c e l l s to secrete

cintibody. The other l i n e of evidence showing 1/B c e l l cooperation

came from hapten-carrier experiments. A hapten i s a small, chemically

defined substance (e.g. trinitrophenyl (TNP)) which can combine

s p e c i f i c a l l y with antibody, but cannot by i t s e l f induce an immune

response; that i s i t i s antigenic but not immunogenic. To induce

an immune response a hapten must be coupled to a macromolecular

c a r r i e r to form an immunogenic complex. To obtain a good

restoration of secondary anti-hapten IgG antibody response i n

i r r a d i a t e d mice, i t was necessary to have present two separate

lymphocyte populations, one responsive to the c a r r i e r and one

to the hapten (Mitchison, 1971). C a r r i e r - s e n s i t i v e c e l l s were

subsequently shown to be thymus-derived, whereas hapten-sensitive

lymphocytes were bone marrow-derived (Raff, 1970). The precise

mechanism of T c e l l help i s s t i l l under debate, but i t undoubtedly

involves one or more signals from the T lymphocyte that triggers

the B c e l l d i r e c t l y or i n d i r e c t l y v i a macrophages (see Miller, 1979).

In more recent years, extensive research has shown that

mammalian effector T c e l l s (unlike B c e l l s , which seem functionally

f a i r l y homogeneous) are a functionally heterogeneous family of

lymphocytes. Cytotoxic (Miller, Brunner, Sprent, Russell and

Mitchell, 1971), helper and suppressor (Gershon and Kondo, 1972)

T c e l l sub-populations, as well as those involved in delayed-type

hypersensitivity reactions, have been i d e n t i f i e d and, furthermore,

characterised by both t h e i r surface differentiation antigens

(e.g. the Ly antigens described by Cantor and Boyse, 1977) and by

t h e i r expression of antigens encoded by the major histocompatibility

gene complex (MHC). I t i s now beginning to be appreciated that

MHC antigens play an important role i n T lymphocyte r e a c t i v i t y .

Thus i n inbred rodents, and possibly also i n humans and chickens,

r e a c t i v i t y of helper and cytotoxic T c e l l s are believed to be

r e s t r i c t e d by the MHC. I t has been known for some time that MHC

antigens are molecular markers of i n d i v i d u a l i t y : thus when

transplants are performed between MHC-disparate individuals,

graft r e j e c t i o n w i l l occur rapidly, in contrast to the much

better transplant s u r v i v a l i f MHC identity e x i s t s between donor

and host. The more recent finding i s that the MHC i s also of

v i t a l importance i n T c e l l recognition of foreign antigens where

the MHC i s not serving as the immunising difference (Miller, 1 9 7 9 ) .

Thus T helper c e l l collaboration with resting B lymphocytes w i l l ,

i n general, occur only i f these l3^phocytes express the same,

lymphocyte-defined MHC antigens (Katz and Benacerraf, 1975;

Andersson, Schreier cuid Melchers, 1980), and T cell-mediated

c y t o t o x i c i t y s p e c i f i c for virus-altered (Zinkeamagel and Doherty,

1 9 7 4 ) , or hapten-modified c e l l s (Shearer, Rehn and Garbarino, 1975)

requires serologically-defined MHC identity of T lymphocytes with

alt e r e d target c e l l s . Although i t has seemed l i k e l y from mouse

chimera studies that i t i s within the thymus that T c e l l s learn

to become MHC r e s t r i c t e d during development (Zinkemagel, 1978 ) ,

recent experiments suggest that t h i s 'learning' within the thymus

may not alone be responsible for determining the range of MHC

r e s t r i c t i o n (see Howard, 1 9 8 0 ) .

Concomitant with the discoveries of the functional complexity

of the thymus-dependent lymphocyte population i n mammals, and the

possible role of the thymus i n MHC r e s t r i c t i o n , have been investigations

to determine the precise role that the thymus plays i n T lymphocyte

development and d i f f e r e n t i a t i o n (see review by Jordan and Robinson,

1981 and by Owen et a l . 1 9 7 7 ) . i t has become apparent that the

thymus does not play a ' s t a t i c ' role i n t h i s context but, rather.

i s involved i n a precise ontogenetic sequence. Early work by

Moore and Owen (1967) on parabiosed chick embryos showed that

there was an input of haemopoietic stem c e l l s (T and B lymphocytes

are believed to derive from a common stem c e l l source) into the

embryonic thymus. Owen and R i t t e r (1969) showed (using an organ

culture technique) that stem c e l l inflow to the, thymus begins a t

a precise time. Thus explants of 6-day chicken thymuses f a i l e d

to become lymphoid in^ v i t r o , whereas 7-day old thymuses displayed

lynphocyte development when placed i n cultiore. The quail-chick

chimera system has subsequently proved valuable i n analysing the

b r i e f periods of development when the thymus becomes attractive

for stem c e l l s (see Le Douarin, 1977). After entering the thymus,

stem c e l l s p r o l i f e r a t e i n the thymic cortex and then lymphocytes

migrate towards the medulla (Borvim, 1968). Lymphocytes are thought

to leave the thymus through the medullary venules (Saint-Marie, 1973).

I t has been suggested that intrathymic MHC r e s t r i c t i o n of mammcdian

T c e l l s i s dependent on contact of thymus lyn^jhocyte with the thymic

epithelium during ontogeny, and i s determined by the haplotype of

the thymic e p i t h e l i a l c e l l s rather than the thymus-lymphocytes

themselves (Zinkernagel, 1978). This r e s t r i c t i n g property of thymus

e p i t h e l i a l c e l l s i s believed to extend through ontogeny at l e a s t

u n t i l juvenile stages (see Triesman, 1981). Organ culture studies

on the thymus (see reviews by Robinson, 1980; Robinson and Owen,

1976, 1977; Mandel and Kennedy, 1978) and sequential thymectomy

experiments (Shimamoto, 1980) reveal that there i s a sequential

emergence of T lymphocyte populations i n mammals.

Phylogeny of T and B equivalent lymphocytes

The central importeuice of ontogenetic events i n the establishment

of the immune system i n mammals and birds makes the study of the

phylogeny of the immune system p a r t i c u l a r l y important. The

relationship between the development of the individual and the

evolution of species and lineages has been an enduring question

i n evolutionary biology. Ontogeny recapitulates phylogeny was

Haekel's answer i n the nineteenth century (see Gould, 1978).

Although a b e l i e f i n recapitulation i s no longer tenable, further

understanding of the relationship between these two phenomena

i s of continuing i n t e r e s t . Heterochronic differences i n the

ontogeny of diverse inmiune systems highlight the importance of

phylogenetic study, of not only vertebrates but invertebrates

too. Developmental and comparative immunology i s now a well

established area of research, and several symposia (see Hildemann

and Benedict, 1975; Wright and Cooper, 1976; Solomon and Horton,

1977; Horton, 1980; Manning, 1980; Solomon, 1981), review a r t i c l e s

(Du Pasquier, 1973, 1976; Borysenko, 1976; Cohen, 1977) and books

(Meuining and Turner, 1976; Cooper, 1976; Gershwin and Cooper, 1978;

Marchalonis, 1977), have defined the central issues of this research

f i e l d . Primordial cell-mediated immunity, characterised by

cytotoxic r e a c t i v i t y following a l l o g r a f t s e n s i t i s a t i o n and by

s p e c i f i c a l l y inducible memory, has been demonstrated in parazoans,

coelenterates, annelids and echinoderms. Furthermore, mixed

lymphocyte culttare (MLC) r e a c t i v i t y has been demonstrated i n

tunicates. These findings suggest that immunocytes functionally-

equivalent to c e r t a i n mammalian T lymphocytes can e x i s t i n the

absence of a thymus. Thus, although invertebrate immunocytes

often develop within s p e c i a l c e l l u l a r aggregations (such as the

a x i a l organ of echinoderms and the g i l l - a s s o c i a t e d nodules of

tunica tes) , no true thymus e x i s t s i n einy backboneless animal.

Although htamoral factors are known to augment phagocytosis i n many

invertebrates, no immunoglobulin (Ig) molecules have yet been found.

Functionally-equivalent T and B c e l l s , s p e c i f i c immunological

memory and Ig's e x i s t i n a l l vertebrates, including agnathan fishes

which are the only vertebrates where there i s no convincing evidence

for the presence of a thymus. (Good, Finstad, Pollara and Gabrielsen,

(1966) have described cui aggregation of lymphocytes i n the l a r v a l

lamprey which they think may be a protothymus, and Riviera, Cooper,

Reddy and Hildemann (1975) suggest the presence of a protothyimis

within the velar muscle complex of the P a c i f i c hagfish). T c e l l -

equivalent functions, such as acute graft rejection and strong

MLC reactions, are two of the functional assays associated with

dispa r i t y a t the MHC. The presence of these reactions i n 'advanced'

amphibians (anurans), 'advanced' fishes ( t e l e o s t s ) , birds ('advanced'

r e p t i l e s ? ) and mammals, but t h e i r absence i n 'primitive' fishes

(e.g. agnatheuis, chondrichthyans and chondrosteans), 'primitive'

amphibians (urodeles and apodans) and reptileis, has led Cohen and

Co l l i n s (1977) to suggest that the MHC may have evolved independently

on four separate occasions during vertebrate phylogeny, and may

r e f l e c t convergent gene evolution. I t seems l i k e l y , however, that

components of the MHC also e x i s t i n some of the 'primitive' forms,

but a l l e l i c polymorphism may be minimal i n these.

The functional dissociation of the ectothermic immune system

into heterogeneous populations of lymphocytes has been a major

consideration of conparative immunology, and a variety of techniques

have been used for t h i s purpose. D i f f e r e n t i a l mitogenesis has been

of importance i n t h i s respect. I n mammals different mitogens

s p e c i f i c a l l y stimulate either T or B lynphocytes and studies using

8

these mitogens have now been performed i n a l l vertebrate groups

(see Chapter 2 for review).' A second potent method for study of

lymphocyte heterogeneity i n poikilotherms has involved the use of

hapten-carrier experiments (see Chapter 5 for review). Lymphocyte

cooperation (suggesting at l e a s t two different lymphoid populations)

i n himioral immune responses heis been suggested i n both teleost

f i s h and amphibians, but seems to be absent i n agnathans ( F u j i i ,

Nakagawa and Murakawa, 1979).

The extent to which immune maturation i s dependent upon the

thymus throughout the vertberates has been examined i n thymus

ablation experiments. Thymectomy i n the teleost f i s h , the

Mozambique mouth brooder ( S a i l e n d r i , 1973) reveals a slow ontogeny

of T-dependent immune functions i n t h i s species. Thymectomy of

4 month old f i s h s t i l l caused extended surv i v a l of scale a l l o g r a f t s ,

while at 2 months i t suppressed humoral immune r e a c t i v i t y to

subsequent challenge with sheep erythrocytes. The e f f e c t of

thymectomy on T-independent antigens was not examined by Sailendri,

so we cannot conclude that inpaired humoral immunity i s due to

deletion of T c e l l s . I t i s possible that both T and B c e l l s are

spawned i n the thymus of some f i s h (see Chapter 2) . Adult thymectomy

has no e f f e c t on the a l l o g r a f t response of rainbow trout (Botham,

Grace and Manning, 1980), as i n the l i z a r d , Calotes versicolor

(Manickavel, 1972), although a v i t a l role of the r e p t i l i a n thymus

i n lymphocyte maturation i s suggested by the use of a n t i r '-thymocyte

serum i n t h i s species, which r e s u l t s i n a profound lynphopoenia i n

thymus (and spleen) and suppresses a l l o g r a f t rejection.

Amongst the ec to thermic vertebrates, i t i s the amphibian

c l a s s , i n p a r t i c u l a r the anuran order, which has received most

attention with respect to the e f f e c t of thymectomy on immvme

maturation. The thymus i s c l e a r l y the major source of graft

r e j e c t i o n c e l l s i n both urodeles (Charlemagne and Bouillon, 1968;

Cohen, 1969, 1970; Toumefier, 1972, 1973; Charlemagne, 1974;

Fache and Charlemagne, 1975) and anurans (Cooper and Hildemann,

1965; Du Pasquier, 1965; Curtis and Volpe, 1971; Horton and

Manning, 1972; Tochinai and Ka t a g i r i , 1975; Rimmer and Horton,

1977). Although the role of the anuran thymus i n antibocty

production seems broadly oompaurable to that occurring i n mammals

(see below), the situation i n urodeles seems more uncertain in

t h i s respect. Thus responses to mammalian T-dependent antigens

are e i t h e r minimally impaired (Toumefier and Charlemagne, 1975)

or increased (Charlemagne and Ttoumefier, 1977) following thymectomy.

Possibly humoral immune regtilation i n urodeles i s not effected

by helper T c e l l s . Indeed i t may be that urodeles recognise

a l l cintigens as T-independent (see Ruben and Edwards, 1980, for

discussion).

The clawed toad (actually a pipid frog - see Deuchar, 1972),

Xenopus l a e v i s , has proved p a r t i c u l a r l y useful i n the study of

functional dichotomy of the anuran immune system into T-dependent

and T-independent con4>onents (see Cohen and Turpen, 1980, for review).

Thymectomy early i n l i f e can e a s i l y be achieved i n this species, which

does not runt a f t e r the operation (Horton and Manning, 1974).

I t has been shown that early thymectomy impairs a l l o g r a f t rejection

(Horton and Manning, 1972), abrogates humoral immune r e a c t i v i t y to

10

c l a s s i c a l T-dependent antigens (K>chinai and Katagiri, 1975;

Turner and Manning, 1974; Horton, Rinnner emd Horton, 1976) and

abolishes i n v i t r o mixed leucocyte culture (MLC) r e a c t i v i t y

CDu Pasquier and Horton, 1976) and responsiveness to the T c e l l

mitogens phytohaemagglutinin (PHA) and Concanavalin A (Con A)

(Du Pasquier and Horton, 1976; Manning, Donnelly and Cohen, 1976;

Donnelly, Manning and Cohen, 1976), whereas i t has no apparent

e f f e c t on antibody production to c l a s s i c a l T-independent antigens

( C o l l i e , Turner and Manning, 1975; Tochinai, 1976) or on i n vitro

lyii5)hocyte stimulation by the putative B c e l l mitogens

E . c o l i lipopolysaccharide (LPS) and p u r i f i e d protein derivative

of tuberculin (PPD) (Manning e t a l . , 1976). Horton and Sherif's

(1977) sequential thymectomy studies have suggested the p o s s i b i l i t y

of sequential establishment of different T c e l l subsets i n Xenopus

(see Chapter 4 ) .

The work i n t h i s Thesis atten5>ts to gain further insight into

the development of thymus function and lymphocyte"' heterogeneity

i n Xenopus. Chapter 2 examines the culture conditions necessary

for obtaining good mitogen responses i n thymus and spleen to putative

T and B c e l l mitogens, and forms the basis for work i n the next

Chapter. Chapter 3 investigates the ontogeny of mitogen reactive

c e l l s i n thymus and spleen, to gain d i r e c t insight into the time

when lymphocytes responsive to putative T and B c e l l mitogens f i r s t

appear, and to follow t h i s responsiveness over the immunologically-

i n t e r e s t i n g period of metamorphosis. The p o s s i b i l i t y of T lymphocyte

heterogeneity i n Xenopus i s assessed i n thymectony experiments

performed i n Chapter 4. This work centres on the i n v i t r o

11

p r o l i f e r a t i v e c a p a b i l i t i e s (mitogen and MLC r e a c t i v i t y ) of

lyaaphocytes taken from toadlets thymectomised i n early or mid-

l a r v a l l i f e . Chapter 5 looks a t the i n vivo ontogeny of antigen-

reactive T-and B-eq\iivalent c e l l s i n the spleen with the aid of

T-dependent and T-independent hapten-carrier coii5>lexes. As i n

Chapter 3, i t i s the l a r y a l and perimetamorphic periods which

are the main focus of study. The ontogeny of splenic antibody-

secreting c e l l s , and the p o s s i b i l i t y of antigen production within

the thymus are also b r i e f l y considered i n Chapter 5. The

penultimate Chapter i s concerned with the h i s t o l o g i c a l changes

occurring i n the thymus over metamorphosis. I t also examines

the h i s t o l o g i c a l outcome of organ culturing the l a r v a l thymus.

The major findings from the work i n t h i s Thesis are outlined

i n Chapter 7.

12

CHAPTER 2

Lymphocyte r e a c t i v i t y to 'T' and 'B' c e l l mitogens ; Studies on Thymus

and Spleen of the adult

Introduction

Hie d i f f e r e n t i a l responsiveness of lyn^jhocytes to mitogens has

been a major means of distingviishing T and B c e l l s i n mammals and birds

Greaves and JanOssy, 1972). The phytomitogens PHA, an extract of

the red kidney bean Phaseolus vulgaris (Nowell, 1960), and Con A,

an e x t r a c t of the Jack bean Canavadia ensiforois (Powell and Leon,

1970), exclusively stimulate T c e l l s . In contrast LPS, an extract

of the bacterium Escherichia c o l i , euid PPD induce proliferation

exclusively i n B c e l l s . Pokeweed mitogen (PWM), an extract of the

stem of Phytolacca americana (Borejeson et a l . , 1966), seems to

stimulate both T and B lyn5)hocytes.

The use of these mitogens i n a variety of endothermic vertebrates

has demonstrated the presence of both T- and B-mitogen responsive

c e l l s i n the peripheral lymphoid system. In contrast, the thymus

was shown to contain lymphocytes responsive only to T c e l l mitogens

(Robinson and Owen, 1976) suggesting that t h i s central lymphoid organ

i s e x c l u s i v e l y a T c e l l domain. Furthermorer the iu^xjrtance of

the thymus early i n ontogeny for the subsequent establishment of

functional T c e l l s was confirmed by the absence of T mitogen

responses i n the periphery following early thymectomy.

The phylogeny of lymphocyte heterogeneity and, in p a r t i c u l a r ,

the role of the thymus i n the establishment of functionally-distinct

lynphocyte populations, i s an important question in immunology.

13

D i f f e r e n t i a l mitogenesis i s a useful means of detecting lymphocytes

functionally-equivalent to mamnalian T and B c e l l s throughout the

vertebrates. Of the agnathan f i s h e s , both the hagfish (Riviere

e t a l . , 1975) and Icuiprey (Cooper, 1971) possess peripheral

lyn^hocytes that are stimulated by PEA. Amongst the elasmobranch

f i s h e s , nurse shark peripheral blood leucocytes (PEL) respond

rather poorly to T c e l l mitogens. They vuidergo blastogenesis to

Con A, but only when large amounts of mitogen (lmg/10^ c e l l s ) are

used, and the response to PHA i s found only i n a population of

gradient-separated PEL (Lopez, Sigel and Lee, 1974; Sigel, Lee,

McKinney and Lopez, 1978). I t has recently been shown, however,

that nurse shark PEL are stimulated by PHA, Con A and LPS to become

cytotoxic to xenogeneic target c e l l s (Pettey and McKinney, 1981).

In the teleosts (Osteichthyes) responsiveness to both T and B

mitogens can readily be demonstrated i n the periphery. Thus

lymphocytes from spleen and pronephros of the bream respond to

PHA, Con A and LPS (Cuchens, McLean and Clem, 1976). These l a t t e r

workers also foiond that the bream thymus contains lymphocytes reactive

to both T and B mitogens (see also Cuchens and Clem, 1977). In

contrast, the rainbow trout behaves more l i k e the mammalian thymus

i n that i t responds to Con A but not to LPS (Etlinger, Hodgins

and C h i l l e r , 1976; Chilmonczyk, 1978).

Recent studies on mitogen responsiveness i n anuran amphibians

have revealed similourities between poikilotherm and endotherm lymphoid

systems, to the extent that normal mitogen r e a c t i v i t y of Xenopus

lymphocytes to PHA and Con A i n the periphery i s dependent on the

presence of the thymus early i n development, i n contrast to the

thymic independence of LPS and PPD responsiveness (Du Pasquier and

Horton, 1976; Manning, Ebnnelly and Cohen, 1976). The work i n this

14

Chapter was designed to conplement these studies by investigating

the i n v i t r o p r o l i f e r a t i v e behaviour of advilt Xianopus lymphocytes

from the thymus to mammaliem T and B c e l l mitogens. Additionally

i t compares thymocyte responses with those of lynphocytes from

the spleen, the l a t t e r being the major peripheral lynphoid organ

i n t h i s species. The experiments investigate optimal culture

conditions, i n p a r t i c u l a r mitogen concentration and l e v e l of serum

supplementation - for mitogen r e a c t i v i t y of thymocytes and splenocytes.

Materials and Methods

(a) Rearing and care of animals

A l l animals used i n t h i s Thesis were bred and reared i n the

laboratory. Spawning was induced by in j e c t i o n of chorionic

gonadotrophin (Xenopus Ltd.) into the dorsal lymph sac of male

and female Xenopus l a e v i s (Daudin). Animals mated overnight and

the eggs l a i d were transferred to aerated, standing water and

maintained at 23°C - 2°C. After hatching, the tadpoles were

distributed a t a density of about 10 per tank (one per l i t r e ) and

were fed on nett l e powder twice a week up u n t i l metamorphosis,

afte r which toadlets were kept 4 per tank and were fed on Tubifex

worms and l a t e r on minced meat ( l i v e r or heart) twice a week.

A l l animals used i n t h i s Chapter were aged 10-15 months.

(b) Lymphocyte cultxire

Thymus and spleen were removed from the animal a s e p t i c a l l y

i n a Icuninar flow hood, following anaesthesia with MS 222 (Sandoz) .

Organs were transferred to watch glasses containing modified Leibovitz

L-15 culture medium (Flow Laboratories). For each 58 cm^ of L-15

medium the following additions were made:- 24 cm^ double-distilled 3 2- 3 water, 1.6 cm 2mM mercaptoethanol, 0.8 cm IM Hepes buffer.

15

3 3 0.8 cm penicillin/streptomycin (Flow), 0.5 cm fungizone (Flow)

and 0.5 cm^ L-glutamine (Flow). This amphibian medium was

s t e r i l i s e d by f i l t r a t i o n through a 0.22 ym 'Millex' f i l t e r (MLllipore)

Tissues were teased apart under a steremicroscope using watchmaker's

forceps. The teased tissues were then transferred to glass

centrifuge tubes and the contents allowed to s e t t l e for a minute.

The supernatant c e l l suspension, devoid of a l l large clunks, was

washed twice (using a refrigerated centrifuge at 350 x g for 10 mins)

cuid resuspended i n medium. Lymphocytes were counted i n a Neubauer

American Optical counting cheimber and their concentration adjusted

to 5 X 10^ cm .

T r i p l i c a t e lymphocyte cultures prepared from individual

adults were set up i n microtest plates (M25 ARIL, S t e r i l i n ) , which

have V-shaped wel l s . Forty y l c e l l suspension (2 x 10^ lyii5)hocytes)

were distributed to each well. Ten y l mit:ogen (or medium in control

cultures) and f i n a l l y 10 y l heat-inactJLvated feetal c a l f serum

(FCS, batch 427006, Flow) were then added (both mitogen and serum

being freshly diluted with an^jhibian medium). The extent to which

the l e v e l of serum supplementation affected lymphocyte v i a b i l i t y

and r e a c t i v i t y to various mitogens was examined:- FCS was added

to give a f i n a l concentration of either 10% or 1%; serum-free

cultures were also set up, the ten y l being replaced by 10 y l medium.

Plated cultures were placed i n a humidified incubator a t 28°C

for 2 days. At t h i s time the cultures were pulsed with 10 y l (lyCi)

t r i t i a t e d thymidine (" HTdR, s p e c i f i c actJ.vity 5 Ci mmol ,

Radiochemical Centre, Amersham). The c e l l s were then incubated

fcr a further 18-24 hours at 28°C before they were harvested with

a Skatron c e l l harvester (water wash model. Flow). Ihe glass fibre

16

d i s c s containing the recovered c e l l s from individual wells

were placed into separate glass s c i n t i l l a t i o n v i a l s , a i r dried

for 30 minutes, 0.5 cm^ tissue s o l u b i l i z e r (Protosol, New England

Nuclear) was then added to each v i a l and incubated at 55-60°C for

1 hour. F i n a l l y 5 cm" s c i n t i l l a t i o n f l u i d ( 42 cm^Liquifluor

(NEN) per l i t r e of toluene) was added. ^HTdR incorporation was

measured with a Nuclear Enterprise l i q u i d s c i n t i l l a t i o n counter.

Stimulation indices ( S . I . ' s ) for each individual experiment

were measured by dividing the mean counts per minute (cpm) for the

t r i p l i c a t e cultures with mitogen added by the mean cpm obtained

with control (no mitogen) cultures. Stimulation indices from,

on average, 8 separate escperiments provided each mean S.I. given

i n Figures 2.1-2.4. S.I.'s were considered positive only when

they reached 1.5, since the mean (- SE) coeff i c i e n t of variation

(SD/mean) i n cpm for t r i p l i c a t e cultures was 23% - 3.5%, the

highest variation being 47%.

(c) Lymphocyte v i a b i l i t y

The e f f e c t of FCS supplementation on lymphocyte v i a b i l i t y

was examined i n 72 hour mitogen-free cultures. C e l l s from both

thymus and spleen from 5 separate experiments were stadned i n

0.2% nigrosine for 10 minutes and the percentage of dye-excluding

lymphocytes calculated from the mean of t r i p l i c a t e cultures.

(d) Mitogens

The following mitogens were used:- PHA M, Con A, E . c o l i LPS

055 :B5 ( a l l from Difco), PPD 298 (Ministry of Agriculture, Fi s h e r i e s

and Food, Weybridge) and PWM (Flow Laboratories).

17

Results

(a) ^HTdR incorporation and v i a b i l i t y of lyiig>hocytes i n mitogen-

free ciiltures.

The l e v e l of ^HMR uptake after 3 days i n culture by thymocytes

and splenocytes i n the absence of mitogen i s given i n Table 2.1.

Addition of 1% FCS to spleen and thymus cultures increased mean

background cpm 2-3 fold when compared with serum-free cultures.

A further 3-4 fold increase i n cpm occurred when splenocytes

were cultured with 10% PCS. In contrast, thymocytes in 10%

FCS displayed only marginally elevated cpm when cooipared with

1% FCS cultures. Hiymocyte background cpm were noticeably

lower than splenocyte cpm for each FCS concentration. With

respect to 0% and 1% serum supplementation, this covild well be

related to the poorer v i a b i l i t y of thymus lymphocytes (35% and 55%

viable respectively) when compared with spleen lymphocytes

(65% and 79%) at 72 hours of culture. Ten per cent FCS supple­

mentation resulted i n improved v i a b i l i t y of thymus lymphocytes

(84%), d i r e c t l y con5)arable to spleen lymphocyte v i a b i l i t y (86%).

This equally good v i a b i l i l y of lymphocytes from both thymus and

spleen i n 10% FCS does not explain the very hic^i levels of ^HTdR

incorporation seen i n tihe splenocyte cultures at this l e v e l of

servim supplementation. This feature m i ^ t be related to antJ.genic

stimulation of spleen lymphocytes by FCS, a stimulation not readily

apparent i n thymus cultures. Or i t could be that 10% PCS culture

conditions are s t J . l l not optimum for thymocyt:e prol i f e r a t i o n .

(b) P r o l i f e r a t i v e responses of thymocytes and splenocytes to

T and B c e l l mitogens

The dose-response curves (based on mean S.I.'s) of thymocytes

and splenocytes to Con A, PHA and LPS are shown i n Figs. 2.1-2.3

18

respectively. Table 2.1 shows d e t a i l s of cpm recorded i n

these experiments, but only for optimally-stimulated and mitogen-

free cultures.

Con A; With 1% FCS stj^jplementation the response of

thymocytes and splenocytes to t h i s mitogen was almost i d e n t i c a l .

Maximal S.I. 's of j u s t over 12 were recorded with c e l l s from

both orgems when 0.5iig cm Con A was used. With 10% FCS, more

mitogen (2.5yg cm~^) vaa required to optimally stimulate splenocytes

and thymocytes, and maximal S.I.'s were lower. The p a r t i c u l a r l y

low mean S.I. (4.3) found with splenocytes cultured i n 10% FCS

appeared to be primarily related to the high background counts

under these serum conditions. Con A responses i n serum-free

media were not examined.

PHA: In contrast to Con A, where substantial stimulation

occurred over only a narrow dose range of mitogen, a wider range

of PHA concentrations effected good l e v e l s of proliferation.

With 10% FCS supplementation, thymocytes and splenocytes displayed

i d e n t i c a l dose-response curves. For both c e l l types 40yg cm ^ PHA

proved optimal, with meem S.I.'s of around 7. The maximal

stimulation of thymic lymphocytes was unchanged when cultured i n

1% FCS or i n the absence of serum, although under these conditions

8yg cm"" PHA also proved to be 'optimal'. Maximal stimulation of

splenocytes cultured i n 1% FCS or with no serum was almost doubled

when compared with the 10% FCS spleen cultures. Forty }ig cm ^ and

8yg cm~^ proved optimal for the 1% FCS cultures, whereas 8yg cm ^

achieved 'maximal' stimulation of serum-free cultures.

19

LPS: Splenocytes responded vigorously, especicdly i n

serum-free conditions to t h i s B c e l l mitogen, displaying a mean

maximal S.I. of 22 with the highest concentration of LPS used

(2mg cm ^ ) . Addition of FCS resulted i n a s i g n i f i c a n t l y lower

maximal S.I. (7.4). In both serum-free and 1% PCS spleen c e l l

cultures, the more LPS added, the greater the upteike of HTdR.

Thymus lymphocytes of these 10-15 month old animals responded

r e l a t i v e l y poorly to LPS when conpared with spleen cultures. In

serum-free or 1% FCS conditions, mean maximal S.I.'s were only

j u s t greater than 2, with only 4/8 and 8/10 thymus cultures

(respectively) displaying s i g n i f i c a n t S . I . ' s . However, with

10% FCS eill 13 thymocyte cultures set up consistently displayed

significeint S.I.'s with optimal LPS concentration (1 mg cm ^ ) .

The mean S.I. of these maximally-responding cxiltures was 4

(range 2.2 - 8.8) .

The potential of thymus lymphocytes to respond to 2 other

mitogens - PPD and PWM - was also b r i e f l y examined (see Fig.

2,4).

PPD: With 10% ser\am supplementation, the maximal mean

S.I. (3.3) was attained with 1.0 mg cm"" PPD, with only one culture

f a i l i n g to respond. The background mean cpm i n these experiments

was 811 - 145, whereas cpm following PPD stimulation rose to

2,365 - 270.

PWM: With 1% FCS supplementation the maximal mean S.I. of

thymocytes was 4.6 when 25 yg cm ^ PWM was used. Background mean

cpm was 790 - 206 i n contrast to the mean of 2631 - 292

20

cpm recorded i n the PWM stimulated cultures.

Combined PHA and LPS: In order to gather some circumstantial

evidence that the T and B mitogens used here were, i n fact ,

stimulating d i s t i n c t populations of Xenopus lymphocytes, an

additional experiment was carried out with splenocytes.

Uiymocytes were not used because of their r e l a t i v e l y poor

response to LPS. In t h i s expexlmsnt, cultures from the same

spleen were set up with (a) PHA (20 yg cm"" ) alone, (b) LPS

(2 mg cm~^) alone and (c) PHA and LPS combined i n the same well.

One % FCS supplementation was losed. The r e s u l t s are shown

i n Table 2.2. The stimulations achieved when the two mitogens

were combined i n culture proved to be approximately that es^jected

from adding the S.I.'s achieved by use of PHA or LPS i n separate

w e l l s . This suggests that these two mitogens may well be

stimulating separate splenocyte populations, i . e . T and B-

equivalent c e l l s respectively.

Discussion

The experiments have shown that both thymocytes and splenocytes

from 10-15 month old Xenopus respond well to the T c e l l mitogens

PHA and Con A, PHA having a broader dose-response curve than

that of Con A. The p r o l i f e r a t i v e response of c e l l s from these

2 organs to Con A was v i r t u a l l y i d e n t i c a l i n terms of optimal

mitogen dose, l e v e l of stimulation and e f f e c t of serum supplementation.

PHA r e a c t i v i t y was also comparable i n the two organs, although

maximal stimulation of thymocytes was lower than that recorded

i n splenocyte cultures v*ien serum-free or 1% FCS sv^jplementation

21

conditions were used, i . e . under conditions that allow only

poor thymocyte v i a b i l i t y . In terms of mitogen r e a c t i v i t y ,

therefore, i t appears that functionally-mature T-equivalent

c e l l s e x i s t i n good numbers i n both organs i n Xenopus.

Reactivity of anuran lyophocytes to these T c e l l mitogens

has been examined i n some d e t a i l (Bufo marinus, Goldshein and

Cohen, 1972; Rana pipiens, Goldstine, C o l l i n s and Cohen, 1976;

Xenopus l a e v i s , Du Pasquier and Horton, 1976; Manning, Donnelly

and Cohen, 1976; Donnelly, Manning and Cohen, 1976; Horton and

Sherif, 1977; Green and Cohen, 1979). In terms of optimal

mitogen dose, these studies are comparable with mammalian

findings (see for example, Coutinho, Moiler, Andersson and

Bullock, 1973; Janossy and Greaves, 1971). The present

experiments on Xenopus highlight the requirement for higher

doses of mitogen to optimally stimulate lymphocytes when these

are cultured with increasing l e v e l s of PCS. This finding i s

well characterised i n mammals and i s thought to be related to

binding of mitogen to PCS proteins (see Coutinho et a l . , 1973).

The finding of good responsiveness to T c e l l iritogens i n the.

anuran thymus contrasts with the r e s u l t s from the urodele,

Ambystoma mexicanum, where no response to T c e l l mitogens was found

i n the thymus (Collins and Cohen, 1976). This i s a good example

of the d i s t i n c t differences that e x i s t between anuran and urodele

immune systems (Jurd, 1978).

The demonstiration of a subst:antial response of Xenopus

thymocytes to Con A and PHA contrasts to some extent with the

work of Donnelly, Manning and Cohen (1976), Green and Cohen (1979),

22

who reported only minimal stimulation of adult Xenopus

thymocytes with these mitogens. The use of a higher

concentration of lymphocytes per culture i n the experiments

reported here may, to some extent, explain t h i s dispeirity.

Thus experiments reported i n the thi r d Chapter of t h i s Thesis,

attempting to reduce c e l l nvmibers i n microtest plate mitogen

cultures, were not successful i n procuring good S.I.'s.

Donnelly et a l (1976) also noted that detection of residual

Con A r e a c t i v i t y i n spleens of early-thymectomized Xenopus

was dependent upon maintaining a s u f f i c i e n t l y high concentration

of c e l l s i n v i t r o . In contrast to unfractionated thymocytes,

t h e i r experiments did demonstrate an excellen'^ Con A and PHA

response i n the low density fraction of bovine serum albiimin

separated thymus lyii5)hocytes, revealing heterogeneity of T-

equivalent c e l l s i n the anuran thymus, which has been previously

demonstrated i n mammals and birds (Stobo and Paul, 1973).

Work in t h i s Chapter reveals that Xenopus thymocytes

are stimulated by the mitogen PWM, although t h i s response i s

r e l a t i v e l y poor con^jared with that to PHA and Con A. Work on

mammals suggests that PWM stimulates a subset of T lyn$>hocytes

that have helper activity,and that t h i s response can be influenced

by suppressor T lymphocytes (Greaves, 1972; Hirano, 1977;

Keigthley et a l . , 1976). In humans PWM stimulation of helper

T c e l l s can lead to polyclonal B c e l l activation (Hammerstr6m

et a l . , 1979; Lanzavecchia et a l . , 1979).

23

Under optimal conditions, the splenic response of Xenopus

to LPS was better than t±at seen with PHA or Con A. Manning

e t a l . (1976) revealed quite the opposite i n their studies.

However they added 1% FCS i n their experiments, which we have

shown are suboptimal for LPS r e a c t i v i t y . The fact that very

high concent:rations (1 mg cm ^) of LPS are required to 'optimally'

stimulate Xenopus splenocytes was, however, also demonstrated

i n t h e i r experiments. Less LPS (100-400 yg cm ^ 055 : B5

preparation) i s required to optimally stimulate splenocyt:es

i n other anphibians (Goldstine, Collins and Cohen 1976;

C o l l i n s and Cohen, 1976), and fishes (Etlinger, Hodgins and

C h i l l e r , 1976), higher doses of LPS proving to be suboptimal.

The reasons for these differences are unclear. A p a r a l l e l

between the findings i n Xenopus and experiments performed i n

mammals e x i s t s , however. Thus the more LPS (055 : B5) added

to mouse spleen c e l l s (maximum concentration used = 100 yg cm ' ^ ) ,

the greater the degree of induced p r o l i f e r a t i o n (Coutinho

e t a l . 1973).

In contrast to spleen c e l l s , Xenopus thymocytes respond

r e l a t i v e l y poorly to LPS. Nevertheless, the positive stimulation

of mciny thymocyte preparations with t h i s putative B c e l l mitogen

and also with PPD suggests the p o s s i b i l i t y of a population of

B-equivalent c e l l s witJiin the anuran thymus. However, one must

be cautious in drawing t h i s conclusion since the highest leve l s

of stimulation with LPS were seen in those thymocyte cultures

supplemented with 10% PCS. I t could therefore be that the

induced r e a c t i v i t y was achieved by synergistic interaction of

foreign serum components with mitogen, possibly effecting alteration

24

of the s p e c i f i c i t y of t h i s mitogen for B-equivalent c e l l s (see

E t l i n g e r et al^, 1976, for discussion). However, the s i g n i f i c a n t

response to t h i s mitogen i n h a l f of the serum-free thymocyte

cultures implies that the better response of thymocytes i n 10% FCS

might simply be related to increased v i a b i l i t y of LPS-reactive c e l l s

under these culture conditions. I t should also be pointed out

that LPS may be i n d i r e c t l y stimulating Xenopus T lymphocytes. Thus

LPS can, v i a interaction with macrophages, cause triggering of

some T c e l l s i n mice (Roelants, 1978). LPS-reactive Xenopus

thymocytes do, however, appear to be a d i f f e r e n t population to

PHA and Con A responsive thymocytes (see nylon wool separation

experiments performed i n Chapter 3). Although the double mitogen

stimulation experiments with Xenopus also suggest that PHA and LPS

stimulate separate spleen lymphocyte populations, similar studies

with thymocytes would ascertain that t h i s holds for the thymus.

Experiments are currently i n progress (Cribbin, Horton and

Zettergren) i n the laboratory to determine whether B-equivalent c e l l s

can be i d e n t i f i e d d i r e c t l y i n LPS stimulated thymus c e l l cultures.

These experiments involve the use of anti-Xenopus Ig reagents to

detect the appeareince of cytoplasmic-Ig-labelled lymphocytes.

LPS i s used i n mammals to stimulate B c e l l s to develop into antibody

secreting plasma c e l l s (see Melchers, 1979). In the teleost, the

trout, LPS stimulation i n v i t r o does induce B-equivalent c e l l production

i n the spleen (assayed by induction of PFC's and by the appearance of

c e l l s with the u l t r a s true ture of plasma c e l l s ) but not in the thymus

(EUinger, Hodgins and C h i l l e r , 1978) . "The finding of a B c e l l a c t i v i t y

within the anuran thymus would not be novel since functioning a n t i ­

body forming c e l l s have been found i n the thymus of diverse vertebrate

25

species, e.g. mice (Herzenberg and Herzenberg, 1974; Bosma

and Bosma, 1974; Micklem elt a l . , 1976); rabbits (Chou, 1966; Jentz,

1979), chickens (Seto, 1978), sneikes (Kawaguchi, Kina and Muramatsu,

1978), f rogs (Moticka, Brovm and Cooper, 1973; Minagawa, Ohnishi and

Murakawa, 1976) and f ishes (Sai lendri and Muthukkaruppan, 1975;

Ortiz-Muniz and S ige l , 1971).

In endotherms intrathymic antibody forming cel ls may we l l

be spawned i n the periphery and then have migrated to the thymus,

since a clear separation o f T and B c e l l systems i s believed to

ex i s t early i n t h e i r development, the thymus e f f e c t i n g T c e l l

maturation, the bursa o f Fabricius or bursal-equivalent being

the primary s i t e of B lymphocyte d i f f e r e n t i a t i o n . There i s no

d i r e c t bursal equivalent i n mammals since B c e l l d i f f e r e n t i a t i o n

i s m u l t i f o c a l , beginning i n the placenta and then l a t e r emerging

i n other s i t e s , e.g. the f o e t a l l i v e r and spleen and eventually

i n the bone marrow (Melchers, 1979; Owen, Raff and Cooper, 1975).

In lower vertebrates there i s substantial evidence that the

thymus i s the primary s i t e o f T-equivalent lynphocyte d i f f e r e n t i a t i o n

(see the Introduct ion to t h i s Thesis) but uncertainties remain

concerning the o r ig ins o f B-equivalent lymphocytes. This i s

discussed i n the next Chapter i n the l i g h t o f experiments invest igat ing

the ontogeny o f mitogen r e a c t i v i t y to putat ive T and B mitogens i n

thymus and spleen o f l a r v a l and young adult Xenopus, since these

studies provide addi t ional characterisation o f the thymocyte response

to LPS.

26

TABLE 2 .1 Comparison o f cpm obtained i n mitogen-free and

opt imally-st imulated thymus and spleen c e l l

cultvires under d i f f e r e n t conditions o f FCS

supplementation.

Mitogen Organ FCS cone. %

Optimal mitogen cone, yg cm"^

Background mean cpm - S.E.

Stim\^ated mean cpm - S.E.

Spleen 1 0.5 2,555 + 737 . 21,500 + 5,296

10 2.5 10,016 + 1,251 41,000 + 5,489

Con A 1 0.5 823 + 127 8,336 + 3,735

Thymus 0.5

+ 9,831 10 2.5 1,063 390 12,365 9,831

0 8 1,651 + 452 17,302 + 667

Spleen 1 40 3,522 +. 887 32,426 + 6,031

10 40 9,131 + 1,294 56,115 + 6,268

PHA 0 8 496 + 72 4,815 + 3,513

Thymus 1 8 1,677 + 494 7,578 + 1,793

10 40 1,936 + 633 12,726 + 4,030

0 2000 822 + 166 12,568 + 4,085 Spleen + 3,473 Spleen

1 2000 2,341 489 19,206 3,473

LPS 0 2000 302 + 33 540 + 130

Thymus 1 1000 957 + 238 2,217 + 611

10 lOOO 1,073 304 4,065 + 1,084

Although maximal S . I . ' s can be calculated from the Table, such indices

w i l l not agree precisely wi th those shown i n Figs. 2.1-2.3, since the

l a t t e r are based on differences between background and stimulated cpm

recorded i n i nd iv idua l experiments, rather than on pooled cpm data

(see Methods).

27

TABLE 2.2 E f f e c t on spleen lymphocyte p r o l i f e r a t i o n o f

combining PHA and LPS i n the same culture

Spleen 1

Mitogen cpm Mean S . I . ( ind iv idua l cpm

wells)

Spleen 2 cpm Mean S . I .

( in d i v i dual cpm wells)

1,825

2,298

3,497 2,540

1,831 1,129 1,480

29,077

LPS _3 28,389 (2mg cm ) 31,222 29,563 11.6

19,263

23,188

16,221 19,557 13.2

17,358

PHA _2 36,643 (20 yg cm ) 12,547 22,182 8.7

14,100

9,831 10,669 11,533 7.8

2mg cm LPS^ 53,979

-3 72,045 20yg cm

68,316 64,780 25.5

41,328 28,500 30,579 33,469 22.6

Figs. 2 .1 - 2.4

O no FCS supplementation

• 1% "

A 10% "

Each poin t represents the mean S . I . ( - S.E.)

calculated from (an average) 8 separate

experiments.

F i g . 2.1

28

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32

CHAPTER 3

Ontogeny of mitogen responsiveness i n thymus and spleen

In t roduct ion

P r o l i f e r a t i v e r e a c t i v i t y to both T and B c e l l mitogens i s not

an inherent property of embryonic lynphocytes. Indeed the

emergence o f mitogen r e a c t i v i t y i n metmmals i s considered to be a

marker f o r a cer ta in l eve l of funct ional maturity (Janossy & Greaves,

1972; Stobo and Paul, 1973; MDsier, 1974; Kruisbuek, 1979). The

mitogen studies i n ' t h i s Chapter were designed to investigate the

ontogeny o f T- and B-equivalent lymphocytes w i t h i n the anuran

thymus during l a r v a l and post-metcunorphic l i f e . The competence

o f in t ra- thymic l a r v a l and young toadlet lymphocytes to respond i n

v i t r o has been examined by others wi th respect to MLC r eac t i v i t y

(Du Pasquier & Weiss, 1973), but there have been no reports concerning

the development of mitogen-reactive ce l l s w i th in th i s organ. The

emergence of thymocyte r e a c t i v i t y during the f i r s t two years of

l i f e to Con A, PHA and LPS are examined here and p r o l i f e r a t i v e

responses are compared, wherever possible, w i th those occurring

i n the spleen. The experiments on ontogeny of mitogen r eac t i v i t y

pay p a r t i c u l a r a t ten t ion to the thymocyte LPS response, which

proves to be maximal i n the young toadlet . The ce l l s involved i n

the p r o l i f e r a t i v e response to th i s mammalian B c e l l mitogen are

compared w i t h thymocytes stimulated wi th PHA fo l lowing autoradiography

and nylon wool separation. The e f f e c t of metamorphosis on emergence

o f thymocyte mitogen r e a c t i v i t y i s also discussed i n some d e t a i l .

33

Materials and Methods

(a) Miniaturised c e l l cul ture technique

Larval lymphoid tissues possess r e l a t i v e l y few lynphocytes

compared wi th the adul t , so the standard technique of lymphocyte

cul ture described i n Chapter 2 had to be miniaturised. I n i t i a l

experiments wi th toadlet lymphocytes showed that reducing c e l l

nvanbers i n the standard microtest plate reduced the subsequent

s c i n t i l l a t i o n counts to an unacceptably low and variable l e v e l .

However, i t was possible t o reduce c e l l numbers and volume by

4

25% (5 x 10 lymphocytes i n lOyl) i n flat-bottomed Terasaki

microcultvure plates ( S t e r i l i n ) : t h i s yielded S . I . ' s comparable to

the standard technique (2 x 10^ lymphocytes i n 40yl) . These

ejcperiments are described below. Success wi th miniatur isat ion

made i t possible to work wi th ind iv idua l l a r v a l thymuses, but

l a r v a l spleens s t i l l proved to contain too few lymphocytes f o r

i n d i v i d u a l analysis. The standard c e l l culture technique described

i n Chapter 2 was used wi th toadlet t issues.

The miniaturised technique i s i l l u s t r a t e d i n F ig . 3 . 1 .

The l a r v a l thymus was dissected out and placed i n a microtest plate

containing medium (see Chapter 2) where i t was broken up mechanically

under a stereomiciroscope using tungsten needles. Thymocytes were

then washed by cen t r i fuga t ion f o r 10 minutes at 350 x g. The

resuspended ce l l s were counted and adjusted to 5 x 10^ lymphocytes

cm~^. Ten y l of the ce l l s and 2.5 y l o f mitogen (medium i n control

cultures) were added to each we l l o f a Terasaki p la te . F ina l ly

2.5 y l of FCS was added to give a f i n a l concentration of 1%.

34

One per cent FCS supplementation was used throughout a l l the

ontogeny escperiments, since t h i s helped keep background counts

down (althouc^ l a r v a l thymocyte backgrovmd cpm were s t i l l very

high i n the TerasaJci plates - see Results). The mitogen

concentrations used are given i n the Tables. T r ip l i ca t e lymphocyte

cultures were set up.

A f t e r 48 hours at 28°C the ce l l s were pulsed wi th 0.25 yCi

3 -3 3 HTdR (2.5 y l o f a 1 : 10 d i l u t i o n ImCi cm HTdR, spec i f ic

a c t i v i t y 5 Ci mmol ^ ) . 18 - 24 hours l a t e r the contents from

i n d i v i d u a l wel ls were t ransferred to separate posit ions on

harvesting mats of the Skatron c e l l harvester by manual p ipe t t i ng .

The mats were then inserted i n to the head o f the harvester and

the c e l l s washed thoroughly and prepared f o r s c i n t i l l a t i o n counting

as i n the previous Chapter. The c o e f f i c i e n t o f var ia t ion using

the miniaturised technique was comparable w i t h that i n the standard

technique. Therefore S . I . ' s o f 1.5 and above were considered

p o s i t i v e .

(b) Autoradiography

At the end o f the 3 day cu l tu re , instead o f harvesting the

radioact ive wells the l a t t e r were washed wi th eunphibian phosphate-

buf fe red saline (PB) and then lyiii)hocytes counted. Approximately

1 X 10^ thymus lymphocytes were resuspended i n 50 y l FCS and a

smear o f ce l l s prepared using a cytocentr ifuge (Shandon) as fo l lows .

The ce l l s were inserted i n t o cuvettes and centrifuged fo r 4 minutes

a t 600 rpm, which forces the ce l l s onto a c i r cu la r area of a

microscope s l i d e . The sl ides were then f i x e d i n methanol and a i r -

d r i ed . I n the darkrxjom they were dipped i n I I fo rd K5 nuclear

35

emulsion, fo l l owing the technique described by Rogers (1973),

and incubated f o r 1 week at 4°C to expose the s i l v e r grains.

Following photographic development they were stained wi th

Leishman's s t a i n , and a permanent mount prepared.

The method f o r counting labe l led and b l a s t lymphoid ce l l s

was as f o l l o w s : - F i f t y f i e l d s , using the microscope at xlOOO

magnif ica t ion , from the centre o f the smear were examined and a l l

lymphoid ce l l s counted and i d e n t i f i e d . Coded slides were used

and gave consistently-repeatable resu l t s . Cells were in t en t iona l ly

heavily-labelled to f a c i l i t a t e counting.

(c) Nylon wool separation

Thymocytes were separated on a nylon wool column using the

method o f Blomberg, Bernard & Du Pasqxiier (1980) . B r i e f l y 1 gm

nylon wool ("Leuko-Pak", Fenwal Laboratories - obtained from

Travenol) was washed by b o i l i n g 6 times i n d i s t i l l e d water.

I t was then dr ied and teased to remove knots, loosely packed in to

a 10 cm^ syringe and autoclaved. Lyii4>hocyte separation then

proceeded as fo l lows . The column was saturated wi th s t e r i l e

amphibian strength PBS containing 10% FCS f o r at least 1 hour

p r i o r to use. The thymocyte suspension was then added i n 1-2 cm^

medium. Two to three cm^ o f the PBS/FCS solut ion was added and

the column incubated at 30°C f o r 1 hour. A f t e r t h i s time the

non-adherent ce l l s were flushed through wi th about 20 cm^ o f

PBS/FCS, centr i fuged and resuspended i n medium and the lyn$)hocyte

concentration readjusted to 5 x 10^ cm~" f o r cul ture . The nylon

wool-passaged ce l l s were compazed f o r mitogen r e a c t i v i t y wi th

unseparated ce l l s from the same thymocyte suspension. In these

mitogen ej^eriments the miniaturised technique was used throughout.

36

(d) Experimental design

( i ) Preliminary stutiies on minia tur isa t ion

Mitogen responses and background cpm o f ind iv idua l thymus

and spleen c e l l suspensions (taken from 4-8 month o ld toadlets)

were examined using the microtest and Terasaki plates, wi th

standard and miniaturised techniques respectively.

( i i ) Ontogeny o f mitogen responsiveness

This major series o f e3q>eriments was designed to investigate

the ontogeny o f responsiveness o f thymocytes, from thymuses taken

from ind iv idua l larvae o f stage 52 (3 weeks old) and continuing

v m t i l about 2 years o f age, to the T c e l l mitogens PHA and Con A

and the B c e l l mitogen LPS. Figure 3.2 shows the relat ionship

between age and developmental stage, that has been used throughout

the thesis , and fol lows the normal table o f Xenopus laevis

development o f Nieuwkoop & Faber, 1956. As already mentioned,

the very low lymphocyte numbers found i n l a r v a l spleens precluded

mitogen studies on t h i s organ u n t i l a f t e r metamorphosis.

( i i i ) Characterisation o f the thymocyte mitogen response

This involved autoradiography and nylon wool separation

ej^eriments. The autoraciiography provided morphologic evidence

o f s t imula t ion w i t h T and B c e l l mitogens. Autoradiographs were

prepared from thymocyte cultures (taken from 6 month o ld animals)

t reated wi th the mitogens PHA and LPS. The nylon wool experiments

looked at the e f f e c t o f removing nylon wool-adherent ce l l s on

mitogen responses. Thymocyte suspensions from 4 animals (6 or

12 months o l d - see Results) were separated on nylon wool columns.

A f t e r separation, the passaged ce l l s were stimulated wi th e i ther

PHA, Con A or LPS.

37

Results

(a) Preliminary studies comparing miniaturised and standard culture

techniques.

Figiare 3.3 examines mitogen s t imulat ion o f thymus and spleen

ce l l s from ind iv idua l toadlets w i t h standard and miniaturised

techniques. Although levels o f mitogen st imulat ion of thymocytes

proved to be rather poor at 4 months o f age (see subsequent

f ind ings below), the microtest (standard technique) and Terasaki

(microtechnique) systems nevertheless proved comparable. With the

spleens (taken from 8 month o l d toadlets) good and conparable

s t imulat ions were seen w i t h both techniques. Findings wi th

thymocytes suggested that the miniaturised technique could be

en^Jloyed f o r the ontogenetic studies on the larvae.

Analysis o f background counts i s given i n Table 3 . 1 . I t

had been expected that the l eve l o f t r i t i u m counts i n the Terasaki

plates would be 25% o f those wi th the microtest system. This

proved to be the case f o r splenocytes, whereas thymocyte counts

i n the Terasaki plates were, on average, more than s i x f o l d

higher than expected from the counts recorded i n the microtest

p la tes . This d i f ference also occurred when 10% FCS supplementation

was used ( t h i s concentration o f FCS promotes greater v i a b i l i t y

o f thymocytes i n microtest plates compared wi th 1% supplementation -

see Chapter 2 ) . I t i s not known why the miniaturised technique

promotes elevated " HTdR uptake only i n the thymocyte cul tures .

Perhaps thyme cultures are more sensi t ive than spleen cultures

to i n v i t r o conditions (e.g. plate shape and c e l l number per wel l)

because the thymus contains (at the outset o f culture) a much higher

38

propertjanof rapid ly p r o l i f e r a t i n g lymphocytes than does the

spleen.

(b) Ontogeny o f mitogen responsiveness

( i ) Studies w i t h Con A

Thymus. The resxilts are given i n Tables 3.2 and 3.3 and F ig .

3.4. At the f i r s t stage examined (stage 52/3, = 3 weeks old) no

response was found; i n f ac t the wells containing mitogen a l l gave

coiants lower than that found i n control we l l s . The next stage

examined was stage 54/5 (= 4 weeks old) and again the cultures

containing Con A generally gave a depressed cpm conpared wi th

unstimulated c e l l s . Thymocytes from 2 animals i n th i s group

d id display a S . I . o f j u s t >1.5, possibly ind ica t ing the emergence

o f Con A reactive thymocytes a t t h i s time. By stage 56/7

(6 weeks old) a small response to Con A was found i n a l l ciiLtures

tested, w i t h a mean S . I . o f 1.71. This response disappears at

the onset o f metamorphosis (stage 58/9, - 7 weeks old) and recovers

only slowly a f t e r metamorphosis. At 4 months o f age s t imulat ion

was pos i t ive i n only one animal o f 6 tested, but a f t e r th i s age a l l

toadlet thymuses responsed to Con A. The ma-ximum mean leve l o f

s t imula t ion seen (mean S . I . = 10) occurred i n 1 year o ld toadlets .

Spleen. The resul ts are given i n Table 3.4 and F ig . 3.4.

At 4 months o f age, which was the ea r l i e s t tested, 3 out of 4

toadlets responded wi th mean S . I . = 2.3. Splenocyte responsiveness

to Con A remained constant (mean S . I . = 6) i n 6-9 month o ld animals,

higher levels being recorded only i n older animals. The highest

S . I . (17.6) was seen i n a 2 year o l d animal.

39

( i i ) Studies wi th PHA

Thymus. The resul ts are given i n Tables 3.5 and 3.6 and

F ig . 3.5. With t h i s mitogen, no consistent p r o l i f e r a t i v e response

was found i n the 4 l a r v a l stages tested (3-8 weeks of age). Just

one stage 56/7 larva gave a s i g n i f i c a n t ( S . I . = 1.6) response.

At 3^ months o f age ( i . e . 6 weeks a f t e r the end of metamorphosis)

2 out o f 4 animals showed a pos i t ive PHA thymocyte response. As

wi th Con A, the PHA response then increased slowly with age,

g iv ing a maximum mean l eve l ( S . I . = 18) by about 9 months of age.

This l e v e l of response appears to decline i n l a t e r l i f e , since

the mean S . I . at 2 years o f age was jus t under 6.

Spleen. The resul ts are given i n Table 3.7 and Fig . 3.5.

The youngest toadlets tested (3.5 months) gave negative responses,

but a constant pos i t ive PHA response appeared-soon afterwards, by

4 months o f age. A high l eve l of PHA stimulaUon (S.I.'=^ 18)

was seen i n spleens taken from 6-7 month o l d toadlets. In 9 and

12 month o l d animals the mean S . I . o f splenocytes to PHA was reduced,

but some ind iv idua l animals s t i l l displayed S . I . ' s o f > 10.

( i i i ) Studies w i t h LPS

Thymus. The resul ts are given i n Tables 3.8 and 3.9 and F ig .

3.6. There was no mitogenic response to LPS at the ea r l i e s t stages

tested (stages 52-55). The mitogen-treated thymocytes from these

young larvae a l l showed a depressed cpm leve l compared wi th unstimulated

c e l l s . At stage 56/7 a small response was detected i n a l l 3 larvae

tested (mean S . I . = 2.1) . LPS r e a c t i v i t y o f thymocytes could no

longer be detected a t the onset o f and jus t a f t e r the completion of

metamorphosis, but re-emerged i n 2 out of 3 toadlets tested a t 4 months

40

o f age. A f t e r t h i s time c o n s i s t e n t LPS responsiveness o f

thymocytes was seen i n young t o a d l e t s , r i s i n g to a peak mean S.li

o f = 6 iat about s i x months o f age. The thymocyte LPS response

g r a d u a l l y diminished a f t e r 6 months and by 12 ronths o f age i t had

dwindled to a low l e v e l (mean S . I . = 1.6) and t h i s was a l s o fovind

i n l a t e r l i f e . These ontogenetic s t u d i e s notonly confirm t h a t LPS

re s p o n s i v e n e s s o c c u r s i n post-metamorphic Xenopus thymus, but

r e v e a l e d t h a t the response i s found c o n s i s t e n t l y i n young t o a d l e t s ,

r e a c h i n g l e v e l s t h a t compare favouredily w i t h r e a c t i v i t y to T c e l l

mitogens.

Spleen. The r e s u l t s a r e given i n Table 3.10 and F i g . 3.6.

The youngest animals t e s t e d f o r splenocyte responsiveness to LPS

were t o a d l e t s o f 3 3 and 4 months o l d . Only 1 o f the 4 animals

t e s t e d responded. A f t e r 4 months a response r a p i d l y emerged, with

a mean S . I . a t 6 months being 7.8. IMl i k e the thymus, t h i s l e v e l

o f LPS r e s p o n s i v e n e s s i s maintained i n o l d e r animals.

(c) C h a r a c t e r i s a t i o n o f thymocyte mitogen responses

( i ) Autoradiographic evidence o f T and B mitogen s t i m u l a t i o n

These r e s u l t s confirm those obtained by s c i n t i l l a t i o n counting

i n as much as s t i m u l a t i o n with both PHA and LPS r e s u l t s i n i n c r e a s e d

l e v e l s o f ^HTdR uptake and b l a s t c e l l transformation i n thymocytes.

S i m i l a r s t u d i e s on a few spleen c e l l p r e p a r a t i o n s a l s o provided

morphologic evidence f o r enhanced " HTdR uptake i n mitogen-treated

c u l t u r e s . The percentage o f tritium-lc±)elled c e l l s i n the unstimulated

c u l t u r e s was about 1% (see Table 3.11 and F i g . 3.7) . I n the LPS-

stimvilated c u l t u r e s from the 6 month o l d animals, the percentage o f

41

l a b e l l e d c e l l s i n c r e a s e d to 5.5%. The m a j o r i t y o f these l a b e l l e d

c e l l s were l a r g e r than lOym i n diameter. I n the PHA s t i m u l a t e d

c u l t u r e s the percentage o f l a b e l l e d thymocytes i n c r e a s e d to 11.2%.

Again the m a j o r i t y o f l a b e l l e d c e l l s were o f lymphoblast morphology

(>10 m). T h i s v a l u e f o r PHA f i t s q u i t e w e l l w i t h mammalian work

where 15% o f murine thymocytes respond to Oon A (Gerhart e t a l . ,

1976). F i g u r e 3.8 (a-c) shows c y t o c e n t r i f u g e p r e p a r a t i o n s o f

thymocytes ( c u l t u r e d i n medium, LPS or PHA) t h a t have been prepared

f o r autoradiography. I t can be seen t h a t thymocytes are a g g l u t i n a t e d

i n the mitogen t r e a t e d c u l t u r e s , p a r t i c u l a r l y w i t h PHA. P r o l i f e r a t i n g

lymphoblasts were g e n e r a l l y h e a v i l y l a b e l l e d (see F i g . 3.8,d)

although some b l a s t s , w i t h fewer exposed s i l v e r g r a i n s , were a l s o

found.

( i i ) The e f f e c t o f nylon wool treatment on T and B mitogen

is t i m u l a t i o n

These experiments were designed to look a t the e f f e c t o f removing

nylon wool adherent c e l l s on the p r o l i f e r a t i v e response of thymocytes

to p u t a t i v e T and B mitogens. Of the 4 animals used i n these

experiments, 2 were s i x months o l d ( i . e . a t an age when the maximum

response o f thymocytes to LPS o c c u r s ) and 2 were twelve months o l d

(when thymocytes respond w e l l only to T c e l l mitogens) . The r e s u l t s

a r e shown i n F i g . 3.9. I n the younger animals a good response to

LPS was found i n c o n t r o l , non-nylon wool t r e a t e d c e l l s ( S . I . ' s = 6.5

and 3 . 5 ) . Mitogen responses to Con A and PHA were a l s o as expected.

Passage through nylon wool e l i m i n a t e d the response to LPS ( S . I . ' s

now 0.9 and 0 . 3 ) . I n t e r e s t i n g l y responses to both T c e l l mitogens

were a l s o a p p r e c i a b l y reduced, but r e a c t i v i t y to these g e n e r a l l y

remained a t around 50% the l e v e l found i n unseparated thymocytes.

42

I n the thymocyte c u l t u r e s prepared from the 2 o l d e r animals

the l e v e l o f LPS response i n unseparated c e l l s was f a i r l y low

( S . I . ' s = 2.5 and 2.1) but the response to the T c e l l mitogens

was s t i l l c o n s i s t e n t l y good. Nylon wool passaged thymocytes from

these o l d e r animals showed no response to LPS. Thymocyte r e a c t i v i t y

to PHA was now e i t h e r u n a f f e c t e d or i n c r e a s e d by nylon wool passage.

Con A r e a c t i v i t y i n t h e s e 1 y e a r o l d animals was only s l i g h t l y

lowered by nylon wool passage.

D i s c u s s i o n

By about m i d - l a r v a l development, anuran l a r v a e are competent

to r e j e c t s k i n a l l o g r a f t s (Horton, 1969) and respond to c i r c u l a t i n g

thymus-dependent a n t i g e n s (Kidder, Ruben & Stevens, 1973). S i n c e

both o f t h e s e immune mechanisms n e c e s s i t a t e thepresence o f T

lymphocytes (see Horton & Manning, 1972; Horton, Rimmer & Horton,

1977) i t might be expected t h a t f u n c t i o n a l T c e l l s would be found

w i t h i n the thymus i t s e l f during l a r v a l l i f e . I n the mouse the

a b i l i t y o f thymic lymphocytes to respond to T c e l l mitogens occurs

by p a r t i o r i t i o n and i s used as the f i r s t hallmark f o r the d i f f e r e n t i a t i o n

o f f u n c t i o n a l T c e l l s (Robinson & Owen, 1976), However, the r e s u l t s

o f thymocyte mitogen r e a c t i v i t y i n developing Xenopus suggest, a t

b e s t , only a minimal response of l a r v a l thymocytes to Con A,

and no response a t a l l to PHA.

The r e s u l t s p r e s e n t e d here show t h a t an appreciable and

c o n s t a n t T mitogen response i n the Xenopus thymus emerges only a f t e r

metamorphosis and maximum l e v e l s a r e not seen i n thymus o r spleen

u n t i l about 6 months or l a t e r . T h i s seems to be r a t h e r slow when

43

compared to mammals, s i n c e s u s t a i n e d a d u l t l e v e l s of mitogen

repo n s i v e n e s s i n the thymus to Oon A and PWM a r 3 found by 3.5 days

a f t e r b i r t h i n the mouse (Robinson, 1976; Mosier, 1977). Adult

l e v e l s o f r esponsiveness i n the thymus to T c e l l mitogens are

found i n the guinea p i g by mid-gestation ( L e i p e r and Solomon,

1977) as i s a l s o the case w i t h sheep (Leino, 1978) and humans

( S t i t e s e t a l . , 1 9 7 4 ) . I n Xenopus PHA responsiveness (but rot

Con A r e a c t i v i t y ) d e c l i n e s i n o l d e r animals. This d e c l i n e i n

PHA r e s p o n s i v e n e s s w i t h age has been reported f o r mammals (0n(5dy

e t a l . , 1980).

The low and t r a n s i e n t response of l a r v a l Xenopus thymocytes

to Con A and the apparent l a c k o f t h e i r r e a c t i v i t y to PHA could be

due to s e v e r a l f a c t o r s . F i r s t , sub-optimal m i c r o c u l t u r e c o n d i t i o n s

used w i t h the l a r v a l thymus may be r e s p o n s i b l e . As mentioned

i n the R e s u l t s , t h e . T e r a s a k i p l a t e s d i d encourage high thymocyte

backgroimd cpm, which may have tended to mask any p r o l i f e r a t i o n

induced by the T c e l l mitogens. However these p l a t e s were used

s u c c e s s f v i l l y i n s e v e r a l experiments on thymocytes. The use o f an

i n a p p r o p r i a t e dose o f mitogen to s t i m u l a t e l a r v a l thymocytes seems

u n l i k e l y , s i n c e a t the o u t s e t of these ontogenetic' experiments other

doses o f Con A and PHA were t e s t e d (without any g r e a t e r s u c c e s s ) .

Secondly, the f i n a l s t a g e s o f f u n c t i o n a l maturation of T c e l l s may,

i n the l a r v a , occur predominantly i n the p e r i p h e r y , perhaps achieved

by thymus humored f a c t o r s t h a t are known to e x i s t i n amphibians

(Dardenne e t a l . , 1973). Although t h i s may be true for mitogen-

r e a c t i v e T c e l l s , Du P a s q u i e r and Weiss (1973) s t u d i e s on Xenopus

showing t h a t MLC-reactive thymocytes are p r e s e n t a t l e a s t by stage 54,

i n d i c a t e t h a t t h i s i s not the case f o r a l l lymphocyte populations.

44

I n mammals, PHA r e s p o n s i v e n e s s o f spleen c e l l s occurs p r i o r

to PHA r e a c t i v i t y o f thymocytes, suggesting post-thymic maturation

o f t h i s p o pulation (Kruisbeek, 1979). Horton and S h e r i f (1977)

found t h a t an i n t a c t thymus was necessary i n Xenopus u n t i l stage

54 to e s t a b l i s h PHA responsiveness i n the a d u l t spleen. S i m i l a r

f i n d i n g s have a l s o been shown f o r Con A (Manning and C o l l i e , 1977).

P e r i p h e r a l T c e l l s capable o f r e a c t i n g to T c e l l mitogens could

t h e r e f o r e be e j ^ e c t e d to be found i n the spleen during l a t e l a r v a l

l i f e . (Experiments were i n f a c t c a r r i e d out on pooled l a r v a l s p l e e n s ,

but the r e s u l t s were too i n c o n s i s t e n t f o r conclusions to be drawn,

probably due to mixed lymphocyte r e a c t i v i t y between a l l o g e n e i c

c e l l s ) . However, the r e s u l t s w i t h the spleen a f t e r metamorphosis

suggest t h a t i f t h i s i s so, then t h e i r numbers probably remain

s m a l l u n t i l w e l l i n t o a d u l t l i f e . S t u d i e s on s e v e r a l . l a r v a l and

post-metamorphic lymphoid organs are obviously required to i n v e s t i g a t e

t h i s i s s u e , although these e:5)eriments w i l l req'oire the pooling o f

lymphocytes from h i s t o c o n ^ a t i b l e animals to y i r l d s u f f i c i e n t c e l l s

f o r a n a l y s i s . T h i r d l y , Con A and PHA might, i n anurans, s e l e c t i v e l y

a c t i v a t e only h e l p e r T c e l l s , r a t h e r than c y t o t o x i c and MLC r e a c t i v e

s u b s e t s . T h i s i s d e a l t w i t h i n more d e t a i l i n the next Chapter,

but alloimmune lymphocyte populations are known to be e s t a b l i s h e d

i n the p e r i p h e r y e a r l y i n l i f e , whereas those h s l p e r subsets

i n v o l v e d i n humoral immianity are dependent upon an i n t a c t thymus

f o r a much longer p e r i o d o f development - even u n t i l a f t e r metamorphosis

when low doses of antigen are used (Gruenewald and Ruben, 1979) .

Indeed s i g n i f i c a n t l e v e l s o f I g "G" antibody production (which

depends h e a v i l y on T c e l l help) occur only i n the a d u l t anuran

(Du P a s q u i e r & Hainovlch, 1976).

45

D i f f e r e n c e s i n the emergence o f Con A and PHA r e a c t i v e

thymocytes i n Xenop\xs a r e p o s s i b l y i n d i c a t e d from the data

p r e s e n t e d here. I t seems t h a t the PHA responsive population

i s s l o w e r to mature, f i r s t appearing only a f t e r metamorphosis.

T h i s c o u l d be because the two T c e l l mitogens a r e s t i m u l a t i n g

d i f f e r e n t T c e l l svibsets. T h i s has been shown to occur i n

inammals ( gayry e t a l . , 1976). Stobo and Paul

(1973) have shown i n mice t h a t only mature thymic lynphocytes

respond to T c e l l mitogens, w i t h one subset responding e q u a l l y

w e l l to Con A and PHA, whereas another s u b s e t responds almost

e x c l u s i v e l y to Con A, A d d i t i o n a l work (Paetkau e t a l . , 1976;

L a r s s o n e t a l . , 1980) has shown t h a t i n some s t r a i n s o f mice

thymocytes respond w e l l to Con A, but only poorly to PHA.

However i t was found t h a t the a d d i t i o n of Con A to thymocyte

c u l t u r e s causes an i n c r e a s e i n t h e r a t e o f s y n t h e s i s o f a

c o s t i m u l t o r f a c t o r ( I n t e r l e u k i n ) , which i s r e q u i r e d f o r e f f e c t i v e

s t i m v i l a t i o n o f Con A r e s p o n s i v e lymphocytes. Moreover, a d d i t i o n

o f t h i s c o f a c t o r (taken from the supernatant of Con A s t i m u l a t e d

c e l l s ) to thymocyte c u l t u r e s enabled these to become good responders

to PHA. Whether o r not c o f a c t o r s are i n v o l v e d i n anuran lymphocyte

mitogen responses i s not y e t known.

The emergence o f LPS r e a c t i v e c e l l s i n the l a r v a l and

young a d u l t thymus (and t h e i r disappeeurance over metamorphosis)

p a r a l l e l s the ontogenetic p a t t e r n o f thymocyte responsiveness to

Con A. T h i s suggests, perhaps, t h a t the LPS response i s manifested

by c e l l s a c t u a l l y spawned w i t h i n the thymus r a t h e r than through

c e l l s c a s u a l l y m i g r a t i n g through t h i s organ from the periphery.

46

LPS r e s p o n s i v e n e s s i n the thymus reaches a maximum l e v e l a t

about s i x months and then d e c l i n e s , i n c o n t r a s t to the gradual

e l e v a t i o n o f LPS r e a c t i v i t y d i s p l a y e d by splenocytes taken from

o l d e r t o a d l e t s .

Morphologic confirmation t h a t LPS r e s u l t s i n thymocyte

s t i m u l a t i o n i n s i x month o l d t o a d l e t s was provided i n the auto­

r a d i o g r a p h i c experiments presented. Moreover, the l i k e l i h o o d

t h a t liPS r e a c t i v e thymocytes are a subset d i s t i n c t from PHA and

Con A r e a c t i v e thymocytes was suggested by the nylon wool s e p a r a t i o n

s t u d i e s . Thus, under c o n d i t i o n s which are known to s u b s t a n t i a l l y

reduce s\irface I g + ve lymphocytes ( i . e . B - equivalent c e l l s )

from the s p l e e n (Blomberg e t a l . , 1980), thymocyte r e a c t i v i t y

remains o n l y to T c e l l mitogens. I n t e r e s t i n g l y a t s i x months o f

age, b u t not a t 12 months, T mitogen r e a c t i v i t y o f thymocytes i s

cilso a f f e c t e d by nylon wool passage, in5>lying t h a t some T-equivalent

c e l l s a r e a l s o removed by t h i s procedure. T h i s could be explained

by the f i n d i n g t h a t thymocytes o f young a d u l t Xenopus are s t i l l

r i c h i n s u r f a c e I g (Du P a s q u i e r & Weiss, 1973).

Organ c u l t u r e s t u d i e s i n mamnals have c l e a r l y shown th a t B

c e l l d i f f e r e n t i a t i o n i s m u l t i f o c a l (Owen, R a f f and Cooper, 1975).

Thus B c e l l s develop f i r s t i n the p l a c e n t a (Melchers, 1979),

then i n the f o e t a l l i v e r and s p l e e n , and l a t e r i n the bone marrow

i n mice. I n Xenopus, 19Sand 7S immunoglobulins are f i r s t found

a t s t a g e 35 (2 days old) when the l a r v a i s j u s t hatching (Leverone

e t a l . , 1979), before a thymic rudiment has even appeared (Manning

& Hbrton, 1969). Furthermore, cytoplasmic Ig"*^ c e l l s a r e found

i n l a r v a l Rana kidney very e a r l y i n ontogeny (Zettergren e t a l . ,

1980). These and o t h e r f i n d i n g s (showing normal LPS r e a c t i v i t y

47

i n early-thymectomized Xenopus - see Turner and Manning, 1974)

i n d i c a t e an important extra-thymic o r i g i n o f c e r t a i n B -equivalent

p o p u l a t i o n s . B - e q u i v a l e n t lymphocyte development i n anurans

may w e l l be m u l t i f o c a l as i n mammals; i t appears t h a t the bone

marrow i s a s i t e o f B c e l l development i n a d u l t frogs ( Z e t t e r g r e n

e t a l . , 1980).

I t i s p o s s i b l e however, t h a t some B-equivalent c e l l s ubsets

d i f f e r e n t i a t e w i t h i n the anuran thymus. A number o f experimental

o b s e r v a t i o n s support t h i s h y p o t h e s i s . Following e a r l y thymectomy

i n Xenopus, both IgG and IgM antibody production to thymus-dependent

a n t i g e n s i s abrogated and a l s o r e a c t i v i t y to TN P - F i c o l l i s d r a m a t i c a l l y

i m p a i r e d (Horton, Edwards, Ruben & Mette, 1979). This hapten-

c a r r i e r complex was, u n t i l r e c e n t l y , thought to a c t i v a t e a subset

o f mammalian B lymphocytes cuid was considered to be a thymic

independent antigen(Mosier, Mond & Goldings, 1977). However,

r e c e n t s t u d i e s suggest a degree o f thymic-dependence of T N P - F i c o l l

r e a c t i v i t y i n mammals (Mond e t a l . , 1980) . C e r c a i n embryological

experiments, i n which g i l l bud regions ( c o n t a i n i n g thymic rudiments)

were t r a n s f e r r e d to p l o i c ^ - d i s t i n c t h o s t s , suggested the p o s s i b i l i t y

t h a t v i r t u a l l y a l l p e r i p h e r a l lymphocytes i n leopard frogs

n o r m a l l y o r i g i n a t e i n the thymus (Turpen, Volpe & Cohen, 1975;

Turpen & Cohen, 1976). However, more r e c e n t f i n d i n g s on the

p o t e n t i a l i t y o f the g i l l bud region suggest t h a t i n a d d i t i o n to

housing the thymus anlage, i t a l s o c o n t a i n s the pronephric rudiment,

which may be an important source o f lymphoid stem c e l l s , p a r t i c u l a r l y

of B lymphocytes (Volpe e t a l . , 1981; Zettergren e t a l . , 1980).

48

The p o s s i b i l i t y t h a t the mammalicui thymus spawns some B

c e l l s has a l s o been considered. Micklem e t a l . (1976) d i s c u s s

the p o s s i b i l i t y t h a t m u l t i p o t e n t i a l stem c e l l s e x i s t i n the murine

thymus which are capable of d i f f e r e n t i a t i n g i n s i t u i n t o B c e l l s .

J e n t z (1979) has found t h a t , by c e l l t r a n s f e r experiments, B

c e l l s i n the thymus have d i f f e r e n t r a t e s of f u n c t i o n to B c e l l s

i s o l a t e d from ot h e r organs (34 times more productive of antibody

than s p l e e n B c e l l s ) , i n r a b b i t s .

Other experimental o b s e r v a t i o n s suggest t h a t the amphibian

thymus i s not a source o f cintibody-producing c e l l s . Thus

thymocyte r e c o n s t i t u t i o n estperiments on thymectomized Xenopus,

both in^ v i v o ( K a t a g i r i e t a l _ . , 1980) and in_ v i t r o (Ruben e t a l . ,

1977 i n d i c a t e t h a t the thymus provides only helper a c t i v i t y

r a t h e r than antibody producing c e l l s .

One major f e a t u r e o f these experiments has been to demonstrate

t h a t metamorphosis e f f e c t s a temporary d i s r u p t i o n of the emergence

o f thymocyte mitogen r e a c t i v i t y . T h i s i s not s u r p r i s i n g s i n c e t h i s

p e r i o d of development i s a s s o c i a t e d w i t h severe d e p l e t i o n of

thymocyte numbers (Du P a s q u i e r and Weiss, 1973; see a l s o Chapter 6) .

Dvuring metamorphosis a d u l t - s p e c i f i c a ntigens are appearing and

t h e r e c o u l d be a danger t h a t these antigens w i l l be destroyed by

an immunologically mature immune system. However i t has been

shown t h a t t h e r e i s an i n c r e a s e d s u s c e p t i b i l i t y of the metamorphosing

anuran to t o l e r a n c e i n d u c t i o n o f MHC antigens, probably achieved

through the a c t i v i t y o f (T) suppressor c e l l s , which have been

demonstrated a t t h i s time i n Xenopus (Du Pasquier and Bernard, 1980).

A c t i v e suppression of lymphocyte r e a c t i v i t y appears to develop

i n the l a r v a e a t the very time t h a t mitogen responsiveness i s

49

beginning to emerge. The gradual diminution of suppressor

c e l l a c t i v i t y w i t h i n the thymus a f t e r a d u l t antigens are

e s t a b l i s h e d may e f f e c t , i n p a r t , the gradual i n c r e a s e i n thymus

(and spleen) mitogen re s p o n s i v e n e s s seen i n e a r l y a d u l t l i f e .

The c o - e x i s t e n c e o f mdLtogen-responsive and suppressor subsets

i n the same lymphoid t i s s u e has been demonstrated i n r a t s by

d e n s i t y g r a d i e n t s e p a r a t i o n (Rocha e t a l . , 1979). There i s

c i r c u m s t a n t i a l evidence f o r suppressor a c t i v i t y w i t h i n the

thymus of a d u l t Xenopus, s i n c e Donnelly, Manning & Cohen (1976)

were a b l e to d e t e c t T mitogen r e a c t i v e c e l l s only i n a c e r t a i n

f r a c t i o n o f s e p a r a t e d thymocytes, whereas unseparated c e l l s

f a i l e d to respond to Con A and PHA. A c t i v e suppression w i t h i n

the thymus during l a r v a l l i f e i s a l s o suspected (Du Pasquier,

p e r s o n a l communication) and t h i s could account f o r the poor

mitogen responses d i s p l a y e d by thymocytes p r i o r to metamorphosis.

50

TABLE 3.1 comparison o f HTdR cpm obtained i n unstimulated

c u l t u r e s i n T e r a s a k i and m i c r o t e s t p l a t e s

Splenocyte c u l t u r e s from 8 month o l d t o a d l e t s

40Ml i n m i c r o t e s t p l a t e

l O p l i n Terasedci p l a t e

480 3355 2963 3367 1292 1994

260 847 347 647 634

1665

T e r a s a k i m i c r o t e s t

%

Observed Expected

54.2 25.2 11.7 19.2 49.1 83.5 40.4 - 24.6% 25.0%

Thymocyte c u l t u r e s f o r 4 month o l d t o a d l e t s

M i c r o t e s t p l a t e

T e r a s a k i p l a t e

T e r a s a k i m i c r o t e s t %

177 202 501 705 485 249 724

207 116.9 209 103.5

1029 205.4 1789 253.8 754 152.5 344 142.1

1103 152.3 Observed 161.9 - 51.0% Expected 25.0 %

Each h o r i z o n t a l row o f data r e f l e c t s cpm obtained f o r the same c e l l suspension.

51

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60

TABLE 3.11 Autoradlograph Data

% of l a b e l l e d and unlabelled thymocytes in relation

to c e l l s i z e following mitogen stimulation

Proportion (%) of unleibelled c e l l s

Proportion (%) of labelled c e l l s

Control (unstimulated)

LPS

PHA

D-iOym >10iJm O-lOpm >10ym Total

98.9 1.1 0 0 0 99.1 0.9 0 0 0 99.9 0.1 O 0 0 98.4 0.4 0.4 0.8 1.2 78.0 17.8 0.5 3.7 4.2 94.3 4.8 0.9 0 0.9

1.05

96.9 0.6 0 . 2.5 2.5 95.8 0.8 1.1 2.3 3.4 95.5 0.9 0.4 3.2 3.6 92.8 0.8 2.7 3.7 6.4 77.7 11.4 0 10.9 10.9 93.8 0.3 0.5 5.4 5.9

5.45

84.3 0.6 5.3 9.7 15.0 82.4 2.7 5.4 9.5 14.9 86.4 0 6.1 7.5 13.6 95.5 1.2 2.2 1.2 3.4 74.6 14.7 1.1 9.6 10.7 87.7 2.8 2.6 6.8 9.4

11.17

0.55

45 - 1.25

,81

F i g . 3.1

Organs were transferred to a Microtest plate

where a lymphocyte suspension was prepared and

washed by centrifugation. Following counting

and adjusting to a concentration of 5 x 10^

lymphocytes cm ^,the c e l l s were distributed into

a Terasaki plate for culture. After 2 days,

the c e l l s were pulsed for 24 hours and then

hand harvested onto a glass f i b r e mat and

prepared for s c i n t i l l a t i o n coiinting.

61 F i g . 3.1

Technique of cell culture in Terasaki plates

DayJ)

10nl cells 2-5^1 mediun)/

mito>?en

^ y 2 _ 2-5ul " H th>'midine

DayJ . cells harvested

62 Fig. 3.2

I

O c O

HI

i n

i n

00

o

i n rsi

00

I / )

CO Q

63

Fig. 3.3

c

1/)

I CO Cl .

.2 "« % 2

E ^ : cfl U

I s .3 -s

3 E

CM CM

C/5

F i g s . 3.4 - 3.6

Each point represents the mean S.I. (- S.E.)

For number of animals used, see Tables 3.2-

3.10.

64

F i g . 3.4

< c I

I I

CM CN

65

Fig. 3.5

' T

I <

CM

CM

ee

c

I

CM

CM CM

cn

66

Fig. 3,6

in

BO <

C/5

Fig. 3.7

67

12

11

- Total percentage of ^ T d R labelled cells

\ :entage of labelled cells

over 10pm tlittmettr.

Percentage of

labelled cells

4 f

• ( • • • l f i j . r ' i | .- -r-:.. illilillli

Ji''it.j,a|:t4ir.r|{

l«<4')"i-' WiU'irMDii'i"'':!

-.4-. t i J i i C H

i iU i i i ' ;•>' ' | " ( " - I , • •

II <N

illfilll fiiiiiiiiilpi iiiiiii lllllli l i i l i l n i l i Pl i

Control LPS PHA

F i g . 3.8

Autoradiographs of control and mitogen stimulated

thymus c e l l s from 6 month old animals.

(a) Control c e l l s showing background l e v e l s

of l a b e l l i n g i n the absence of mitogsn.

(L-labelled c e l l ) .

(b) Thymus c e l l s stimulated with LPS.

Some clumping of the c e l l s has occurred.

Several thymocytes with exposed s i l v e r

grains can be c l e a r l y seen.

(c) Thymus c e l l s stimulated with PHA.

Considerable agglutination has occurred.

^HTdR incorporation i s extensive, with

many thymocytes s i l v e r - l a b e l l e d .

(d) Higher magnification micrograph of PHA-

stimulated thymocytes.

Note that the majority of labelled lymphocytes

are b l a s t c e l l s . Most b l a s t s are heavily-

la b e l l e d , while others possess fewer exposed

s i l v e r grains.

•* - • • •

1cm=50^d

68

69

F i g . 3.9

A l l mitogens were used a t the optimum doses .

used f o r the ontogenetic s t u d i e s . Cultures

were supplemented w i t h 1% FCS.

Animals 1 and 2 were 6 months o l d .

Animals 3 and 4 were 12 months o l d .

70 C/5

CD

• o

I

o

QTG

3-< g c o n

IS9

• • (It

71

CHAPTER 4

Mitogen and mixed lymphocyte c u l t u r e responses f o l l o w i n g

thymectomy ; Studies i n v e s t i g a t i n g p u t a t i v e T lymphocyte heterogeneity

I n t r o d u c t i o n

I n t h i s Chapter a d i f f e r e n t approach to the study o f the

ontogeny o f th^Tnus f u n c t i o n i n the Xenopus larvae i s used and two

issues, emanating from previous studies on thymectomised Xenopus,

are i n v e s t i g a t e d . A few years ago i t was demonstrated t h a t f o l l o w i n g

thymic a b l a t i o n by microcautery a t 7 o r 8 days (Horton & Manning,

1972; Rimmer & Horton, 1977), and even as e a r l y as 5 days o f age

(Horton and Horton, 1975)(when the thymus contains, i n t o t a l ,

<500 c e l l s ) , a chronic f i r s t - s e t a l l o g r a f t r e j e c t i o n eventually

occurs, u s u a l l y several months p o s t - g r a f t i n g a t 23°C. Moreover,

second-set g r a f t s , a p p l i e d a t various i n t e r v a l s (3-112 days)

a f t e r f i r s t - s e t r e j e c t i o n , were r e j e c t e d r a p i d l y w i t h i n 2-3

weeks by these early-thymectomized ( t x ) Xenopus. I t was

suggested t h a t t h i s r e j e c t i o n capacity i s probably achieved by a

thymus-independent component, p o s s i b l y one t h a t i s not normally

a c t i v e i n the non-tx animal, or o n l y o f secondary importance.

However, thymectomy experiments performed a t even e a r l i e r stages

by others (Tochinai and K a t a g i r i , 1975; Ton?>kins and Kaye, 1981)

p o i n t t o the d i s t i n c t p o s s i b i l i t y t h a t very e a r l y seeding from

the thymus o f a few lymphocytes might have occurred p r i o r t o

thymectomy a t 5-8 days o f age. I f t h i s were t r u e , the memory

response displayed by e a r l y - t x toads could w e l l be achieved by

expansion o f t h i s small T c e l l p o p u l a t i o n , p o s s i b l y induced by

h i s t o c o m p a t i b i l i t y antigens o f the g r a f t e d s k i n . I n an attempt

72

t o i n v e s t i g a t e whether such r e s t o r a t i o n occurs, experiments

were s e t up to look f o r the reappearance o f T c e l l markers,

such as r e a c t i v i t y t o the T c e l l mitogens (PHA and Con A), i n

a l l o g r a f t e d , e a r l y - t x toads, since these markers are v i r t u a l l y

undetectcible i n non-grafted, e a r l y - t x animals (Ho'rton and

S h e r i f , 1977; Manning and C o l l i e , 1977; Du Pasquier and Horton,

1976; Manning, Donnelly and Cohen, 1976; Green and Cohen, 1979).

The i n v e s t i g a t i o n concentrates on s p l e n i c lymphocytes, but

p e r i p h e r a l blood lymphocytes (PBL) are also used, since i t

has p r e v i o u s l y been shown (Horton, Horton and Rimmer, 1977)

t h a t the spleen i s p o s s i b l y not c e n t r a l l y involved i n s k i n

g r a f t d e s t r u c t i o n i n t x animals ( i . e . may not be a s i t e t h a t

houses any r e s t o r e d T c e l l p o p u l a t i o n ) , i n c o n t r a s t t o i t s

c e n t r a l involvement i n alloimmunity i n c o n t r o l toads.

The second p a r t o f t h i s i n v e s t i g a t i o n concerns a r e ­

examination o f the e f f e c t s o f l a t e r l a r v a l thymectomy on in^ v i t r o

p r o l i f e r a t i v e responses. I n a study o f the e f f e c t o f sequential

thymectomy on the a b i l i t y o f Xenopus splenocytes t o d i s p l a y mixed

lymphocyte c u l t u r e (MLC) r e a c t i v i t y and t o respond to T c e l l

mitogens, Horton and S h e r i f (1977) i n d i c a t e d t h a t the thymus may

be r e q u i r e d f o r a longer p e r i o d o f l a r v a l l i f e to e s t a b l i s h

mitogen responsiveness than i s necessary f o r the establishment

o f MLC r e a c t i v i t y . For example they showed t h a t thymectomy

a t 3-4 weeks o f age (stages 52-54) no longer abrogates s p l e n i c

MLC r e a c t i v i t y (compared to the e f f e c t o f 7 day thymectomy) ,

whereas PHA r e a c t i v i t y o f spleen c e l l s taken from other 3-4 week-

t x t o a d l e t s was abolished. They suggested the p o s s i b i l i t y

73

that MLC and PHA reactive lymphocytes i n Xenopus may therefore

be d i s t i n c t T c e l l popiHations. In order to determine more

rigorously whether MLC positive lymphocytes, that are PHA

negative can be detected within the spleen, splenocytes from

individual toadlets thymectomlzed at 3-4 weeks of age were here

tested i n both MLC and mitogen assays. Mitogen studies on

PBL i n these animals were also c a r r i e d out to examine whether

other peripheral lymphocyte populations also require a prolonged

thymus presence i n vivo to permanently establish their PHA

r e a c t i v i t y .

Materials and Methods

(a) Thymectomy

Thymectomy was performed by microcautery, following the

technique described by Horton and Manning (1972), either at

stage 47 (1 week) or at stages 52-54 (3-4 weeks). At one v;eek

of age the thymus i s a small translucent organ, approximately

lOOym i n diameter, thymus lymphocyte differentiation has begun,

as revealed by l i g h t and electron microscope studies (Horton

and Manning, 1972; Nagata, 1977; Rimmer, 1977), but few small

lymphocytes are found. By 3-4 weeks of age the thymus has

enlarged to a diameter of about 1mm, displays cortico-medullary

d i f f e r e n t i a t i o n , and contains large numbers.of small lymphocytes

(p a r t i c u l a r l y in the cortex). Thymic ablation was confirmed

when the animals were k i l l e d . Non-operated animals served as

controls.

(b) Grafting

The grafting technique of Horton and Manning (1972) was

followed. Dorsal skin (2mm^) from a non-sibling donor was transplanted.

74

After grafting the toadlets were kept at 26°C to encourage

more rapid a l l o g r a f t rejection i n the tx animals than occurs

at 2 3 ° C .

(c) Separation of PBL

A mixture of 34% T r i o s i l ("Isopaque," Vestric) and 9%

F i c o l l 400 (Pharmacia) was prepared which gave a f i n a l density

of 1 . 0 9 4 . This mixtvure was then distributed .'.nto aliquots,

autoclaved, stored i n the dark at 4°C u n t i l use. Blood was

colle c t e d under s t e r i l e conditions from anaesthetised animals

by cardiac puncture, and was transferred by pipette into

amphibian L 15 medium containing 20 linits heparin (Roche) cm ^.

The blood/medium mixture was made up to 2 cm^ by adding more medium

and was then c a r e f u l l y layered onto 2 cm^ of tl.e F i c o l l / T r i o s i l

mixture i n a 10 cm^ centrifuge tube. This was centrifuged

for 2 minutes at I60x g. The top 1 cra' of medium was then

discarded and the bottom 1 cm" containing lymphocytes at the

interface was c a r e f u l l y pipetted off. The c e l l s were f i n a l l y

washed 3 times i n medium, prior to th e i r use i n the mitogen .

assays.

(d) Mixed lyn^hocyte culture

Preparation of c e l l s for MLC i s similar to that described

for the standard mitogen culture i n Chapter 2 . Control cultures

contained 40vil spleen c e l l s from one animal at a concentration of

5 X 10^ lymphocytes cm . The mixed lymphocyte cultures used

2 0 p l of c e l l s from each animal and consisted of splenocytes from

either 2 control or 2 tx animals. Control and thymectomized

75

t o a d l e t s belonged t o the same f a m i l y . I n a d d i t i o n to 1%

FCS supplementation, l O y l Xenopus serum ( f i n a l concentration

0.5%} was added. Xenopus serum has been shown to lower

background counts, so r e s u l t i n g i n b e t t e r S.I.'s (Du Pasquier

and Horton, 1976). P i l o t experiments t e s t e d the e f f e c t o f

3 d i f f e r e n t pools o f Xenopus serum on MLC r e a c t i v i t y and the best

pool was used throughout a l l experiments described here. The

c u l t u r e s were pulsed a t 3 days w i t h 1 pCi " HTdR and c e l l s

harvested 18-24 hours l a t e r , p r i o r t o processing f o r s c i n t i l l a t i o n

counting. S t i m u l a t i o n i n d i c e s were c a l c u l a t e d by d i v i d i n g

mean cpm o f mixed c u l t u r e s by the average o f the mean cpm o f

the corresponding c o n t r o l c u l t u r e values. .S.I.'s were considered

p o s i t i v e when they reached 1.5.

(e) Experimental design

( i ) P r e l i m i n a r y studies on mitogen s t i m u l a t i o n o f PBL

Ihe a b i l i t y o f PBL t o respond t o PHA and LPS w i t h our c u l t u r e

technique had t o be assessed p r i o r t o the use o f these c e l l s i n the

2 major i n v e s t i g a t i o n s . The standard c u l t u r e technique was used

and i n v o l v e d separated PBL a t 5 x 10^ cm w i t h 1% FCS supplementation.

Both 7-day-tx and c o n t r o l t o a d l e t s (8-10 months old) were used.

PHA was added a t a f i n a l concentration o f 20 yg cm ^ and LPS

a t 2 mg cm"" . LPS was used p r i m a r i l y t o check f o r f u n c t i o n a l

v i a b i l i t y o f lymphocytes i n t x animals.

( i i ) Mitogen s t u d i e s on lymphocytes from c o n t r o l and

thymectomized alloimmune t o a d l e t s .

C o n t r o l and 7-day-tx t o a d l e t s were g r a f t e d when 8-10 months

o l d . (Grafts f o r t x hosts came from c o n t r o l or t x donors but t h i s

d i d not a l t e r t h e i r f a t e l ) Grafts were inspected every 2-3 days.

76

Eighteen weeks p o s t - g r a f t i n g splenocytes and PBL were examined

f o r mitogen r e a c t i v i t y by standard c i i l t u r e technique. The

f o l l o w i n g concentrations o f mitogens were used:- PHA - 20yg cm -3 -3 Con A - 1 yg cm ; LPS - 2 mg cm . The 18 week i n t e r v a l

between g r a f t i n g and the mitogen e^qseriments Afas selected

because second-set g r a f t s a p p l i e d t o e a r l y - t x animals a t t h i s

p o s t - t r c i n s p l a n t a t i o n i n t e r v a l should be r e j e c t e d r a p i d l y - i . e .

any T c e l l r e s t o r a t i o n o c c u r r i n g should be detectable by t h i s

time.

( i i i ) Mitogen and MLC studies on lymphocytes from t o a d l e t s

thymectomized a t 3-4 weeks o f age.

Animals used i n these studies were 8 months o l d . PBL wCfi used

o n l y f o r mitogen experiments and were c u l t u r e d w i t h the standard

technique. Splenocytes from i n d i v i d u a l animals were u s u a l l y

t e s t e d f o r mitogen r e a c t i v i t y ( m i n i a t u r i s e d technique) and i n

MLC (standard technique). LPS was again used to check f u n c t i o n a l

lymphocyte v i a b i l i t y . Doses o f mitogen used were as i n ( i ) above.

Results

(a) P reliminary studies on mitogen s t i m u l a t i o n o f PBL

Results are i l l u s t r a t e d i n Table 4.1 and Fig. 4.1. They

show t h a t although the response o f PBL from c o n t r o l boadlets t o

PHA was ge n e r a l l y low (mean S.I. - S.E. = 5.5 - 2.8), a l l the

cultxares gave p o s i t i v e S.I.'s. I n c o n t r a s t PHA stimulated

PBL c u l t u r e s on thymectomized animals gave negative S.I.'s

throughout (mean S.I. = 0.9 - 0.2. With LPS both c o n t r o l and

t x PBL gave p o s i t i v e S.I.'s (means o f 6.0 - 3.4 and 12.0 - 3.0

r e s p e c t i v e l y ) . These r e s u l t s confirm t h a t responsiveness t o T

c e l l mitogens i s abrogated by e a r l y t x , vAiereas r e a c t i v i t y to B

77

mitogens; i s l e f t i n t a c t . The data i s i n agreement with recent

findings (Green and Cohen, 1979) on mitogen r e a c t i v i t y of tx

Xenopus PBL.

(b) Mitogen studies on lymphocytes from alloimmune toadlets

Counts per minute are given in Table 4.2. S.I.'s and graft

r e j e c t i o n times are i l l u s t r a t e d i n Fig. 4.2. At 26°C the 7

control toadlets on average rejected allografts in 16 - 0.8 days.

The 7 tx animals took, on average, nearly three times as long

(43 - 8.3 days).

Following graft r e j e c t i o n , splenocytes from controls

displayed good l e v e l s of r e a c t i v i t y to both PHA and.Con A

( S . I . ' s were 15.4 (-4.4) and 7.4 (-3.2) respectively. PBL

from these animals responded well to both PHA (mean S.I. =6)

and LPS (mean S.I . = 12.2). A l l the spleens from early-tx

allografted toadlets f a i l e d to respond to Con A (mean S.I.

= 0.5 (-0.2)). Reactivity to PHA was also abolished in spleen

c e l l cultures from 4 out of 5 animals (mean S.I. = 0.9 - 0.3) ,

the other showing a dramatically impaired response ( S . I . = 1.8).

The p o s s i b i l i t y that s i t e s other than the spleen could contain

restored populations of T-mitogen-reactive c e l l s seems unlikely

i n view of the r e s u l t s with PBL from 2 tx allografted animals.

Thus PBL from these f a i l e d to respond to PHA, although they showed

good mitogen r e a c t i v i t y to LPS. I t appears, then, that a l l o g r a f t

r e j e c t i o n occurs i n tx animals in the absence of lymphocytes able

to respond to T c e l l mitogens.

78

(c) MLC and mitogen studies on lymphocytes from toadlets

thymectomized a t 3-4 weeks o f age

MLC datcK. f o r splenocytes o f c o n t r o l and t x animals i s given

i n Table 4.3. A l l 9 alloge n e i c combinations between c o n t r o l s

r e s u l t e d i n enhanced ^HTdR uptake compared w i t h background

(autologous) c u l t u r e s (mean S.I. - S.E. = 3.4 - 0.5; range

1.5-5.9). I n c o n t r a s t , only h a l f (6/12) MLC combinations

between the 11 t x animals used displayed p o s i t i v e S.I.'s. ( o v e r a l l

the mean S.I. was 1.6 - 0.3, ranging from 0.6-3.4). I t appears

t h a t i n these ea^periments thymectony during m i d - l a r v a l l i f e

has impaired the normal a a t i i r a t i o n o f MLC-reactive lymphocytes

i n some, but not a l l , t o a d l e t s .

Mitogen responses o f splenocytes and PHA o f 8 o f these

same t x animals are given i n Table 4.4. Only 3/8 splenocyte

c u l t u r e s responded t o PHA (mean S.I. = 2.2 - 0.6), the best S.I.

being 6.0. Likewise only 2/7 PBL c u l t u r e s displayed PHA

r e a c t i v i t y (mean S.I. = 1.1 - 0.3). R e a c t i v i t y o f splenocytes

and PBL t o LPS was, as expected, unaffected by m i d - l a r v a l

thymectomy (mean S.I.'s were 7.6 - 1.9 and 9.4 - 1.9 r e s p e c t i v e l y ) .

Since only one i n d i v i d u a l case o f p o s i t i v e MLC r e a c t i v i t y

(animals 10 x 11) but negative PHA r e a c t i v i t y o f both partners

occurred i n the t x group, i t seems t h a t t x a t 3-4 weeks o f age

has here impaired both PHA and MLC r e a c t i v i t i e s t o such an ex t e n t

t h a t the experiments provide no c l e a r evidence t h a t these functions

are a f f o r d e d by d i s t i n c t T lymphocyte populations.

Discussion

One major o b j e c t i v e o f t h i s Chapter was to determine

whether lymphocyte r e a c t i v i t y t o T c e l l mitogens occurs i n e a r l y -

79

t x animals t h a t have r e j e c t e d s k i n a l l o g r a f t s . Responsiveness

t o T c e l l mitogens i s o f t e n considered t o be a hallmark o f a l l

T c e l l popvilations, one which transcends t h e i r s p e c i f i c

inmunologic f u n c t i o n . Indeed T c e l l mitogens are widely used

as an i n d i c a t o r t h a t competent T lynphocytes are present i n an

i n d i v i d u a l (see Chapter 3 ) . I n the present experiments on t x

Xenopus, these mitogens have been used t o i n v e s t i g a t e the

p o s s i b i l i t y o f a general r e s t o r a t i o n o f T-equivalent c e l l s .

Considering f i r s t the speed o f g r a f t r e j e c t i o n i n the

7-day-tx t o a d l e t s , i t i s i n t e r e s t i n g t o note t h a t keeping the

animals a t 26°C r a t h e r than 23°C a f t e r t r a n s p l a n t a t i o n considerably

reduces the time taken t o e f f e c t g r a f t r e j e c t i o n ( c f . Rimmer and

Horton, 1 9 7 7 ) . E l e v a t i n g the temperature by 3°C r e s u l t s

i n a more r a p i d and uniform alloimmune r e j e c t i o n i n e a r l y - t x

toads. Perhaps acute g r a f t r e j e c t i o n a t 23°C i s c r i t i c a l l y

dependent on a n p l i f i e r T c e l l s (MLC-positive lymphocytes are

absent from t x , non-grafted animals - see Du Pasquier and Horton,

1 9 7 6 ) , whereas a t higher temperatures these c e l l s assume less

inportance.

E a r l y - t x animals t h a t have r e j e c t e d g r a f t s were shown

t o be unresponsive t o 2 T c e l l mitogens, a t l e a s t w i t h respect

to t h e i r splenocytes and PBL. As these p e r i p h e r a l popialations

o f lymphocytes responded w e l l t o LPS, the n o n - r e a c t i v i t y seems

s p e c i f i c f o r T, r a t h e r than B c e l l mitogens. The mitogen

assays were performed 18 weeks p o s t - f i r s t - s e t g r a f t i n g - i . e .

a t a time when second-set g r a f t s should be r e j e c t e d r a p i d l y -

80

t h e r e f o r e i t seems l i k e l y t h a t even acute a l l o g r a f t d e s t r u c t i o n

i n t x animals i s e f f e c t e d by lymphocytes t h a t are not able t o

respond t o PHA o r Con A. This i n t e r p r e t a t i o n f i t s w e l l w i t h

previous sequential t x data, where thymic a b l a t i o n as l a t e as

30 days s t i l l abrogates s p l e n i c PHA r e a c t i v i t y , but leaves

a l l o g r a f t r e j e c t i o n capacity t o develop q u i t e normally (Horton

and Manning, 1972; Horton and S h e r i f , 1977; Manning and C o l l i e ,

1977).

I t w i l l now be important t o determine whether MLC r e a c t i v i t y

( p a r t i c u l a r l y towards donor MHC antigens) i s restored f o l l o w i n g

a l l o g r a f t d e s t r u c t i o n i n t x animals. I f i t i s not, then i n

order t o argue t h a t a l l o g r a f t immunity i n e a r l y - t x t o a d l e t s i s

mediated by a thymus-dependent lymphocyte subset ( r a t h e r than

a thymus-independent component), one would have to suggest

t h a t t h i s i s achieved by an early-seeded T-equivalent c e l l

p o p u l a t i o n t h a t i s PHA and MLC negative. This l i n e o f research

should be a u s e f u l approach i n determining whether c y t o t o x i c T

lymphocytes ( r e c e n t l y denonstrated i n Xenopus by Bernard,

Bordmann, Blomberg and Du Pasquier, 1979) represent a separate

T-equivalent subset from MLC- and PHA-responsive lymphocytes

i n amphibians. I n t e r e s t i n g l y i t has been shown i n mammals t h a t

MLC r e a c t i v e populations can respond t o PHA and Con A, but t h a t

c e r t a i n a l l o r e a c t i v e T c e l l s are non-responsive t o these T c e l l

mitogens (Watenabe e t a l . , 1977). Indeed Hayry e t a l . (1976)

have found, by long term s e l e c t i v e c u l t u r e , t h a t there i s only

a small overlap between these f u n c t i o n s i n mice.

The second major i n v e s t i g a t i o n performed i n t h i s Chapter

i n v o l v e d a f r e s h look a t the p o s s i b i l i t y t h a t thymectomy i n mid-

l a r v a l l i f e could reveal t h a t T-mitogen-reactive c e l l s are, a t l e a s t

81

to some extent, a population of lymphocytes d i s t i n c t from

MLC-reactive c e l l s . However the studies on in vitr o p r o l i f e r a t i v e

behaviour of lymphocytes from toadlets tx at 3-4 weeks of age

have provided no c l e a r d i s t i n c t i o n between these two populations

i n terms of their degrees of thymus-dependency. The establishment

of both PHA- and MLC-reactive lymphocyte populations in the

periphery would appear to require a prolonged thymus presence

i n vivo compared with the more rapid, but equally gradual,

establishment by the thymus of i i i vivo alloimmune r e a c t i v i t y

(with respect to the l a t t e r , see Tompkins and Kaye, 1981).

Although a d i s t i n c t i o n between the duration of thymus-dependence

of MLC and PHA reactive populations was suggested by Horton and

Sherif (1977) (as noted i n the Introduction), these workers

did indicate some impairment of MLC responses regularly occurs

following thymectomy at the stages of development studied here.

Further investigations on sequential thymectomy and MLC

r e a c t i v i t y should make use of defined strai n s of Xenopus, using

MLC combinations where the mixed lymphocytes come from toadlets

with strong MHC differences. Under such cond-.tions MLC r e a c t i v i t y

of control c e l l s would be far more uniform than seen here in

experiments which, unfortunately, had to make use of si b l i n g

combinations. In Xenopus, antigens stimulating in the MLC

reaction segregate as i f controlled by a single genetic locus,

and acute graft r e j e c t i o n seems to be governed by t h i s same region.

This polymorphic genetic locus has been c a l l e d the XLA MHC system

(Du Pasquier and Kobel, 1977) and bears certain homology with

mammalian MHC's. In Xenopus, some erythrocyte antigens segregate

with MLC determinants, while others belong to different linkage

groups (Du Pasquier and Kobel, 1977). As i n endotherras, there

82

must also be minor HC euitigens i n Xenopus, since MLC identical

toadlets are s t i l l capable of rejec t i n g grafts exchanged between

each other. Du Pasquier and Kobel (1977) have shown that

graft r e j e c t i o n times can be predicted by MHC haplotype differences,

and that the l e v e l of MLC r e a c t i v i t y i s also related to t h i s

MHC d i s p a r i t y (2 haplotype differences giving greater MLC

S.I.'s than 1 haplotype difference). This linkage betv/een

MLC r e a c t i v i t y and speed of a l l o g r a f t rejection i s discussed

by Cohen and C o l l i n s (1977) who conclude that there I s some

correlation between these two functions throughout a l l vertebrates .

so far investigated, although Cohen and Horan (1977) found no such

correlation i n the newt.

83

TABLE 4.1

Controls

Mitogen stimulation of peripheral blood lymphocytes i n non-grafted toadlets ; the effect of 7-dav thymectomy

-3 PHA 20ygcm Background counts per min.

124 377 78

630 593 626 146

Stimulated counts per min. SI

LPS 2 mg cm -3

680 146 593

284 1604 1370 1037 1147 2067 464

Mean - S.E,

2220 1885 1213

2.3 4.3 22.0 1.7 1.9 3.3 3.2

- 5 7 5 ^

3.2 12.8 2.1

2.8

Mean - S.E. 6.0 ± 3.4

7-day thymectomized -3 PHA 20ygcm

599 1241 1315 1180 457 657

675 195 1196 1159 726 558

Mean S.E.

1.1 0.2 0.9 1.0 1.5 0.9 0.9 3 0.2

LPS 2 mg cm 599

1241 183

1315 1180 457 1967

16852 8570 1928 17108 v3350 2399 2945

Mean - S.E.

28.1 6.9

10.3 13.0 5.4 5.2

12.0 - 3.0

Controls

84

TABLE 4.2 Mitogen stimulation of splenocytes from control and 7-day thymectomized toadlets following graft rejection

Unstimulated cpm PHA Stimulated cpm Con A stimulated cpm

1 4034 42765 30176

2 3193 16818 13308

3 438 11960 7120

4 841 20665 nd

5 1428 13261 2122

7 day thymectomized

Unstimulated cpm

188 1

2

3

4

5

2807

2790

1426

327

PHA stimulated cpm Con A stimulated cpm

185

1427

802

1639

597

181

593

623

287

253

85

TABLE 4.3 The e f f e c t of thymectomy at stage 52-54

on MLC r e a c t i v i t y of splenocytes

CONTROLS THYMECTOMIZED

3275 20272 18612 1 271 709 794 (4.7) (3.7) (3.1) (0.9)

5328 9260 2 182 1466 (1.5) (1.9)

6919 3 1356

5 6 4 5 6

4 897 3027 2793 (2.5) (2.8)

5 1560 4903 (3.7)

6 1101

7 8 9

7 2938 56921 8361 (5.9) (4.5)

8 16295 13428 (1.6)

9 802

4 1053

7 133

10

11

5035 6512 (3.4) . (1.1)

1930 . 8283 (1.3)

10677

8 9

21.9 nd (0.7)

446 nd

5257

10 11

nd 1780 (0.9)

nd 1159 (0.6)

8074 7321 (2.0) (1.5)

2428 5702 (1.8)

3864

S.I.'s given i n parentheses

86

TABLE 4.4 The e f f e c t of thymectomy at stage 52-54 on

r e a c t i v i t y of splenocytes and PEL to PHA and LPS

Animal No. Spleen Blood PHA LPS PHA LPS

4 1.9 11.9 0.9 3.3

5 1.2 8.4 2.6 17.7

6 1.1 5.9 0.8 10.6

7 3.9 0.5

.8 1.1 1.3 1.6 5.2

9 6.0 17.6 0.2 10.3

10 1.3 7.8 0.9 13.0

11 1.3 7.6 1.0 5.4

Animals 4-11 are the same tx toadlets as i n the MLC experiments

i n Table 4.3.

F i g . 4.1

Mitogen responsiveness of peripheral

blood leucocytes (PBL) from control and

thymectomized animals. PHA was at 20iigcm

and LPS 2.0mg cm^. One per cent FCS

supplementation was used throughout.

87 CO

CO

CO

-o V 1/1

1

£

ee CM

r

eo

c o

CM CMI

a.

< X Ou

Q-

<

F i g . 4.2

Mitogen responsiveness of control and

thymectomized animals that have rejecte'^.

a l l o g r a f t s .

m PHA at 20Mgcm~'

^ Con A at l.Oygcm ^

I I LPS at 2 mg cm"'

88

E c

E

89

CHAPTER 5

Ontogeny of i n vivo lymphocyte r e a c t i v i t y to the hapten t r i n i t r o -

phenyl conjugated to a thymus-dependent and thymus independent c a r r i e r .

Introduction

Central to the fvinctioning of a l l immune systems i s the a b i l i t y

to recognise antigenic determinants, and c r u c i a l to this i n i t i a l

recognition phase i s the binding of antigen to s p e c i f i c receptors

on the surfaces of c e l l s of the immune system. An immune response

to c i r c u l a t i n g antigen can be characterised by an increase i n both

number of lymphocytes capable of binding antigen and the a f f i n i t y

of t h i s binding capacity. Although some antigens stimulate B c e l l s

d i r e c t l y to produce antibody, most ('thymus-dependent') antigens

require the involvement of helper T c e l l s before a response can be

e l i c i t e d . The importance of t h i s collaboration between T and B

c e l l was f i r s t investigated i n thymectomized and restored mice,

using xenogeneic erythrocytes as a thymus-dependent antigen (Claman

et. a l , 1966; Davies, e t a l , 1967; Mitchell and Miller, 1968).

Hapten-carrier experiments have also been centrally involved in

t h i s respect (see Mitchison, 1971). Lymphocyte collaboration has

since been demonstrated (through hapten-carrier studies) on diverse

vertebrates that include teleost f i s h (Yocum et^ a i . , 1975; Stolen

& Makela, 1975, 1976; Ruben, Warr, Decker & Marchalonis, 1977),

urodeles (Ruben, van der Hoven & Dutton, 1973) and anuran amphibians

(Ruben, 1975; Ruben & Edwards, 1980).

This chapter exploits the hapten-carrier system to investigate

the i n vivo ontogeny of T-helper c e l l a c t i v i t y and B-equivalent

90

lymphocytes i n the Xenopus spleen. In the presenc studies sheep red

blood c e l l s (SRBC) have been used as a thymus-dependent c a r r i e r , and

LPS as a thymus-independent c a r r i e r for the hapten trinitrophenyl

(TNP). Recent studies on Xenopus (Horton, Edwards, Ruben and Mette,

1979) have confirmed the thymus-dependent/independent status

respectively of these two c a r r i e r s . Carrier pre-immunisation i s

required to achieve good l e v e l s of anti-TNP responses following

TNP-RBC i n j e c t i o n s . In contrast TNP-LPS requires no such c a r r i e r

pre-immunisation and so one can examine functional B c e l l ontogeny

d i r e c t l y .

While at metamorphosis many anuran immune responses seem

to decline or disappear (e.g. thymocyte mitogen r e a c t i v i t y ) (Chapter

3) i t has recently been reported that i n vivo induced antigen-binding

splenocyte r e a c t i v i t y to a thymus-dependent hapten-carrier complex

act u a l l y increases a t t h i s time (Ruben eit al^., 1980). These authors

suggest that t h i s elevation i n 'humoral' immune r e a c t i v i t y may be

caused by an enhancement of helper T c e l l a c t i v i t y at metamorphosis,

necessary perhaps as a counterbalance to the suppressed c e l l u l a r

immunity found at t h i s time (Du Pasquier and Bernard, 1980). The

present experiments further investigate the influence of metamorphosis

on the ontogeny of humoral immune r e a c t i v i t y , and examine the p o s s i b i l i t y

that both T- cuid B-equivalent lymphocyte r e a c t i v i t y i s elevated at

t h i s time.

The work begins with a comparison of plaque-forming c e l l (PFC)

responses i n spleens taken from dif f e r e n t aged animals to three antigens •

SRBC, TNP-SRBC and INP-LPS. The ontogeny of splenocyte r e a c t i v i t y

to the 2hapten-carrier conjugates (using the more sensitive immuno-

91

cytoadherence (ICA) assay) i s then considered in d e t a i l . The

chapter ends with a consideration of the thymus as a s i t e containing

antibody-forming c e l l s , since mitogen studies e a r l i e r i n the

Thesis suggest t h i s organ contains some B-equivalent lymphocytes.

Materials and Methods

(a) Antigens and i n j e c t i o n s

Sheep red blood c e l l s (SRBC) and horse red blood c e l l s (HRBC)

i n Alsever's solution (Flow Laboratories) were washed three times

i n s a l i n e p r i o r to use i n i n j e c t i o n and/or assays).

Toadlets received a single i n j e c t i o n of 10% SRBC, 50yl per gram

body weight, v i a the intraperitoneal (i.p.) routT. Larvae v;ere

immxanized with a single i n j e c t i o n of Syl 50% SRBC i.p. Larval

i n j e c t i o n s were performed with the aid of a micropipette, drawn

from a 50yl microcap (Shandon). The t i p of the micropipette was

inserted through the ventral t a i l musculature into the peritoneal

cavity. When the pipette was withdrawn, the muscle prevented

leakage of injected material from the peritoneal cavity.

Haptenation of erythrocytes with INP followed the procedure

of Rittenberg and Pratt (1969). TNP-SPBC for i n j e c t i o n were prepared

by adding 2.5 cm^ of packed SRBC to 7 cm^ cacodylate buffer (pH 6.9),

containing 0.065g TNBS (BDH). The mixture was s t i r r e d constantly

i n a foil-covered f l a s k for 30 minutes at room temperature, and then

15 cm^ s a l i n e were added to stop the reaction. These 'heavily'-

conjugated c e l l s were then washed i n glycyl-glycine (4.5 x 10 'M) to

remove any unbound TNP, and following t h i s were washed three times

i n s a l i n e . These haptenated c e l l s were injected at the same

92

concentration and dose as the unhaptenated SRBC. Carrier

pre-immunisation was necessary to obtain a good RFC response to

TNP-SRBC (see R e s u l t s ) . Here 0.0025% SRBC were injected 2 days

p r i o r to the TNP-SRBC i n j e c t i o n , 50yl (g.b.w.) of this low c a r r i e r

dose for toadlets, 5yl for larvae.

TNP-LPS was prepared as described elsewhere (Jacobs and

Morrison, 1975) and was injected at a concentration of SOyg cm ^

for toadlets (50yl g.b.w.) and O.Smg cm for larvae (5pl).

For the PFC and ICA assays, HRBC were used as TNP-carrier,

but with a l i g h t e r haptenation protocol than used for TNP-SRBC

in j e c t i o n ; only O.0025g 1NBS was used and the reaction was stopped

a f t e r 10 minutes.

(b) PFC assay

The s l i d e haemolytic plaque assay, o r i g i n a l l y described

by Cunningham and Szenberg (1968) and modified by Du Pasquier (1970)

for amphibian studies, was used. C e l l suspensions were prepared

i n culture medium as described i n Chapter 2, but here c h i l l e d on

i c e , and adjusted to a maximum of 5 x 10^ lymphocytes cm ^. Each

PFC assaly mixture consisted of 200JJ1 spleen c e l l s mixed with 20IJ1

of 25% assay RBC and 50yl 1:10 guinea pig serum (Wellcome) as a source

of complement. Tests of several different complement batches were

made and the best was selected for use throughout a l l the experiments.

Control assays were also set up for each experiment, using 50ul

medium instead of complement; these assays were uniformly negative.

The assay mixtures were prepared on i c e , but then allowed

to reach room temperature to prevent formation of a i r bubbles when

inserted into the PFC chambers. After thorough resuspension, duplicate

lOOyl assay samples were gently pipetted into PFC s l i d e chambers

93

(Wild and Dipper, 1974). Chambers were sealed with molten

par a f f i n wax and vaseline and were then incubated for 2-3 hours

at 28°C. After t h i s time plaques could be seen macroscopically

as d i s t i n c t c l e a r areas, but they were checked under the microscope.

Only when a central Xenopus lymphoid c e l l could be seen surrounded

by mammalian red c e l l ghosts was a PFC scored. PFC were expressed

as the number 10 ^ lymphocytes. Anti-TNP PFC were estimated

from the difference i n numbers of PFC v. TNP-HKBC mini& HRQC (stC

Resultis).

Cc) ICA assay

C e l l suspensions were prepared exactly as described for

the PFC assay. Two tubes were set up for each assay, each txibe.

containing lOul t e s t RBC ( 1 % ) , and 50]il of the lymphocyte suspension.

Tubes were coded (to avoid bias when counting RFC's) and incubated

overnight at 4°C. Following gentle resuspension, the assay

suspensions were scanned microscopically using the whole .9 mm

of an American Optical Neubauer coxinting chamber. RFC were scored

when more than 3 t e s t RBC adhered to the lymphocyte membrane

(see F i g . 5.3). The number of RFC, eacpressed as RFC/10^ lymphocytes,

was calculated from at l e a s t 4 haemacytometer counts (2 from

each duplicate tube s e t up).

However, anti-TNP RFC were usually estimated from the difference

i n numbers v. TNP-HRBC-HRBC. In a few ICA experiments (see Results),

the s p e c i f i c i t y of anti-TNP r e a c t i v i t y was checked by incubating

the assay suspensions i n either TNP-glycine (10 M) or glycine (10 M)

alone.

94

(d) Experimental design

( i ) Ontogeny of induced splenocyte PFC r e a c t i v i t y

The k i n e t i c s of the PFC response of toadlets to a single

i n j e c t i o n of SRBC were examined f i r s t to ascertain optimum times

to assay. Injected animals were kept at the elevated temperature

of 26°C to t r y and increase the uniformity of the antibody

response (which had been r e a l i s e d with the a l l o g r a f t rejection

studies performed i n Chapter 5 ) . Sixteen animals, aged 9-15

months, were given a single i n j e c t i o n of SRBC and spleen c e l l

suspensions from a t l e a s t 2 separate animals were assayed on

days 4,6,7,8,9,12 and 14.

These i n i t i a l k i n e t i c experiments were preliminary to a

second s e r i e s of experiments (again carried out at 26°C), which

compared ontogeny of PFC responsiveness to the T-dependent antigens

SRBC and TNP-SRBC with that occurring to the T-independent antigen

TNP-LPS. For each l a r v a l assay i t was necessary to pool 10-20

individual spleens (depending on age) due to the very low lymphocyte

nvmibers. In these experiments some of the assays on toadlet

spleens were performed on pooled spleen suspensions, to ensure

that the l a r v a l r e s u l t s were not an a r t i f a c t of the pooling of

allogeneic c e l l s i n the PFC assay. These pooling experiments

are marked i n the Tables.

( i i ) Ontogeny of induced antigen-binding c e l l s in the spleen

La r v a l assays consisted of a pool of 10-20 spleens. I n i t i a l

experiments on larvae excunined TNP-SRBC immunisation schedules

required for good an t i TNP-HRBC binding a c t i v i t y in the spleen.

These studies included an experiment to check whether or not

95

low-dose priming with the erythrocyte c a r r i e r was necessary

to e f f e c t elevated l e v e l s of RFC's over background (assays from

sa l i n e injected animals). Ontogenetic ICA experiments ( a l l carried

out a t 23°C) with TNP-SRBC and also with TNP-LPS were then examined,

with p a r t i c u l a r emphasis on events at metamorphosis. Again some

of the toadlet assays were performed on pooled splenocyte suspensions,

to ensure that l a r v a l antigen binding r e s u l t s were not an a r t i f a c t

of pooling allogeneic lymphocytes.

( i i i ) Antibody forming c e l l s within the thymus

I n the t h i r d study, the thymus i t s e l f i s b r i e f l y examined as

a s i t e of PFC a c t i v i t y following immunisation with TNP-SRBC.

Procedures were exactly as for the spleen, but each assay was

prepared from an individual thymus. The thymuses came from

6 month old toadlets, which had been kept at 26°C after i n j e c t i o n .

Results

(aI Ontogeny of induced splenocyte PFC r e a c t i v i t y

The r e s u l t s showing the k i n e t i c s of the toadlet response to a

single i n j e c t i o n of SRBC show (see Table 5.1) that moderate le v e l s

of PFC were induced i n the spleen. In contrast to the absence of

splenic PFC recorded i n non-injected toadlets kept at 26°C, by 4 days

post-SRBC i n j e c t i o n PFC had appeared. Maximum PFC l e v e l s were

found by 6-8 days post-injection, after which time the l e v e l of

response declined. The k i n e t i c s and l e v e l of PFC responsiveness

correspond quite well with PFC r e s u l t s from other poikilotherms

(e.g. cyprinid f i s h , R i j k e r s and Muiswinkel, 1977).

Table 5.2 shows the r e s u l t s of experiments comparing the

PFC responses of different aged animals to the three antigens SRBC,

96

TNP-SRBC and TNP-LPS. No splenic PFC to any of the antigens

were seen here before 3 months of age - i . e . u n t i l post-metamorphic

l i f e . By 4 months of age (two months after the end of metamorphosis)

small numbers of PFC were fovmd to SRBC, and by 8 months, adult

anti-SRBC PFC l e v e l s were seen. With regard to TNP-SRBC, a n t i -

TNP PFC were found i n 7 month old animals, although the numbers were

rather low. The assay on the 12 month animals was performed on a

pool of spleen c e l l s and these again showed a (low level) anti-TNP

PFC response, suggesting that the lack of PFC responses to TNP-

SRBC before and over metamorphosis i s not an artefact of pooling

c e l l s . The r e s u l t s with TNP-LPS are similar to those with the T-

dependent antigens, i n that up to 3 months of age no splenic PFC

response was found. At s i x months, however, reasonable l e v e l s of

anti-TNP PFC were found after TNP-LPS inje c t i o n , as they were at

12 months.

I t appears from these r e s u l t s that a s i g n i f i c a n t change in

some immune componentts) takes place after the end of metamorphosis

which allows the subsequent detection of splenic PFC. However,

a second s e r i e s of ontogenetic experiments was undertaken using the

more s e n s i t i v e ICA assay.

(b) Ontogeny of induced antigen-binding c e l l s i n the spleen

Antigen-binding r e a c t i v i t y to TNP-SRBC and TMP-LPS during l a r v a l ,

metamorphic and adult l i f e i s shown i n Tables 5.4 and 5.5, also i n

Figures 5.1 and 5.2. Table 5.3 shows the r e s u l t s of i n i t i a l

experiments investigating the TNP-SRBC response with pooled l a r v a l

spleen c e l l suspensions. These experiments confirmed the need for

low dose c a r r i e r priming (as demonstrated by Rviben and Edwards, 1977)

for adult Xenopus, given 2 days before the main injection of TNP-

erythrocyte complex to produce good l e v e l s of TNP-HRBC splenic RFC.

97

Lack of such c a r r i e r pre-immunisation - i . e . giving TNP-SRBC

alone did, however, appear to give a low l e v e l aati-TNP-HRBC response

Cc.f. s a l i n e injected c o n t r o l s ) . Furthermore, in j e c t i o n of 50%

SRBC alone f a i l e d to induce TNP-HRBC binding above background

l e v e l s . Background l e v e l s of an t i TNP-HRBC RFC's following NaCl

i n j e c t i o n were found to be low throughout ontogeny with a mean l e v e l

of 782 - 204.

Table 5.4 and F i g . 5.1 show the r e s u l t s of the complete

ontogenetic study on anti-TNP-SRBC responses, using the standard

immunisation ( i . e . low dose priming) schedule. Anti-TNP binding

lymphocytes are found i n the e a r l i e s t - i n j e c t e d animals (injected

a t stage 52/3). I n these larvae modest RFC numbers are also seen

with HRBC: high background a c t i v i t y to erythrocyte antigens early

i n ontogeny i s expected i n view of Du Pasquier's findings on

Alytes obstetricans (1970). The mean l e v e l of anti-TNP RFC's

following TNP-SRBC i n j e c t i o n increased through ontogeny, reaching

maximum l e v e l s of 6418 - 3641 and 7684 - 3776 RFC/10^ lymphocytes

a t stages 56/7 and 58/9 respectively. Animals injected a t stage

65/6 (which i s a t the end of metamorphosis, when lymphocyte numbers

are at th e i r lowest) showed a lower mean l e v e l of anti-TNP response

than did those larvae injected a t stages 56-59 (although S.E.'s

are very high at these stages of maximal response, which precludes

any s t a t i s t i c a l significance of this, apparent difference). The

anti-TNP RFC l e v e l C3402 - 260) at stage 65/6 TNP-SRBC injected

animals i s similar to the mean anti-TNP l e v e l s found in the juvenile

and adult animals assayed.

98

Table 5.5 and F i g . 5.2 show the ontogeny of splenocyte

RFC a c t i v i t y to TNP when the hapten i s coupled to the T-independent

c a r r i e r LPS. Again a reasonable anti-TNP response i s seen i n the

e a r l i e s t stage larvae injected (52/3) 4622 - 755 RFC's). A rather

high backgrovind RFC count against HRBC was again seen in one spleen

pool at t h i s stage. By the onset of metamorphosis (stage 56/7)

ttxe l e v e l of anti-TNP RFC following TNP-LPS inj e c t i o n was s i g n i f i c a n t l y

elevated (11,937 - 3579) and t h i s high l e v e l was sustained right

through metamorphosis. At stage 65/6 mean anti-TNP RFC numbers

were 13,203 - 357. Anti-TNP RFC l e v e l s i n juvenile and adult stages

tested were 7276 - 554 and 8396 - 462 respectively, which were

s i g n i f i c a n t l y lower than the l e v e l s found over metamorphosis. Some

assays were incubated with TNP-glycine (see Tcible 5.4) to demonstrate

the s p e c i f i c i t y of the anti-TNP RFC response.

(c) Antibody forming c e l l s within the thymus

Table 5.6 shows the r e s u l t s of a b r i e f investigation of the

thymus as a centre of PFC a c t i v i t y following TNP-SRBC injection.

A low l e v e l of response was consistently found i n thymocyte suspensions

assayed 10 days af t e r low dose c a r r i e r priming, although the PFC

response was afforded almost exclusively to the c a r r i e r (SRBC).

Thus there were no PFC's i n 5/6 thymic suspensions to TNP-HRBC

and no thymic PFC a t a l l to HRBC. The thymic, a n t i - c a r r i e r PFC

response appeared to have disappeared by 14 days. Other preliminary

findings (not reported) with the ICA assay support these PFC r e s u l t s .

Thus low l e v e l s of a n t i - c a r r i e r RFC's are seen on day 10, but l i t t l e ,

i f any, response to the hapten occurs.

Discussion

The i n i t i a l experiments reported i n t h i s Chapter demonstrated

that splenic PFC can consistently be induced i n 9-15 month old toadlets

99

following i n j e c t i o n with the thymus-dependent antigen SRBC.

However, when PFC responses to SRBC, TNP-SRBC and TNP-LPS were

examined i n younger toadlets and larvae, no antibody forming c e l l s

were found i n the spleen vintil 1-2 months after metamorphosis.

This seems rather surprising, since servmi immunoglobulins in Xenopus

have been reported to be present from a very early stage of

development (stage 35 - see Leverone et al^., 197S) and because PFC

have been found i n other anuran larvae (Alytes oustetricans, Du

Pasquier, 1970); Rana catesbeiana, Moticka et ca,, 1973).

There are a number of possible reasons why PFC could not be

detected i n spleens of yoving Xenopus. F i r s t , i t i s possible that

the spleen i s not established as a s i t e of antibody production u n t i l

a f t e r metamorphosis, but t h i s seems unlikely as RFC's are found i n

t h i s organ from an early age, and antigen binding i s a prerequisite

for PFC a c t i v i t y . Perhaps the k i n e t i c s of PFC production d i f f e r

before metamorphosis and hence the l a r v a l assays were performed

a t the 'wrong' times a f t e r i n j e c t i o n . Secondly, i t seems that

metamorphosis i n Xenopus signals major changes with respect to

immunoglobulin production; these changes include alterations i n both

surface Ig expression on thymocytes and the capacity for Ig 'G'

antibody production. With respect to the l a t t e r , i t has been found

that although l a r v a l serum contains Ig'G'-like antibodies, such

antibody i s not detected as r e a d i l y as i n the adult (Du Pasquier

and Haimovich, 1976). Although deficient Ig'G' antibody production

would not appear to be a causative factor for the lack of PFC' s

encountered here (the assays measured d i r e c t plalques, presiomably

IgM-secreting c e l l s ) i t i s possible that l a r v a l Xenopus IgM antibody

i s , for some reason, unable to f i x mammalian complement e f f i c i e n t l y .

100

Possibly amphibian complement could be used successfully here,

although i t may be necessiary to use adult serum. I t has been

shown that some genes of the mammalian MHC code for, and control

expression of, complement components (see Schreffler, 1978 for

review). I f the Xenopus MHC also controls some aspects of

complement activation, i t could be that the complement genes

involved are activated only i n the adult. In t h i s respect

Du Pasquier £t al_ (1979) suggest that serologically-detectable

MHC antigens i n Xenopus are detected at or only af t e r metamorphosis.

Although no PFC were found u n t i l after metamorphosis, antigen

binding c e l l s can readily be induced i n the Xenopus spleen from

ea r l y i n ontogeny. Thus Kidder et a l . (1973) demonstrated

anti-SRBC binding c e l l s i n lairval spleens from animals injected

at stage 50 onwards. The experiments reported here employed

the hapten-carrier system to examine the ontogeny of the RFC

response of spleen c e l l s to TNP-SRBC and to TNP-LPS. Good levels

of induced antigen-binding c e l l s i n the larva to TNP after TNP-

SRBC i n j e c t i o n required low dose c a r r i e r priming. In the adult

amphibian such c a r r i e r - r e a c t i v e lymphocytes are believed to

represent T-helper c e l l s i n view of t h e i r antigen-binding c h a r a c t e r i s t i c s

(they are minimal-binding c e l l s of the non-secretory type - see

Rxiben, 1975) and t h e i r thymus-dependency (Horton £t al_., 1979).

Therefore i t would appear from the present experiments with TNP-

SRBC that some T-helper c e l l populations are fvinctioning at the

e a r l i e s t stages injected (stage 52/3). This finding i s i n

agreement with the work of Ruben, Welch and Jones, 1980. On the

other hand i n order to explain the deficient Ig'G' antibody

responses displayed by anuran larvae (see above), f u l l establishment

of T helper c e l l a c t i v i t y may develop only af t e r metamorphosis.

101

The experiments here reveal that stage 52/3 Xenopus larvae also

contain antigen-reactive, B-equivalent, splenic lymphocytes,

since antigen binding r e a c t i v i t y to TNP occurrec following TNP-LPS

administration to such smimals.

Maximum anti-TNP RFC l e v e l s following low dose SRBC priming

and TNP-SRBC i n j e c t i o n 2 days l a t e r were seen i n the spleen at the

onset of metamorphosis. This finding i s i n agreement with similar

experiments on Xenopus reported by Ruben, Welch and Jones (1980).

These workers suggest that the elevated TNP response found i n the

spleens of metamorphosing animals may be due to the effect of

metamorphosis on a population of c e l l s that normally suppresses

helper function i n l a r v a l and adult Xenopus: i f t h i s suppressor

population was s e l e c t i v e l y l o s t or blocked early i n metamorphosis,

then higher l e v e l s of anti-TNP binding c e l l s i n the spleen following

TNP-RBC in j e c t i o n might be expected, due to unrestrained T c e l l

help. However, the elevated response to TNP-LPS at metamorphosis

demonstrated here does not altogether f i t with t h e i r suggestion.

Thus the TNP-LPS r e s u l t s suggest that elevated B c e l l r e a c t i v i t y may

be occurring at metamorphosis. Alternatively, their could simply

be a temporary increase i n proportion of (antigen-binding) B c e l l s

over T c e l l s i n the small spleen found at metamorphosis (Du Pasquier

and Weiss, 1973), possibly related to reduced T c e l l migration from

the thymus that may be occurring at t h i s time (see Chapter 6) .

Whether the RFC's recorded i n the spleen against TNP-HRBC represent

B- rather than T-equivalent antigen-binding lymphocytes i s uncertain,

although i t i s pertinent to point out that the majority of such

RFC's were of the secretory type which, i t has been suggested

elsewhere (Ruben and Edwards, 19 80), are antibody-secreting

lymphocytes. f<f S C I E N C E

SECTION LibrarV

102

Very recently Jones and Ruben (1981) have re-examined the

e f f e c t of metamorphosis on the splenic antigen-binding response

to TNP when conjugated to an erythrocyte c a r r i e r . They reveal

that stage 57/8 i s a unique period i n which a (low level) a n t i -

TNP RFC response (3.5 x 10^ RFC*s/10^ lymphocytes) can occur

i n the absence of c a r r i e r (erythrocyte) pre-immunisation.

(Interestingly a low l e v e l anti-TNP response was also seen here

i n stage 57/8 euiimals injected with TNP-SRBC without low dose

SRBC priming (see Table 5.3). Jones and Ruben suggest that

an i n t e r n a l histoincompatibility between l a r v a l and adult lymphoid

c e l l s , which i s l i k e l y to be occurring at t h i s time (see Du Pasquier,

Blomberg and Bernard, 1979), provides a substitute for c a r r i e r

priming ) i . e . overrides the absolute necessity for T c e l l help).

I n t e r e s t i n g l y t h e i r experiments provide no evidence for elevated

B c e l l r e a c t i v i t y at metamorphosis, since although anti-TNP

splenic RFC numbers following TNP-LPS in j e c t i o n at stage 57/8

were higher =10,500 RFC/10^ lymphocytes) than i n the adult =4,000

RFC/10^ lymphocytes), they were no different from RFC numbers i n

the group of animals injected with TNP-LPS at stages 51-54 (10,000

RFC) . This contrasts with the findings presented here, where larvae

injected with TNP-LPS at stage 52/3 yielded s i g n i f i c a n t l y fewer

TNP-binding splenocytes than those animals injected a t stage 57/8.

The stage 52/3 larvae used i n t h i s Chapter were a l l 3 weeks old,

whereas the ages of the stage 51-54 larvae used by Jones and Rviben

were not given. I f the i r stage 51-54 animals were i n fact older

than the stage 52/3 larvae used here- i . e . had a more developed

lymphoid system (closer to metamorphosis?) than t h e i r external

stage suggested (see Ruben, Stevens and Kidder, 1972 for discussion

of t h i s age/stage effect) - t h i s could conceivably provide an

explanation for the discrepancies encountered.

103

The f i n a l experiment i n t h i s Chapter revealed that

( i n vivo) the young adult thymus contains anti-SRBC PFC (and RFC)

a f t e r TNP-SRBC immunisation, whereas anti-TNP plaques i n t h i s

organ were not detected. Although further experiments are

obviously required to investigate the significance of positive

a n t i - c a r r i e r , but negative anti-hapten antibody production, the

findings do suggest that antigen administration can achieve

PFC appearance i n the thymus. This has previously been demonstrated

i n thymuses of several vertebrate species (see Discussion i n Chapter

2 ) . The experiments here do not define whether the thymus

i s i t s e l f a s i t e for reaction to the hapten-cairier complex,

but the r e s u l t s do suggest that c e l l u l a r antibody production

i n t h i s organ i s not achieved simply by a random recirculation of

PFC, as only PFC against the c a r r i e r were found in the thymus.

Recent findings (Hsu and Du Pasquier, 1981) demonstrating the

high l e v e l s of PFC production in^ v i t r o by Xenopus thymocytes

Cfollowing i n vivo priming with DNP-KI£ and reimmunisation

i n vitrc> point again to the thymus as a major s i t e of B-equivalent

c e l l s i n t h i s species.

104

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111

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Cytocentrifuge preparations of c e l l s from

an ICA assay using splenocytes from toadlets

immunised against sheep erythrocytes (SRBC).

Rosette forming c e l l s (RFC) can be seen xn

each micrograph and comprise a central

lymphoid c e l l (small or large lymphocyte)

binding one or additional layers of SRBC.

XRBC = Xenopus erythrocyte.

XL = Xenopus lymphocyte Scale 1cm = 25vim

112

113

CHAPTER 6

Thymus histogenesis over metamorphosis ana i n organ culture

Introduction

U n t i l quite recently, studies on the ontogeny of the immune

system i n anurans have concentrated on events during embryonic and

l a r v a l l i f e . Both c e l l u l a r and humoral immunity develop prior to

metamorphosis, the acquisition of immunocompetence being concommitant

with the d i f f e r e n t i a t i o n of lymphoid ti s s u e s , p a r t i c u l a r l y the

thymus (see Horton, 1969; Kidder et a l . , 1973). In Xenopus early

thymus histogenesis has been studied i n considerable d e t a i l .

Thus u l t r a s t r u c t u r a l investigations have shown the appearance of

large basophilic c e l l s within (between the thymic e p i t h e l i a l c e l l s )

and immediately adjacent to the thymus at 3 days (stage 40) (Nagata,

1977) . Recent experimental embryological evidence on ploidy-

l a b e l l e d Xenopus reveal that the extra-thymic origin of these stem

c e l l s may well be the nephric region of the embryo (Volpe et^ a l . 1981) .

The basophilic stem c e l l s gradually increase i n number within the

thymus and are thought to be the precursors of the f i r s t small lymphocytes

seen a t around 7 days of age (Manning and Horton, 1969; Rimmer,

1978) . An e x t r i n s i c o r i g i n of thymic lymphocytes in other vertebrates

has also received considerable experimental support (see Le Douarin,

1977).

In Xenopus the immune system as a whole i s thought to undergo

major chajiges at metamorphosis (which occurs about 7-8 weeks after the

f i r s t appearance of small lymphocytes i n the thymus and the i n i t i a t i o n

of immunocompetence). Thus the number of recoverable thymocytes

i s known to drop dramatically at t h i s time (Du Pasquier and Weiss,

114

1973), alloimmune r e a c t i v i t y i s 'suppressed' (Chardonnens, 1976)

eind regulatory T c e l l a c t i v i t i e s (e.g. help and suppression) are

altered (see Discussion i n Chapters 3 and 5 and also Du Pasquier

and Bernard, 1980). Details of histologic changes in the thymus

over metamorphosis are, however, not available (see Manning and

Horton, 1969) . The f i r s t part of t h i s Chapter therefore looks

at the e f f e c t of metamorphosis on thymus histogenesis and attempts

to correlate the s t r u c t u r a l changes observed during l a r v a l ,

metamorphic and early post-metamorphic l i f e with the numbers of

thymic lymphocytes that can be recovered when the organ i s teased

apart iri v i t r o .

The second part of t h i s Chapter i s a preliminary investigation

on the e f f e c t of organ culture on l a r v a l thymus lymphopoiesis.

Organ culturing of the amphibian thymus should, i f technically

f e a s i b l e , prove to be a powerful tool to examine development and

d i f f e r e n t i a t i o n of thymic lymphocytes (e.g. i n early l a r v a l l i f e and

at metamorphosis) i n the absence of in^ vivo c e l l u l a r migrations or

humoral e f f e c t s . I t has been shown i n mice (Robinson, 1980;

Mandel and Kennedy, 1978) and chickens (Sallstrom and Aim, 1973) that

transfer of the thymus early i n ontogeny into organ culture does not

prevent p r o l i f e r a t i o n and d i f f e r e n t i a t i o n of lymphocytes. Precursor

c e l l s , present i n the embryonic thymus before removal, continue to

d i f f e r e n t i a t e normally with respect to the i r surface antigenic

markers and T c e l l functions, t h i s d i f f e r e n t i a t i o n p a r a l l e l i n g the

developmental sequence observed i n vivo.

Materials and Methods

Ca) Thymus morphology

The f i r s t experiment considered the number of thymic lymphocytes

recoverable from animals of various stages of development, including

115

the period over metamorphosis. The c e l l s were prepared as

described i n Chapter 3. For each stage examined, an average of 10

animals was used.

For the h i s t o l o g i c a l studies, 3 animals were selected at

the following stages; stage 54/5 (4 weeks), stage 58 (7 weeks),

stage 60 (8 weeks) , stage 66 (9 weeks, the end of metamorphosis),

one week post-metamorphosis, 2 weeks post-metamorphosis and 3 weeks

post-metamorphosis. The selected animals were heavily anaesthetised

i n MS 222 then fixed i n Bouins f i x a t i v e . After dehydration, the

specimens were wax embedded cind Bum sections cut and stained with

haematoxylin and eosin. Thymuses from two additional stage 66 animals

were prepared for l\im sectioning. These thymuses were fixed in cold

gluteraldehyde (5% gluteraldehyde i n O.IM sodium cacodylate) overnight,

then washed i n O.lM cacodylate buffer for 24 hours and post-fixed

i n osmium tetroxide for half an hour. After washing for ha l f an

hour i n O.IM cacodylate buffer, the specimens were dehydrated i n

methanol, cleared i n propylene oxide and then transferred to a

1:1 mixture of epon/epoxypropane overnight. They were f i n a l l y

embedded i n epon, the p l a s t i c being allowed to polymerise at 60°C

for 48 hours. lym sections were cut using a Reichert ultramicrotome

and were stained i n toluidine blue. To assess the location of

p r o l i f e r a t i n g lymphocytes within the young adult thymus, two 3-week

post-metamorphic toadlets were injected with t r i t i a t e d thymidine

(S.A. 22.4 Ci/mmol) 4 hours before k i l l i n g . Thymuses were then

prepared for autoradiography as described in Chapter 3.

Obi Thymus organ culture

The technique used followed that developed by Robinson and

Owen (1976) for mammalian thymus organ culture. Thymuses were

116

dissected out of anaesthetised larvae a s e p t i c a l l y , care being

taken to remove as much connective tissue as possible from around

the thymic capsule. Thymuses were placed in a watchglass containing

amphibian culture medium (see Chapter 2) but with the addition

of sodium bicarbonate (0.085 g cm • ) . Ten cm^ culture medium .

was added to a Falcon s t e r i l e p l a s t i c P e t r i dish, which was

supplemented with 1.5 cm^ decomplemented FCS. Several batches

of FCS were t r i e d i n preliminary organ culture experiments,

but the batch (purchased from Gibco) showing maximum lymphocyte

recovery was selected and used i n a l l experiments presented here.

On the surface of the medium were floated 13mm diameter Nucleopore

polycarbonate f i l t e r s ( S t e r i l i n ) with d.4iim pore s i z e . These had.

been boiled i n d i s t i l l e d water, with 3 changes, and then autoclaved

before use. Each pair of thymuses was placed on the prepared

f i l t e r , as i n the diagram below.

thymuses on f i l t e r .

Such thymus organ cultures were incubated i n a humidified incubator

at 28°C with 5% CO2 i n a i r . Two stages of development were selected

for experimentation; stage 52, as a young thymus where lymphocyte

numbers are rapidly increasing, and stage 58, when metamorphosis

has j u s t begun. At both stages 9 animals were used. Thymocyte

suspensions were prepared d i r e c t l y from 3, as described i n Chapter

3, except that a f t e r the mechanical disruption the thymus was broken

up by incubation i n trypsin/EDTA for half an hour. (Trypsinisation

117

proved to be necessary for releasing lymphocytes from organ-cultured

thymuses). Three animals were kept as controls and allowed to

develop normally to determine the extent of i n vivo changes i n

thymus morphology over the experimental period. The f i n a l 3

animals provided thymuses for organ culture. On day 7 the organ

cultured thymuses were taken off the f i l t e r s and c e l l suspensions

prepared by teasing and try p s i n i s a t i o n . Thymocytes were then

counted i n a haemacytometer and a cytospih preparation made (see

Chapter 3 ) . The cytospins were used to count the numbers of

lymphocytes and e p i t h e l i a l c e l l s present. Thymocyte cytospin

preparations from the 3 control animals were also made at 7 days.

One pair of 7-day organ-cultured thymuses from a stage 52 larvae

were plastic-embedded, sectioned at 1pm and stained with toluidine

blue (as described i n Ca) above).

Results

(a) Thymus morphology

Figure 6.1 shows the number of thymic lymphocytes that

can be recovered a t various stages of development from stage 5o/51

(2 weeks old) up to 3 weeks a f t e r .the end of metamorphosis ( i . e .

12 weeks t o t a l age) . The data show that i n the larva a maximum

l e v e l of about 10^ thymic lymphocytes occurs at the very beginning of

metamorphosis (stage56/7(6 weeks)) . . This l e v e l i s similar to that

found by Du Pasquier and Weiss (1973) at t h i s stage. Du Pasquier

and Weiss recorded that thymocyte numbers decline to 3 x 10^ over

the peri-metamorphic period, whereas the r e s u l t s here show a sharper

f a l l to only 3.5 x lo'^ a t 9 weeks (the end of metamorphosis).

Thymocyte numbers increase dramatically within 3 weeks post-metamorphosis,

when 1.2 x 10^ lymphocytes are recoverable.

118

The histology of the thymus i n l a r v a l , metamorphosing

and post-metamorphic Xenopus i s shown in Figs. 6.2-6.5. At

stage 54 (Fig. 6.2a) and stage 58. (Fig. 6.2b) the thymus i s

highly lymphoid. At stage 60 the thymus (Fig. 6.2c) i s s t i l l

large and continues to be an extremely lymphoid structure.

Cortex and medulla are c l e a r l y defined at a l l the above 3 stages,

with a greater density of lymphocytes i n the cortex. Lymphoblasts

can be seen i n the subcapsular region of the cortex (Fig. 6.3a)

but these become l e s s evident by stage 60 (Fig. 6.3b). By the.

end of metamorphosis (stage 66) the thymus i s not reduced i n

s i z e but h i s t o l o g i c a l l y now looks quite different (Fig. 6.4a).

There i s a large, e s s e n t i a l l y e p i t h e l i a l , medullary region,

i n which many basophilic granulocytes are found. The cortex

appears narrower than at early stages. Overall the number of

small lymphocytes appears greatly reduced. Furthermore, there

i s now no indication of a subcapsular layer of lymphoblasts (Fig.

6.4a). By one week post-metamorphosis, the thymus appears to be

s l i g h t l y reduced i n s i z e and i s again sparsely populated with,

lymphocytes (Fig. 6.4b). However, there now appears a broad and

d i s c r e e t band of lymphoblasts beneath the thymic capsule (Fig. 6.5a).

Towards the medulla there i s a small c o r t i c a l band of lymphocytes,

and lymphocytes are now seen again i n the medulla. Two weeks after

the end of metamorphosis, the discreet peripheral band of lymphoblasts

i s p a r t i c u l a r l y noticeable (Fig. 6.4c and 6.5b)., By t h i s

time the area occupied by c o r t i c a l small lymphocytes has enlarged

and the thymus as a whole has grown. Increasing numbers of

lymphocytes are v i s i b l e i n the medulla. At 3 weeks post-metamorphosis

the thymus has enlarged considerably (Fig. 6.4d) and takes on i t s

'adult' form; i t i s highly lymphoid i n both corcex and medulla.

119

with a narrow band of lymphoblasts around the periphery of

the cortex. Autoradiographs of such thymuses (Fig.

6.6) show that i t i s the lymphoblasts of the peripheral cortex

which are a c t i v e l y synthesising DNA. This zone therefore

represents the region where thymic lymphocyte pro l i f e r a t i o n i s

occurring.

(b) Organ culture experiments

Development of the stage 52 thymus i n organ culture does not

mirror develoEment occurring i n vivo (see Table 6.1). After

culture, the proportion of lymphocytes to e p i t h e l i a l c e l l s decreases

when compared with the thymuses prepared d i r e c t l y from the control

larvae. Many large e p i t h e l i a l c e l l types are noticeable i n the

7r-day cultured thymus (see Fig.6.7-6.8) . The t o t a l number of thymocytes

also declined after 7 days in culture, i n rela t i o n both to the

nvunber at the s t a r t of the culture period ( i . e . at stage 52) and the

number i n the control thymuses, which remained in vivo for the

duration of the experiment. However, the c e l l s remaining aft e r

7 days i n culture showed good v i a b i l i t y (70-80%, as assessed

by dye exclusion).

The r e s u l t s were e s s e n t i a l l y the same when stage 58 thymus was

cultured. Although thymocyte nxambers i n the in vivo developing

controls declined (as expected) at t h i s time from 0.94 x 10^ to

0.40 X 10^, i n organ culture the decline was greater (0.17 x 10^

c e l l recovered at 7 days of c u l t u r e ) . The proportion of lymphocytes

to e p i t h e l i a l c e l l s was only marginally decreased in organ-cultured

thymuses compared with i n vivo development.

120

Figure 6.7 shows a lum section of a 7-day organ cultured

thymus taken from a stage 52 animal. I t can be seen that the

cortico-medullary structure becomes rather i n d i s t i n c t . Further­

more, compared with the i n vivo picture expected at t h i s time

(see F i g . 6.Xa), there are large v e s i c l e s i n the medulla, an overall

reduction i n number of lymphocytes and a flattening of the thymus

onto the f i l t e r . Melanin deposits are noticeable following organ

culture.

Discussion

The work i n the f i r s t part of t h i s Chapter investigated

morphologic events occurring i n the thymus at metamorphosis.

The data on c e l l recovery confirms previous findings (Du Pasquier

and Weiss, 1973) that t h i s period of development i s associated

with a considerable diminution of thymocyte niambers but highlights

the end of metamorphosis as being the most dramatic period i n t h i s

respect. The h i s t o l o g i c a l study of thymus ontogeny also reveals

that the end of metamorphosis i s associated with signi f i c a n t

s t r u c t u r a l a l t e r a t i o n s i n t h i s organ. Thus thymic medullary

lymphocytes are v i r t u a l l y absent at t h i s time and the subcapsular

lymphoblast zone, normally found i n the cortex, has disappeared.

Int e r e s t i n g l y these morphologic events occur at about the stage

when serologically-detectable MHC antigens can f i r s t be detected

on leucocytes i n Xenopus (Du Pasquier, Blomberg and Bernard, 1979).

I f the thymus i s c e n t r a l l y involved i n MHC r e s t r i c t i o n of certain

T lymphocyte subsets i n Xenopus (see Bernard, Bordmann, Blomberg

and Du Pasquier, 1981) then perhaps substantial reduction in

thymocyte nvmibers, and discontinuity of normal lymphopoiesis,is

an e s s e n t i a l prerequisite to reprogramming a new population of T

121

c e l l s to i n t e r a c t c o r r e c t l y with, and become tolerant to, the

new adult MHC antigens emerging at, or soon afte r , metamorphosis.

Certainly -the data presented here suggest a new wave of thymus

lymphocyte p r o l i f e r a t i o n occurs rapidly j u s t a f t e r the completion

of metamorphosis, since the peripheral c o r t i c a l lymphoblast zone

reappears and lymphocyte numbers throughout the thymus increase

at t h i s time. Thymus lymphopoiesis i n Xenopus may well be similar

to that seen in mice, to the extent that lymphocytes at the periphery

of the cortex divide rapidly and produce a maturing population

which moves toward the centre of the organ (Weissman, 1967).

Whether or not the surge i n thymus lymphopoiesis seen immediately

afte r metamorphosis i s dependent.upon a new wave of stem c e l l

input into the organ at t h i s time (from newly emerging bone marrow?)

i s unknown (see below).

The organ culture experiments on Xenopus thymus, revealing

r e l a t i v e l y low lymphocyte/epithelial c e l l r a t i o s and low overall

thymocyte nxambers compared to thymuses developing i i i vivo, appear

to c o n f l i c t with findings i n birds and mammals, which show that

removal and organ culture of the embryonic thymus does not prevent

p r o l i f e r a t i o n and d i f f e r e n t i a t i o n of functional T lymphocytes

(see Introduction). Before considering the p o s s i b i l i t y of r e a l

physiologic differences, technical factors must be considered.

Although many amphibian organs have been cultured (Monnickendam and

B a l l s , 1973), t h i s i s the f i r s t time that intact Xenopus thymus has

been kept i n v i t r o and examined h i s t o l o g i c a l l y after several days

in organ culture. Although the culture technicjue used was adapted

from a successful mammalian system (Robinson and Owen, 1976), and it

one used successfully for culturing the chicken thymus (Sallsffom

and Aim, 1973), i t may be unsuitable for the amphibian thymus.

122

I t must be remembered that i n i t i a l attempts at thymus organ

culture i n the mouse led to largely e p i t h e l i a l cultures. Improved

cultvire techniques led to the f i r s t demonstration of thymic

lymphopoiesis i n v i t r o (see Robinson, 1980, for review). In

the present experiments on the Xenopus thymus, lymphocytes were

found at the end of the culture period and t h e i r v i a b i l i t y was

quite good. However, the decrease i n thymus lymphocyte numbers

revealed a f t e r culture might suggest that a constant input of

precursor c e l l s into the amphibian thymus i s required. Proliferation

of a small number of stem c e l l s that enter the thymus early in

ontogeny may be s u f f i c i e n t for lymphopoiesis i n mammals but not

in amphibians. Although t h i s hypothesis could help to explain

the rather gradual establishment of T-dependent functions in Xenopus,

further work on the organ culture system i s obviously required.

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124

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Fig. 6.2

Thymus histology during l a r v a l and e a r l y

metamorphic l i f e showing the increase i n

s i z e and the highly lymphoid structure

of cortex (c) and medulla (m)

(a) Stage 54

(b) Stage 58

(c) Stage 60

8pm sections

Stain H and E

125

m

5^

F i g . 6.3

(a) This micrograph shows the band of

lymphoblasts (L) found at the periphery

of the cortex at stage 54.

(b) By stage 60, although the thymus i s s t i l l

lymphoid, t h i s layer of large, rapidly

dividing lymphocytes, i s no longer so

read i l y apparent.

8vim

Stain H and E

126

a

/

F i g . 6.4

Thymus histology during l a t e metamorphosis

and early post-metamorphic l i f e .

Ca) Stage 66 - the end of metamorphosis, lym

(b) 1 week a f t e r the end of metamorphosis

(c) 2 weeks "

(d) 3 weeks "

I I I I

I I I I

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H and E

127

•

5^.

128

F i g . 6.5

Micrographs of the thymus

(a) 1 week and

(b) 2 weeks a f t e r the end of metamorphosis.

A discrete band of lymphoblasts (L) can

be c l e a r l y seen i n the peripheral c o r t i c a l

region. Lymphocyte numbers are increasing

i n both cortex and medulla.

8ym

129

F i g . 6.6

Autoradiograph of thymus from 3-week

toadlet following in^ vivo i n j e c t i o n

of t r i t i a t e d thymidine.

LL= Labelled lymphocytes i n peripheral

cortex.

130

F i g . 6.7

lym p l a s t i c section of a thymus ( s t a i n

toluidine blue) from a stage 52 animal

following orgcui cultvire for 7 days.

The organ has reduced lymphocyte numbers

with many large non-lymphoid elements i n

the medulla (M) membrane to which thymus

i s attached. n»^. 3C (OO

Fi g . 6.8

Cytospin preparations of thymocytes taken

from larvae that were a t stage 52 at the

s t a r t of the experiment.

(a) control c e l l s prepared d i r e c t l y from

the l a r v a a f t e r 7 days i i i vivo.

(b) c e l l s following organ culture

i n v i t r o for 7 days. Although lymphocytes

(L) can be seen, there are many large

e p i t h e l i a l c e l l s (E) present.

7%

•

132

CHAPTER 7

Concluding remarks

The major findings of t h i s Thesis are outlined in

Figure 7.1 and can be considered i n four sections, representing

the main technical approaches used.

CD Mitogen r e a c t i v i t y of lymphocytes i n v i t r o

I n i t i a l culture experiments investigated the capacity of

adult Xenopus thymus and spleen c e l l s to p r o l i f e r a t e following

treatment with an array of mitogens which, i n mammals, s e l e c t i v e l y

activate either T or B lymphocytes. Good p r o l i f e r a t i v e responses

to putative T c e l l mitogens were recorded (using s c i n t i l l a t i o n

counting) for both thymocytes and splenocytes.. These c e l l s required

comparable optimal PHA eind Con A doses and displayed similar l e v e l s

of stimulation. Splenocytes responded well to high doses of the

B c e l l mitogen LPS. Interestingly thymocytes also displayed

a consistent, a l b e i t low l e v e l , p r o l i f e r a t i v e response to LPS

and PPD, suggesting the p o s s i b i l i t y that a population of B-

equivalent lymphocytes resides within the adult anuran thymus.

4

The use of a miniaturised culture technique (5 x 10 lymphocytes

per well) allowed, for the f i r s t time, mitogen experiments to be

performed on thymocytes taken from individual larvae and

metamorphosing Xenopus. During these stages of development

thymocytes appear to display l i t t l e sign of enhanced ^HTdR uptake

following Con A, PHA or LPS stimulation. A low l e v e l response

to Con A and LPS was, however, recorded j u s t prior to metamorphosis,

when the thymus reaches i t s maximal l a r v a l s i z e . Metamorphosis

133

effected a temporary disruption of the emergence of thymocyte

mitogen r e a c t i v i t y . Appreciable and constant T and B mitogen

responses i n the Xenopus thymus and spleen gradually

emerge a f t e r metamorphosis. Maximiim l e v e l s of LPS responsiveness

of thymocytes were seen at 6 months of age (when the mean S.I.

reached 6) . LPS r e a c t i v i t y of thymocytes at t h i s time was confirmed

by autoradiography; moreover, nylon wool separation experiments

showed that t h i s responsiveness l i e s within, or i s dependent upon,

a nylon wool adherent population of thymocytes. The d i s t i n c t

ontogenetic pattern of thymocyte responsiveness to LPS suggests

that these B-equivalent c e l l s may actually be spawned within the

thymus rather than be casually migrating through t h i s organ from

the periphery.

The capacity of l a r v a l lymphoid c e l l s other than those from the

thymus to react to mitogens was not examined i n rny d e t a i l here.

Such experiments are needed to determine the s i t e s where functionally-

equivalent T and B lymphocytes f i r s t emerge i n the anuran. These

experiments w i l l require the use of histoccanpatible animals where cell

pooling w i l l allow study of very early stages . (and during metamorphosis),

when lymphocyte numbers are small. In Xenopus some hybrid l a e v i s / g i l l i

females lay a proportion of diploid eggs, which can be activated

by i r r a d i a t e d sperm into gynogenetic development (Kobel and Du Pasquier,

1975) . LG hybrids from a p a r t i c u l a r clone produced in t h i s way have

been shown to be i d e n t i c a l to one another i n a variety of immunological

assays (e.g. grafting, MLC r e a c t i v i t y and antibody patterns (see

Kobel and Du Pasquier, 1977). Gynogenetic development of anuran eggs

through pressure (Tompkins, 1978) or cold shock treatment (Kawahara,

1978) following activation with irradiated sperm are other procedtares

that are being successfully used to procure (by suppressing elimination

134

of the second polar body) MHC compatible animals for immunological

study (see discussion i n Kobel and Du Pasquier, 1977) .

Histocompatible colonies of Xenopus occurring naturally have

also been described i n some laboratories (Katagiri, 1978). The

increasing a v a i l a b i l i t y of histoconpatible s t r a i n s of Xenopus

increases the value of t h i s model system for future immunologic studies,

C2) Thymectomy

Thymectomy was used i n conjunction with lymphocyte culture

and grafting studies to investigate the extent to which different

T c e l l functions are dependent upon an i n t a c t thymus. Xenopus

thymectomized at 7 days of age are often s t i l l capable of rejecting

skin a l l o g r a f t s , a l b e i t i n very chronic fashion. Spleen and blood

lymphocytes taken from such thymectomized and alloimmune animals were,

however, shown to be unresponsive to the T c e l l niitogens Con A and

PHA, although they responded normally by p r o l i f e r a t i o n following

LPS treatment. Lymphocytes involved i n graft rejection and T-mitogen-

reactive c e l l s might therefore represent d i s t i n c t T c e l l populations

in Xenopus, a suggestion which f i t s with previous sequential thymectomy

studies (Horton and Sherif, 1977). Experiments i n t h i s Thesis

involving thymectomy during mid-larval l i f e f a i l e d to provide support

for Horton and Sherif"s suggestion that T mitogen reactive c e l l s and

MLC reactive lymphocytes represent separate T c e l l lineages.

E a r l y thymectomy i n Xenopus w i l l continue to be a valuable

technique with which to probe the developing immurs system. With

isogeneic animals, reconstitution experiments are readily feasible

to further d i s s e c t possible T lymphocyte heterogeneity. The role

135

of the thymus i n MHC r e s t r i c t i o n Ceui also be profitably examined

i n t h i s anuran: thus thymuses of known MHC haplotype can be implanted

to early-thymectomized animals, whose subsequent cytotoxic T c e l l

r e a c t i v i t y and T-B c e l l interactions can be assessed. The lack

of runting or wasting disease i n early-thymectomized Xenopus

suggests, perhaps, that natural k i l l e r (NK)-like c e l l s assvmie

considerable importance i n t h i s anurah. This i s certainly an

inte r e s t i n g area for future work.

C3) Hapten-carrier immunisation

Three main findings came from these experiments, which

employed the hapten TNP conjugated eo either a T-dependent (SRBC)

or T-independent c a r r i e r (LPS). F i r s t l y , i t was shown that

responsiveness to these injected antigens can be detected (by

antigen binding) i n the spleen from an early l a r v a l age, revealing

that T-helper c e l l s and B-equivalent lymphocytes are functioning

from t h i s time. The l e v e l of induced RFC's against TNP reached

adult l e v e l s by the onset of metamorphosis, irrerpective of whether

the iiapten was presented on the T-dependent or T-independent c a r r i e r .

I n f a c t the RFC response to TNP-LPS during metamorphosis was elevated

when compared with the adult. Secondly, antibody-secreting PFC to

injected SRBC, TNP-SRBC and TNP-LPS were detected in the spleen only

aft e r the end of metamorphosis. Thirdly, small numbers of anti

SRBC PFC were found i n the adult thymus following TNP-SRBC administration.

This work suggests that metamorphosis i s associated with

altered humoral immune regulation. Further experiments are required

to characterise the TNP respqnse at metamorphosis to determine the

basis for the enhanced splenic r e a c t i v i t y recorded at t h i s time.

The apparent lack of PFC response u n t i l after metamorphosis i s

136 intriguing and also warremts further investigation.

(4) Histologic studies on thymus development at metamorphosis

The h i s t o l o g i c a l studies highlighted the dramatic drop i n

lymphocyte numbers that occurs at the end of metamorphosis. A

dearth of lymphocytes throughout the thymus occurs at th i s time,

j u s t p r i o r to a rapid reappearance of lymphocytes i n the peripheral

cortex. Three weeks a f t e r the end of metamorphosis the thymus i s

considerably enlarged and i s again highly lymphoid. These

findings correlate with altered immune r e a c t i v i t y demonstrated

both i n t h i s Thesis and elsewhere during metamorphosis.

The concept of s p e c i f i c suppressor c e l l s as a discrete T

c e l l s\ibpopulation, which faced some resistance from immunologists

when f i r s t proposed, has now come of age (Golub, 1981), and advances

i n vmderstanding immunoregulation i n the next few years w i l l , i n

part, focus on the central involvement of these c e l l s i n modulating

immune responses. Amphibian metamorphosis (a time when an internal

histoincompatibility i n the form of the emergence of new adult-

s p e c i f i c antigens i s presented to a competent immune system)

presents a unique opportvinity for examining immunoregulatory T c e l l

populations, which may have significance outside of purely

phylogenetic consideration.

137 Figure 7.1 Summary of major findings i n Thesis concerning the development

of the iiamune system i n Xenopus -laevis.

Chronic a l l o g r a f t r e j e c t i o n c a p a c i t y but not r e a c t i v i t y to T c e l l mitogens — call develop i f the thymus i s removed a t t h i s stage. Antigen binding responsiveness to T-dependent and T-independent antigens i s — found i n the spleen. ' Thymectomy a t t h i s stage causes impairment of both T mitogen r e a c t i v i t y and HLC responsiveneiss Small response to both T and B c e l l mitogens f i r s t found: the thymus. I j a r v a l thymic lymphocyte numbers reach maximum l e v e l .

Age (weeks

0

1

Thymic lymphocyte n-jmbers are declining-Mitogen responsiveness no longer •observed i n the thymus. Elevated level.s of RFC r e a c t i v i t y to TNP-SHBC and TNP-LPS are found i n the spleen.

Thymic lymphocyte numbers are a t a minimum. No mitogen responsiveness observed i n the thymus. El e v a t e d l e v e l s of antigen binding to TNP-LPS found i n the spleen.

Thymic lymphocyte numbers reach maximum adult l e v e l s . Good mitogen responsiveness to both T and B c e l l mitogens nov? found i n both thymus and Spleen. iMaximum l e v e l s of thymus response to LPS , found. PFC responsiv'sne.Ga to T-dependent and T-independent antigens i s e s t a b l i s h e d . PFC'a detectable i n thymus.

24

138

ACKNOWLEDGEMENTS

I should l i k e to express my thanks to toy supervisor

Dr.J.D. Horton for h i s unstinting guidance and help during

the course of t h i s study and i n part i c u l a r my appreciation

of h i s enthusiasm and commitment to t h i s work and develop­

mental and comparative immunology i n general.

Thanks are due to Professor D. Barker and the

Department of Zoology for providing the f a i c i l i t i e s to make

t h i s study possible. i • •

I would also l i k e to thank those who have provided

technical assistance at various times throughout t h i s study,

i n p a r t i c u l a r Miss Judith Chambers, Miss Margaret I'Anson

and Mrs. Christine Richardson. f

|I am es p e c i a l l y indebted to Mrs. Pauline Bransden

and Mr, David Hutchinson for the es^pert help received in

the preparation of t h i s t h e s i s .

F i n a l l y I should l i k e to thank Durham University

for providing my Research Studentship for the duration I

of these studies. I

139

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