Irnmunoloi^y Tixiay. vol- 3. .\'o. 1. I'JHI

The role of H-Y as a minor transplantation antigen

Elizabeth Simpson Transplantation Biolos y Section, fllinical Research Ccnire, Harrow. Micidlesex. HAl 3UJ, U.K.

H-Y antigen i\ pxprcsseil in mammals (inly by males, sn Ihal grafts of nude tissue are rejeeled by females uulhin certain highly inbred strains. H-}" antigen a/ifears la be a simfile, nem-polymiirfihie antigen and the genetic ciinlriil nf anli-H-T rcspimses has been extensively sinihed. In this article Elizabeth Sunfisim discusses the many insights obtained.

H-Y was discovered as a transplantation antigen by Eichwald and Silmser in 195.5'. They reported that when skin grafts from certain strains of inbred mice were exchanged in all possible syngeneic donor/recipient combinations, i.e. male to female (M - - F ) , M - * M, F - • F, F - » . \ I , only male grafts on syngeneic females were rejected. Since the only genetic difference between the males and females was the male's possession of the Y chromosome, Snell deduced that the transplantation antigen involved in the rejection of such grafts was coded or controlled by a gene(s) on the Y chromosome - hence the name H-Y antigen (see Ref. 2 for review). Data showing that female mice treated with testosterone did not become H-Y positive and that castrated males remained H-Y positive^ suggested that the expression of H-Y antigen was not likely to be under hormonal control.

Experiments done soon after the original report showed that not all inbred strains responded to H-Y by rejecting syngeneic male skin. This strain variation might indicate cjuantitative or qualitative differences in H-Y expression by donor male tissue, or variable recipient female responsiveness. On the question of differences in H-Y expression, [I-Y antigen is ex­pressed l)y males of both responder and non-responder strains, and there is only one allelic form of H-Y-' ' . Nevertheless, genes of the major his­tocompatibility complex (MHC:), //-2 as well as non-H-2 genes, can modify the presentation of H-Y by donor cells" . On the question of female responsive­ness to H-Y, it is now known that there are immune response (Ir) genes, both within the //-2 complex and unlinked to //-2, which control the graft rejection

response-H-Y ant

and all other 'I'-cell responses to the

igen'

Expression of H-Y Hauschka suggested'", that the lack of response to

H-Y in females of some strains might be due to

translocation of part of the Y' chromosome to an auto­some or the X chromosome, .'\ccording to such a hypothesis, the females of non-responder strains should be H-Y positive. This was not found to be so. Their cells did not tolerize neonatal females of the responder strain C57 to H-Y, while male cells of non-responders could do so'"; and female F, hybrid progeny of a non-responder female (e.g. CB.4) and a responder male (e.g. C57) could reject male skin from a non-responder parent (i.e. CBA), so no putative translocated H-Y gene had been transferred to the progeny ' ' " ' ' . This also illu.strates that male skin from a non-responder (e.g. CB.A) does express H-Y' antigen. In fact, for reasons that are not altogether understood, CB.A male skin is rejected much faster by ( ( 3 A X C57) F| females than C.S7 male skin". Pre­sentation of the H-Y antigen together with H-2 antigens of the H-2^ haplotype (CBA is H-2^) apfiears to be a factor"" although non-H-2 donor genes are now known to influence the rejection of male skin". I'he demonstration of H-Y' on the male tissue of non-responder strains is consistent with the evidence that male cells of non-responders are killed in H-Y specific cytotoxicity tests", results that are discussed in more, detail later.

The same H-Y antigen is expressed by all strains of mice, regardless of the ability of the females of these strains to respond to H-Y'. Several lines of evidence support this conclusion. Firstly, females of the responder strain, C.S7. could be made tolerant to H-Y by neonatal injection of both syngeneic and allogeneic male cells, including those of such non-responder strains as A,(;.3H and CB.A'". Because of the very high degree of specificity characteristic of immunological tolerance, this was taken as evidence for the identity of the H-Y' antigen in the strains tested. Secondly, reciprocal F male grafts, each deriving the Y chromosome from a different inbred strain, were not rejected'. 'Fhis is a particularly sensitive test for

98 Immunology Today, vol. 3, JVo. 4, 1982

differences in transplantation antigens. The question of possible alleles of H-Y in wild strains of mice was approached in a later study"" using both skin grafting and H-Y specific cytotoxic T cells. Again, H-Y appeared to be identical in wild mice and in the laboratory strain C57BL/10 within the limits of sensitivity of the assay systems used.

H-Y antigen is present on almost all tissues of the body. Recent good evidence for this has been obtained by Johnson'* in sublethally irradiated C57BL/6 males repopulated with female bone-marrow cells. In these chimeras, the entire lymphoreticular and haemo-poietic tissue is of female origin. Tissues from such mice were used to sensitize normal C57BL/6 females for a popliteal lymph node enlargement assay elicited with male spleen cells after sensitization. H-Y antigen thus was demonstrated on skin, testis, pancreas, lung, spleen, liver, thymus and kidney. Skeletal muscle, heart and probably red blood cells were apparently negative.

Responsiveness to H-Y It was realized early on that the response to H-Y as

manifested by skin grafting was an immunological one, of the classical transplantation reaction type. The evidence for this, reviewed and discussed in Ref. 2 was that: (a) females that had rejected syngeneic male skin grafts rejected subsequent grafts in an accelerated 'second set' fashion; (b) responder strain females, e.g. C57, could be rendered tolerant of syngeneic male grafts by exposing them to male cells in the neonatal period; and (c) histologically, the rejection of H-Y incompatible grafts was indistinguishable from that of allografts bearing H-2 plus non H-2 incompati­bilities.

It was also an early finding that not all female mice could reject primary syngeneic male grafts^. Females of some strains did not reject such grafts at all, whilst in other strains, the rejection of H-Y incompatible grafts was late and occurred in only a small proportion of females. It was then found that the ability to reject primary syngeneic male skin grafts was largely deter­mined by the //-2haplotype'*"^", see Table I. Thus all the females from strains of the H-2^ haplotype (BIO, B6, C3H.SW, A.BY) rapidly rejected syngeneic male skin grafts, whilst the corresponding H-2 congenic strains BIO.BR, C3H(//-2i<), B10.A(//-2a), B10.D2, B6.//-2d(//-2d) did not^". The H-2 Ir gene controlling rapid primary graft rejection was mapped to the /Tend of the //-2 complex when it was discovered that the H-2i> recombinant strain B10.A(5R) {hbbddd) could respond but the strain B10.A(2R) (kkkddh) could not*. The discovery that females of the H T G (dddddh) strain could also make rapid graft rejection responses'* was attributed to the presence of non-H-2 Ir genes*". The role of such non-H-2 Ir genes in other types of T-cell response to H-Y will be discussed later. The mapping of the //-2b Ir gene for rapid primary graft rejection of male skin was taken further by the finding that not

only B10.A(5R)(AWrfrfrf) females but also }iW./K{AK){kkhbbb) females rejected male skin'". Since these two strains share the non-//-2 background genes, but only the / /Jbpart of the //-2b haplotype, this result suggests that the Ir gene controlling rapid primary graft rejection of H-Y incompatible skin lies within the IB subregion of the H-2 haplotype. As will be discussed later, the ability to mount a delayed-type hypersensitivity (DTH) response to H-Y is also deter­mined by the presence of IB^, and strains showing DTH to H-Y are B10(5R)(AAArWrf), BIO.A (4R) (kkbbbb) and V)Z.G\){ddbbbbY''-K

Tolerance to H-Y Tolerance to H-Y can be induced in //-2b responder

strains (e.g. C57, C57BL/6, C57BL/10) by a variety of procedures, viz- injection into neonatal females of small numbers (10") of syngeneic male or allogeneic male spleen cells, injection into adult females of very large numbers (> 10*) of male spleen cells, multi-parity and grafting of adult females with neonatal male skin (reviewed in Ref. 2). Tolerance in these earlier studies was measured by the inability of the female to reject syngeneic male skin grafts. Billingham et al.^~ reported that tolerance to H-Y could be transferred to naive recipients when the donors were C57 females tolerized by syngeneic male cells, but not if the donors were tolerized with allogeneic male cells. Subsequently, Smith and PowelF^ showed that tolerance to H-Y induced by multiparity could also be

TABLE I. MHC(H-2) of mouse : regions, sub-regions and II-2 haplotype of strains mentioned in the text^

Stra in Region

Sub-region

C,S7, C57BL, C 5 7 B L / 1 0 ( B 1 0 ) , C 5 7 B L / 6 ( B 6 ) , A.BY, BALB.B, C 3 H . S W , 129/J

C B A , C 3 H , BIO.BR,

A . B I O . A

A K R

B 1 0 . D 2 , B A L B / c , D B A / 2 ,

BIO.S, A.SW, SJL

B10.A(.SR)

B10,A(4R)

B10.A(2R)

D 2 . G D

H T G , B I O . H T G

B I O . H T T

A . T H

C.3H.OH

A Q R

BIO.G

BIO.M

, BALB.K

B6.H-2d

K

h

k

k

d

V

/) k

k

d

d

s

,, d

']

<l

J

A

h

k

I

I B

h

k

k

d

> b

h

k

h

d

( , d

k

'/ ./

C

b

k

d

d

> (/ * d

h

d

k

s

d

d

'1

I

S

/; k

d

d

V

d

h

d

h

d

k

s

d

d

?

I

D

b

k

d

d

V

d

b

b

b

b

d

d

k

d

<1

I

11-2 hap lo typ

H-2l'

H-2''

11-2"

H-2''

H-2'

H-2''''

H-2'''

H-2''^

H-2S^

H-2S

H-2''^

H-2>'-

H-2"-

H-2y'

H-21

H-2f

^ H - 2 i histacom

is on ch romosome 17, see Klein, J . (1975) Biology of the T mpatihility-2 complex. Spr inger Verlag, New Yorlc

Immunology Today, vol. J..\'o. /, !0,S2

adoptively transferred. The ability to transfer toler­ance suggests that it is being maintained by an active mechanism such as suppression. Another interpreta­tion could be placed on the transfer ol tolerance induced neonatally since these donors arc likely to be chimeric with respect to the syngeneic male tolerizing cells and, therefore, tolerance might be induced in the recipients by male cells in the transferred inoculum. This possibility was investigated by Weissman-' who estimated the level of chimerism in tolerant donors by giving graded doses of their spleen cells to: (a) adult female C57BL mice to sensitize them for second set graft rejection of syngeneic male skin; and (b) neonatal female C57BL mice to tolerize them to H-Y, and comparing them with a standard dose response curve using male cells alone. He found a large (up to 1000-fold) discrepancy between these two methods in the estimation of the level of chimerism of male cells in the transferred inoculum. Abnormally low numbers of male cells appeared to induce tolerance in the neonates, i.e. it seemed that in adcfition to any male cells a population of female cells of donor origin were responsible for transfer of tolerance to H-Y. Further­more, adoptively transferable tolerance in multi-parous females, who are unlikely to be chimeric, also argues for transfer by an active suppressor mechanism.

Recent studies-" have extended tfie assessment of tolerance to H-Y following various regimens of tolerance induction to the H-2 restricted, H-Y-specific cytotoxic T-cell res[)onse. The findings accord with earlier results: C.57BL/10 female mice tolerized neonatally, either with syngeneic or allogeneic male cells, not only failed to reject syngeneic male skin grafts but also generally failed to generate anti-H-Y cytotoxic T-cell responses. It was possible to trans­fer tolerance from syngeneically tolerized females, measured by the failure to make H-Y specific T-cell responses, but not from C57BL/1() females tolerized with allogeneic male cells. However, in some individuals, both donors and recipients, there was a discrepancy between tolerance measured by skin grafting and that measured by the cytotoxic T-cell response, and this will be discussed later.

Antibody responses to H-Y Minor transplantation antigens characteristically

provoke good T-cell responses (e.g. graft rejection, cytotoxic T cells) but poor antibody responses. H-Y is no exception to this, although pioneering work has been done by several groups-"- '"•" using low titre antibodies to detect H-Y antigen. To a large extent, these experiments have addressed the sex-determining role of H-Y and its tissue and species distribution. It appears that H-Y has been extraordinarily well pre­served during evolution, as it is [jresent on mosi lissues in members of the heterogametic sex in all vertebrates. However, these experiments have not been easy to reproduce in other laboratories, mostly because of the

difficulty in raising anti-H-Y' antisera, and the low titres obtained. It has proved extremely difficult to make the high titred, high affinity anti-H-Y mono­clonal antiloody needed to resolve cjuestions of re|)roducibility and to characterize H-Y' antigen bio­chemically. However, the sex-determining role of H-Y will not be reviewed here; suffice it to say that low antibody titres have been reported in Ijoth responder and non-res[)onder strains of mice-" suggesting that they do not play a role in rejection of H-Y' incompat­ible grafts or in the generation of cytotoxic T-cell responses. An interesting observation has been made of a human antibody with specifiicity for male, but not female, cells carrying the HLA specificity A-2-". This is the first report of an MHC-restricted antibody (the M H C restriction of cytotoxic T cells, are described below).

The H-Y determinant detected by antibody may not be the same as that identified by T-cell responses such as graft rejection and the cytotoxic T-cell response, since there are reported discrepancies in H-Y typing with these different methods in certain individuals. Thus, Melvold </ al.-'' described a pheno-typically male mutant mouse which lacked H-Y antigen on the basis of skin graft testing but which was positive for H-Y on serological testing. More recently, it has been reported that mice lacking a Y' chromo­some (XO) are H-Y positive by serological testing"'. This is in contradistinction to earlier reports of X O mice being H-Y negative by the criterion of graft rejec-ti(m- and current findings that they are negative by cytotoxic T-cell testing since they can themselves make a cytotoxic T-cell response to H-Y^". It has also been reported that some human X O (Turner's syndrome) patients arc H-Y positive by serological testing'". Another finding which introduces dis­crepancy between sex determination and the pres­ence of H-Y' is that Richer and colleagues" '- who have located a mutant gene on the Y chromosome. Mice bearing this mutant gene are either H-Y' posi­tive, phenotypic females or XY' hermaphrodi te phenotypic males". The XY females are H-Y' positive by the criteria of skin graft rejection, H-Y'-specific cytotoxic T cells and host-graft (HVG) response". There are examples where H-Y antigen detected in karyotypically abnormal individuals by serology, skin graft criteria and H-Y-specific cytotoxic T cells are concordant. XX mice carrying the gene Sxr are pheno­typic males and have been shown to be H-Y jxjsitive by typing with antibody and graft rejection cri teria" as well as by H-Y-specific T cells". These findings are consistent with a translocation of a piece of the Y chromosome.

However, the apparently paradoxical findings above not only put in cjuestion whether the TI-Y' deter­minant detected by T cells and the products of B cells is the same, but also the exclusive primary sex deter­mining role of H-Y. This interesting field will be the subject of a separate review.

100 Immunology Today, vol. 3, No. 4, 19H2

Cytotoxic T-cell responses to H-Y in H-2^ mice

Targel-ailspecijuily H 11-2KjD reminded H-Y-specific cytotoxic T cells were first found at

low levels in females of tine responder strain C57BL/6 among cells from lympfi nodes draining tlie site of a rejecting syngeneic male graft' '. Subsequently, Gor­don el a / ." '" found that very high levels of H-Y-specific cytotoxic T cells were induced when spleen cells from C57BL/10 female mice that had been immunized i.p. with male spleen cells or by grafts of male skin were restimulated in vilro in mixed lympho­cyte culture (MLC) with syngeneic male cells. The finding, remarkable at that time, was that the target-cell specificity of these H-Y-specific cytotoxic T cells was H-2 restricted, in the manner previously reported for virus-specific T cells by Zinkernagel and Doherty'" and for haptens by Shearer". Concurrently, Bevan^"

reported that cytotoxic T-cell responses to other minor transplantation antigens were also H-2 restricted.

The essence of the H-2 restricted cytotoxic T-cell response is that the extrinsic antigen, i.e. H-Y, virus or hapten, is recognized by the cytotoxic T cell in the context of a self H-2K and/or H-2D molecule. Thus, H-Y sensitized spleen cells from C57BL/10 (BIO) (//-2b) mice will kill only male target cells which carry the Db molecule"' ' \ They fail to kill male target cells of other H-2 haplotypes, or of intra-//-2 recombinant haplotypes carrying the A and not the D end of the 11-2^ haplotype, e.g. B10.A(5R) {bhhddd) male cells not killed by H-Y-specific cytotoxic cells from BIO {bbbhbb) females whereas those of B10.A(2R) {kkkddb) are (Table 1, Table II exp. A). It is unclear at the moment whether the Db and H-Y molecules are recog­nized as two distinct entities or whether their non-covalent association in the cell membrane creates a

TABLE II. H-Y responses in H-2 homozygotes, heterozygotes and recombinants

Exp. Responder (fromref.) female H-2

Priming and boosting antigen

•farget cell H-2 Corrected % lysis ^

Restricting specificity

A( l l ) BIO

B(l l )

C( l l )

b h h h b h B10<^

b b b b b h CBA<?

^^^-^""^ k k k k k k

B10<? B109 C3H0 C3H.SW<J B10.A(2R)C? B10,A(2R)Q B10.A{5R)O

CBAfJ CBA9 BIO.AO

h b b b b b 3.3.3 + 2.5 h h h b b b 2..S + 0.2 k k k k k k 7.3 + 1.3 b b h h b b 38.5 + 3.8 k k k d d h 30.6 + 2.1 k k k d d b 2.2 + 0.2 b b b d d d 3.9 + 0.3

k k k k k k 31.1 +0.8 k k k k k k 2.4 + 0.5 k k k d d d 4.6 ±0.9

H-2l9'

(B10xCBA)F|

(B10xBALB/c)F, b b b h b b BIQC

d d d d d d

CSH.OHC? d d d d d k 35.1 + 1.9 BlOf b b b b h h 1.2 ±0.3

BIQC? h b b b b b 10.5 ± 1.6 BIO9 b b b b b b 2.0 ± 1.3 B10.A(2R)<^ k k k d d b 15.8 ±1.8 B10.A(5R)<^ b b b d d d -1.6 ± 1.2 BALB/c d d d d d d 2.3 ± 0.8

/ / -2»*

11-21)"

{B10xBALB/c)F, h b b b BALB/c S

D(12)

E(64)

B10.A{5R) B10.A(4R) (B10.A(5R)x BALB/c)F,

(B10.A(4R)x BALB/c)F,

(BlOxBlO.S)F,

d d d d d d

b b b d d d B10.A(5R)C? k k b b b b B10.A(4R)<^

b b b d d d BALB/cC?

d d d d d d

k k b b h b BALB/cC?

d d d d d d

b b b b b b (B10xB10..S) BIO^ " F,t? B10.S<^

BIO.S9

BALB/c(^ BALB/c9 D2.GD(? BlO.HTTcJ BlOcf

B10.A(5R)<? B10.A(4R)(?

BALB/cC^ BALB/C9 D2.GDd' BIO.A*^

BALB/c<?

d d d

b

b k

d d d k

d

d d d

b

b k

d d d k

d

d d b

b

b b

d d b k

d

d d b k b

d b

d d b d

d

d d b k b

d b

d d b d

d

d d b d b

d b

d d b d

d

14.9 + 2.9 T -1.7 ±0.3 25.0 ±3.0 V

2.6 ±0.3 -0.6 ± 0.2 J

0.1 +0.5 T 1.0 ±0.6 J

22.3 + 2.3 -1 0.8 ±0.2 1

14.7 ±1.3 ( 3.8 ±0.1

-0.9 ± 1.2

H-2K''

no response

H-2K'I

no response

b b b b b b 2.7 ±2.1 ,( I ( i f ,f 39.8 + 2.7 y //-. ,t ,( ,1 1 .1 .1- -2.7 ±0.1

per cent specific lysis of target ceils at A ; T 4 ; 1 as determined from a four point regression curve

Imnmmhsiy Tmlriy. r<,l. .1 \ „ . I. I<IS2

single nco-anligenic component which is rccogni/ed as a single entity by the V cell.

In the initial experiments '" ' using iri-rivn i/p priming followed by in-rilm boosting in MIX: to generate anti-H-^' cytotoxic T cells, only the //-_"' strains and their P", hybrids made a response. .\s in the graft rejection response, there ap])eared to be a dominant //-i-linked Ir gene for responsiveness in the //-2'> haplotype""" '" . .Xs in the earlier skin lirafting experiments, failure ol non-responder strains to make cytotoxic T-cell responses to H- \ ' were not attribut­able to the failure of such strains to exf)ress the I l - \ ' antigen, since F, hybrid females with one FI-2'> |)arent, e.g. (BIO X CBA)F|, (BIO x BALB/c)F| , when stimulated with male cells of CB.A (ll-2k) or B.\LB/c (11-2'^) respectivelv, could make H-Y-specihc responses. These were restricted to the self \\-2 antigens of the stimulating cell, i.e. H-2'< and H-2rf respectively. When intra-//-2 recombinant mouse strains were used to map the restriction antigens, they were found to be D^ and A'<l, respectively (Table II, ex[)s, B and C),

The implication from target-cell specificity experi­ments that anti-H-Y cytotoxic I cells recognize H-Y in the context of self K/1) molecules is contirmcd by the blocking experiments with monoclonal anti-//-2 antibodies reported by Fischer I.indahl and Lemke' . They used three anti-//-_'l< monoclonals which blocked the //-2'< associated H-Y siieeilic cytotoxicity of (B6 x CB.'\) F( spleen cells stimulated with C:B.\ male cells. //-2l' restricted H-Y killing of (B6 x CBA)F| spleen cells stiinulated by B6 male cells were not blocked by anti-//-2k antibodies.

Hell) /or Ihf cyliiloMi resfxitisi' nKiji.s In 11-21

The mapping ol the dominant / region gene ol the //-2'> haplotype came from studies using F, mice with one parent of the appropriate //-2l' recombinant strains as respondcrs'-^ ", i.e. B 1 0 . , \ ( . T R ) (hl)h<l(li[) and B10.,'\(4R) (kkhhlili). ,Such an experiment is shown in Table n (exp, D). Neither B10.A(,SR) or BI0.A(4R) make anti-H-Y cytotoxic responses themselves: how­ever, (BIO,A(,SR) X BAI,B/c)F, hybrid females do and (B10,A(4R) x BALB/c)F, hybrid females do not. 'The failure of homozygous BI0.A(5R) mice to respond can be attributed to their jjossession of the A'' and /> ' alleles, which apparently cannot) associate with H-Y' for target-cell killing ('Table II, exps. ,\ and C). However, BIO..A(-TR) mice have the Aand lA sub-region of the / / -2 ' ' haplotype. whilst BI0. , \(4R) does not, since it derives its A and IA regions from the 11-2^ ha[)lotype. Thus, it would apjiear that A''' and/or /.T' are necessary for resjionsiveness to H-Y following i/p priming, 'The definitive mapping of this Ir gene to /.T' rather than A'' comes from more recent studies of Michaelides <•! al.'^ who showed that the bm 12 //-_^'' mutant (which has the normal H-2Ki> molecule, biu a mutation affecting TX'') fails to respond to \\-\ . 'Thus, a non-responder recombinant possessing IA^\ such as BIO. . ' \ ( .TR) , can complement a non-responder strain

lacking /.T' but possessing K/I ) antigens 'associative' for H-Y'. 'This type of (T\ ' ' x \ R ) F | has also been reported as responsive to H-Y by von Boehmer rl a!.". Thev have also used radiation chimeras of the type BIO.A(.SR)—(CBA x B6)F, to show that B10.A(,SR) cells could respond to H-Y' when matured in an environment where they could recognize both TV*' and associative K/1) molecules (I)'', I)**) as self".

The target-cell specificity of anti-H-Y cytotoxic cells (Tc) implies that for Tc H-Y is recognized together with self K or D antigens. The identification of the need for hV> implies that 'T helper cells (Th) necessary for the H-Y cytotoxic response recognize H-Y' in association with ItV' antigens. Further indirect evidence for this comes from the exjierimenls of von Boehmer and Haas^' who used, as putative Th and Tc responding cell populations, I" cells educated in a chimeric environment where they would either recog­nize as self I,\i' (= Th) or Uk (=Tc), Each of these two populations was transferred either separately or together into an irradiated host bearing both types (I.'\l> and Dk) of self antigen. When challenged to make an anti-H-Y response in vilrn after appropriate in-vivn immunizations, neither of the two populations (puta­tive Th and 'Tc) alone could respond, but together they could, suggesting that one could help the other. Direct evidence supporting the separate role of Tc and Th comes from the experiments of .Simon et nl.'"- who used the fluorescence activated cell sorter to separate Ly I +2+-3+ Tc and Ly I +2-3- Th cells from the spleens of B6 females primed in rirn with B6 male cells. The ca[)acily of these cells, separately and together, to generate H-Y-specific cytotoxic cells after in-vilru restimulation with B6 male cells was tested, .Again, neither population alone generated H-Y cytotoxicity but together they made an H-Y-specific response ec]ual to that of the unsorted T-eell population, ,\ further confirmatitm of these sets of findings comes Irom limiting dilution analysis of anti-H-Y [jrimed Tc cells from BIO female mice placed in culture with BIO male cells as antigen, plus or minus a source of irradiated H-Y primed Th cells. It was found that H-Y-specific cytotoxic responses could only be generated from these limiting number of Tc in the pre­sence of irradiated 'Th, which by themselves could not generate a cytotoxic response (Ashman and .Simpscm, unpublished).

Cytotoxic T-cells responses in non H-2'' mice: /'"i hyhnils, chimeras nnd uilra U-2 reciinihinanls

The first indication that the IA^> gene was not mandatory, presumably for T-helper cell function, for the generation of CTL to H-Y', came from experiments showing that certain non //-2b (NR x NR) F, hybrids could make H-Y' specific cytotoxic responses to male cells of one or both (jarcntal haplotypes ' - ' - ' " "' and that both non-//-2' ' NR-«-^NR tetrajiarental chimeras and NR, —» (NR, x NR,) F, irradiation chimeras could likewise respond to H-Y"'"' (Table III, exps.

102 Immunology Today, vol. 3, No. 4, 1982

A - F ) . T h e (not u n r e a s o n a b l e ) ob jec t ion w a s raised*"' t l ia t b e c a u s e u n i r r a d i a t e d m a l e p a r e n t a l cells h a d b e e n u s e d t o i m m u n i z e t h e female F , h y b r i d s a n d c h i m e r a s , a n a l logene ic effect was t h e r e b y i n d u c e d by b a c k s t i m u l a t i o n , a n d this a c t e d to r ep lace t h e T h e l p e r cells o t h e r w i s e neces sa ry for g e n e r a t i n g H - Y -specific T cells, i.e. t h a t t h e r e sponses so g e n e r a t e d were he lper -ce l l i n d e p e n d e n t . W h i l e it is no t poss ib le to s ay w h e t h e r o r no t s u c h effects o p e r a t e d for t h e resu l t s exempl i f ied in T a b l e I I I (exps . A - F ) , exp . G, T a b l e I II shows t h a t ( N R x N R ) F | females p r i m e d a n d b o o s t e d w i th F , m a l e cells i / p c a n also g e n e r a t e H-Y-speci f ic cy to tox ic r e sponses , a resul t w h i c h cou ld no t d e p e n d on a l logeneic effects.

T h e i n t e r p r e t a t i o n m a d e of posi t ive a n t i - H - Y r e sponses in n o n H-2^ ( N R x N R ) F | h y b r i d s was t h a t

/ r e g i o n a n t i g e n s of t h e F , were different f rom e i the r of those of t h e p a r e n t a l h a p l o t y p e s . T h u s , / - / c o m p l e ­m e n t a t i o n o c c u r r e d , w h e r e b y t h e F , l a a n t i g e n s b e c a m e ' a s soc i a t ive ' for H - Y ( w h e r e a s t h e p a r e n t a l ones were no t ) a n d cou ld effectively p r e s e n t H - Y to t h e h e l p e r cell. The re fo r e , a n a n a l o g y w a s d r a w n b e t w e e n t h e ' a s soc i a t ive ' K / D a n t i g e n s i m p l i e d by t h e ta rge t -ce l l specificity d a t a a n d ' a s soc i a t i ve ' l a a n t i g e n s n e e d e d to ac t iva te T h e l p e r c e l l s ' ^ ^ ^ ' ' ' " . T h e r e su l t s w i th t h e c h i m e r a s were i n t e r p r e t e d a l o n g s imi la r l ines, by sugges t ing t h a t t he l a a n t i g e n s of c e r t a i n h a p l o ­types , e.g. / / -2k, whi ls t i n a d e q u a t e l y assoc ia t ive w i t h H - Y to ac t iva te t h e / /-2 '<Th cell cou ld never the less ac t iva te , for e x a m p l e , t h e //-2"^Th cell. T h e i n t e r p r e ­t a t i on of the r e s p o n s e of F , h y b r i d s fol lowing i / p i m m u n i z a t i o n as / - / c o m p l e m e n t a t i o n is not

T A B L E III. H-Y responses in non- H-2b F, hybrids and chimer

Exp. Responder (fromref.) female

,, , Primins and ^ ,, U-2 , . ' Target cell

boosting ^ antigen

n-2 Corrected % lysis

Restricting specificity

AdD

B(12)

C(12)

D(53)

E(53)

F(53)

(CBAxBlO.S)F, k k k k k k CBAC?

J J ,1 ,1 V i B10.S<?

(B10 .D2xB10 .S)F | r f d d d d d BlO.Sc^"

B10.D2C?

( C B A x B l O . G ) F i k k k k k k BIO.CJC?

q q q I] q q

CBAJ

(B10.MxB10.D2) / / / / / / B10. \4C?

(/ d d d d d

B10.i:)2c?

( B l O . S x B l O . M ) 1 .V ,1 >• ,( ,< BlO.Sc?

./ .f / / .1 ./• B10.M<J

(BALB/c • CBAC? (CBAxBALB/c) * * * * * * (chimera) F,

dddddd » d d d d d d

CSHOHC?

Biased A.THc?

BlO.Sc? B10.S9 A.TH<J

B10,D2cJ B10.D29

BlO.Gc? BIO.G9 AQRc?

CBA(J CBA? B10.A<? C3H.OH(?

B10.M<J BIO.M9 BlOd"

BIO.D2t5'

BlO.Sc? BIO.S9 BlOcJ

BlO.Mc? BIO.M9 BlOc?

CBAC?

CBA9

k k k d d d 22.7 + 0.6 " d d d d d k 12.5 ±2.3

I I .( .1 s s 14.1 ± 2 . 2 '

J ,1- ,1 i f d 0.0 + 0.1

,1- 42.6 + 1.4 ,>• - 0 . 2 ± 0 . 1 d - 1 . 8 + 0.3

H-2KK fl*'

H-2iy

d d d d d d d d d d d d

-3.9 + 0.4 -3.7 ±0.1 }

II-21Y

no response

(j q q q q q 22.1 + 1.1 ^ q q q q q q 3.9 ± 0.2 V I!-2I)'l q k k d d d - 2 . 9 ± 0.7 J

k k k k k k 16.0 ± 0 . 5 •] k k k k k k 3.8 ± 0.3 I , . t k k k d d d 13.7 ± 0 . 4 r d d d d d k 12.5 ± 1.4 J

f f f f f f 27 .0 ± 3 . 4 1 f J J f f f f 6.1 ±1.2 ^ " n d l r / / * h h h h h 2,2 ± 0.8 J " " " / ' " "

d d d d d d 0.8 ± 1.5

/ / / / / / 26.7 ±1.6 J f J f f .f 1.2 ±0.1

* /) /) /) /) /; - 2 . 2 ± 0 . 6

k k k k k k 32.8 ± 1.5

k k k k k k 1.4 ± 2 . 0

no response

,1 ,1 ,( ,v ,( ,f 1 5 . 2 ± 1.5 V V .( J i .f 1.9 ± 0 . 2 ) - If-.

h b b b b h 2.1 ± 3 . 8

H-2f

(BALB/cxCBA) d d d d d d (BALB/c F, ^ ^ xCBA)F,c?

k k k k k k

(BALB/c xCBA)F,c? (BALB/c xCBA)F,$

16.8 ±2.1 T //-i*' Y and/or

5.5 ± 1.3 J H-2''

Im/niinoloi^] Today, vol .1. .\'a. I. I9S2 103

invalidated by suljscquent findings that certain non-//-2b strains can be induced to respond following footpad immunization (see next section), since none of tfie parental strains of the l", hybrids used could respond after i/p immunization. While we are in con­tinued ignorance of the detailed nature of the T-cell receptor, it is very difficult to evaluate the likelihood of this 'association model" hypothesis to explain H-2-linked Ir gene control of responsiveness to H-Y. It is still an attractive hypothesis to explain certain aspects of H-2-linked control, but it cannot be the whole story, since subsequent experimental results have since revealed non-H-2 //• genes which apparently interact with //-2 genes to determine responsiveness.

HiiiiKi.zygoli'x III (ilhfr }l-2hiiplnlypes

Kli Sercarz c/ rtlr", showed that H-2-linked geneti­cally controlled non-responsiveness to hen egg lysosyme in //-2b mice could be overcome by immunizing mice in the footpad (fp) rather than via the usual i/p route. Following this lead Miillbacher and Brenan'" showed that CB.A (11-2^) female mice given syngeneic male cells via the footpad (fp) could generate excellent H-Y-specilic, H-2-restricted C TL responses. These responses were subsecjuently shown to be helper-cell de|)endent, because they could be blocked in the in-ntm induction phase by a combina­tion of two monoclonal anti-la antisera, one anti-I.X*^ and one anti-IE/C*"". The contrast between the failure ot CBA mice to respond after i/p in rivn immunization and their excellent responsiveness after fp [jriming is not completely understood, but may be due to the preferential induction of T suppressor cells (Ts) after i/p |)riming, as reported by Araneo el nl."'' for egg lysosyme. or with some special property ol antigen-[)resenting cells (e.g. Langerhans cells) in the footpads''".

Additional factors have to be invoked to explain the strain distribution pattern of res|)onsiveness to H- \ ' following fp priming found in subsec]uent studies by Fierz li tii.'\ They fovmd that the //-2 hapiotxpe alone does not predict responsiveness: some 11-2^ strains '(e.g. C^BA.BALB.K) are good responders with over 8()'!ii of the immunized mice making strong res])onses whilst others e.g. AKR. CE/ J ) do not respond. I'here are also strains showing intermediate responsiveness, in that only a proportion of primed mice respond (e.g. C,3H, RF). I'his same pattern is found when strains of the Il-2'i and //-2'^ haplotypes are examined following footpad priming, e.g. for ll-2'K Bl().n2 is a responder, B.\LB/c a non-responder. for / / - 2 \ BHKS is a responder, ;\.SVV is not. However, in the case of //-2b strains, all those examined give excellent H-\'-specific cvtotoxic F-cell res|)onses after either i/|) [triming (C,57BL/10, C.3H.,S\V, A.B\ ' . 129/Ji-) or fp priining (C57BL/IO, BALB.B F P / ) , 129/J) (Ref. FT). Fakcn together, these results stiggest thai whilst the 11-2 Ir genes of the //-2b haploty])e have an overriding effect, allowing resjjonses regardless of non-//-2 genes, thai for all the other haplotvpcs examined. non-//-2 Ir

genes are involved, as well as 11-2 Ir genes. Fhe interaction between these two types of genes is suggested by the fact that a particular set of non-Il-2 genes, e.g. those of the BALB background, do not alone determine responsiveness since B.'\FB/c (H-2'^) are non-responders whilst B.XLB.K (H-2^) are responders to H-Y. Fhe nature and mapping of the non-//-2 gene(s) involved is presently under investiga­tion but it can be speculated that they might have to do with the structure of the T-cell receptor itself. On this hypothesis non-responsiveness could indicate a deficit in the 'l"-cell repertoire for the FI-Y antigen in assoeiatitm with self-H-2 molecules".

Non-responsiveness and natural tolerance .•\ hypothesis has recently been put forward by

Miillbacher"^ to account for some forms of non-responsiveness to both H-Y and viruses. It suggests that H-Y' (or virus) associated with certain self K / D alleles mimics the identity of another self molecule to which the animal is necessarily naturally tolerant. Fhus, because the T-cell clone bearing the appro­

priate receptor has been deleted, or suppressed, e.g. by anti- idiotype suppression, there can be no response. Two observations suggested this hypothesis.

Firstly, a [iroportion of female mice making an FI-Y-specific response generate cytotoxic cells which will also kill allogeneic male and female targets"'^"". Fhese killer cells could either be anti-allogeneic clones polyclonally activated during the iti-ritrn M F C or could be genuinely cross-reaetive, i.e. self K / l ) + FI-Y and alloantigcns may look alike. There is evidence that cross-reactive clones occur from the isolated H-Y + Db specific clone which also kills any targets bearing I)il, reported by von Boehmer I'l iil."". and from the alloreaetivity mediated by herpes simplex virus-specific cytotoxic clones reported by Pfizenmaier el al." . Secondly, the hypothesis was suggested by the [)henomenon of 'parental preference' first reported by CJordon el iil." and further analysed by Brenan and Miillbacher"'"'. It was found that although F, hybrid females with one //-2b parent can make an H-Y-speeilie response to male cells of either parental haplotype when primed and boosted with homo­zygous male cells ('Fable IF exps. B and C), when the response is elicited with F, cells, it is frequently directed against the male cells of only one of the two parental 11-2 haplotypes. In the case of (BIO x C ; B ; \ ) F , hybrids, this is usually the CB. \ male^' "' and in the case of (BIO x B1().S)F| hybrids, it is always the BIO..S male"'. This is illustrated in'Fable IF (exp. K).

Miillbacher"-- argued that these findings could be explained by natural tolerance. 'Fhus. in the case of the example given in 'Fable II (exp. E). Db + H- \ ' cross-reacted with //-2A'» and/or l)^ so that the (/) x OF, female, which had K^ and 1)^ as self-molecules, could not make the response to I)b + H-Y. Certainly no better explanation has been ptit forward for the phenomenon of parental preference in H- \ ' responses

104 Immunolvgy Today, vol. 3, No. 4, 1982

of F | hybrids and it would also account for some similar preferences seen in anti-viral responses.

The prediction which can be made from this is that if homozygous H-Y responderstrain mice are made tolerant of certain alloantigens, they may be rendered specifically non-responsive to H-Y. This has been demonstrated in CBA mice tolerized to BIO allo­antigens by neonatal injection of (BIO x CBA)F| female cel ls ' \ Such mice fail to make an H-Y response after fp priming but can make normal third party and anti-viral cytotoxic T-cell responses.

Whether natural tolerance could account for non-responsiveness to H-Y in normal homozygotes of strains which do not respond remains to be seen. Such an explanation would predict that in F, hybrids non-responsiveness would be dominant between respond-ers and non-responders of the same H-2 haplotype unless there were other regulatory influences con­comitantly involved.

Non-responsiveness and suppressor cells The involvement of T suppressor cells (Ts) in

regulating some aspects of H-Y responsiveness was implied from the results of work on tolerance to H-Y with respect to graft rejection-^ ^ and the cytotoxic T-cell response^'' in the responder strain C57BL. The ability to transfer tolerance to H-Y using spleen cells suggests that such tolerance is maintained by Ts. The occasional finding of individual donor and recipient mice which were tolerant by skin graft criteria, but could still make a cytotoxic response, or vice versor'', indicated that either separate Ts were involved in suppressing anti-H-Y skin graft rejection and the H-Y-specific cytotoxic response, or that the mediators of these two responses were different cell types and had different sensitivities to the effect of Ts, or both. As will be discussed below, there is good evidence that the cells responsible for rejecting syngeneic male skin grafts are not H-Y-specific cytotoxic T cells.

Further evidence for the involvement of Ts in regulating responsiveness to H-Y comes from studies of the secondary host-v-graft response and this will be discussed in a later section.

DTH responses to H-Y D T H responses can be elicited to H-Y following

subcutaneous (s/c) immunization with syngeneic lymphoid cells'^-'. Such responses are given not only by C57BL/10 (//-2b) females but also by all those //-2b recombinant strains which carry the / /? ' ' subregion, i.e. B10.A(5R) {bbbddd), B10.A(4R) (kkbhhh)'and D2.GD (ddbbbb)'^--'. However, H-2b recombinant strains which lack IB^\ e.g. B10.A(2R) (kkkddb) as well as non-//-2b strains, e.g. CBA {H-2^), BALB/c (//-2d) and A.SW (//-2s) do not make DTH responses to H-Y following s/c immunization. In fact, the strain distribution pattern of DTH responses after s/c immunization and that of primary skin graft rejection responses is perfectly correlated,'"'^ suggesting that

the effector cells for skin-graft rejection may be T^ j , cells. Further evidence for this comes from the finding that Ly 1 + but not Ly 2+ (i.e. Ly 1 + 2+) T cells transfer DTH to H-Y'^ and that skin graft rejection involving discrepancies at H-2 is mediated by Ly 1+ but not by Ly 2+ T cells''". What certainly makes it unlikely that //-2-restricted H-Y-specific cytotoxic T cells are crucially involved in rejection of the H-Y-incompatible skin grafts is the existence both of strains which reject such skin grafts but cannot make cytotoxic responses (e.g. B10.A(5R), B10.A(4R)) and of strains which can make cytotoxic responses but do not reject primary H-Y incompatible skin grafts (e.g. many non-H-2^F, hybrids following i/p priming'-). An interesting recent finding has been that following fp priming with syn­geneic male cells some CBA females will reject CBA male skin grafts (Chandler and Simpson, in prepara­tion). However, the time course of this response following immunization is rather different from the time course for the H-Y-specific cytotoxic T-cell response after fp pr iming" in that such secondary graft rejection responses can be dieted in 30—40% of fp immunized mice 14 days after priming, in 90% after 21 days and thereafter only irregularly (Chandler and Simpson, in preparation). This correlates with the ability of CBA females to make DTH responses to H-Y 21 days after footpad immunization, but not at 14 days (Liew and Simpson, in preparation). In contrast, more than 95% of CBA females will give H-Y-specific cytotoxic T-cell responses from 3 weeks until months after p r iming"" ' ' " . Skin grafting of CBA females with syngeneic male skin alone does not immunize such mice to give either secondary skin graft rejection of subsequently placed CBA male grafts, or H-Y cytotoxic or D T H responses (Chandler, Liew and Simpson, in preparation). This might suggest that attempts to immunize CBA females to H-Y by graft­ing or by i/p injection of male spleen cells generates suppression (perhaps via Ts) rather than immunity. Further evidence for this will be considered in the following section.

Host-v-graft (HVG) responses to H-Y Primary HVG responses to H-Y can be measured

by the increase in popliteal lymph node weight of female mice injected 9-12 days previously with 15 x 19'' syngeneic male spleen cel ls '"" ' ' . Not only do C57BL/10 (//-2b) mice give good HVG responses to H-Y but so do CBA (//-2k) and even BALB/c (//-2d) although they make neither skin-graft nor cytotoxic T-cell responses to H-Y. BIO.RIII {H-2'') appear to be non-responders'". Secondary HVG responses are seen 2 weeks after immunization with a peak at 2-4 days after elicitation (Ref. 18; Pole, in preparation). In C57BL/10 (//-2b) females such secondary responses occur following immunization by skin grafting or by injecting 1 x 10' spleen cells by s/c"*, i /p, i/v or fp routes (Pole, unpublished). In contrast, CBA mice give good secondary HVG responses after fp

Immunoh^y Today, ml. J, ,\''. /. /<'S2

TABLE i \ ' . T-ccIl responses lo \\-Y in //-_''' nnd non 11-2" strains

Strain Skill tiral't rejection

Frimar\ Seeondarv

HV(i U-Y si)critic cvtotoxicity \rVU after

Primary Secondary i/p priming tp j)riming

H6. BIO HI0.A(5R) B10.A(4R) B1().A(2R)

CBA AKR BALBK

Bid, 1)2 BALB/t

BIO.S A,SW

/,

k 1. k

,1 •1

'

;, /,

/,

k k k

,1

k> /) /,

k k k

,1 ,k

'

/- /, ;, d ,1 ,1 /) A /,

k k k k k k k k k

,k ,k -/ ,k ,k //

, , .

-M

M M

NT N l

NT Nl

_ -

NT'

-

\JT

NT

NT'

NT

NT

NT

NT

NT

bcsl after l[ ''oniv after Ij) j)rimin^; immunization but i/v immunization sup|)resscs the response. In BALB/c miee immunization \)\ any route abolishes HVfi responsiveness so that it cannot be seen cither at the lime expected for secondary or primary responses (Pole, in preparation). The cell type(s) proliferating in the HVCi responses have not yet been identified but it is possible that they represent helper cells (Th), e.g. those necessary for either the ' re res[)onse and/or the I) TH response. It is upon this Th population that the Is may act, hence the abrogation of such proliferation in CB.A females following inappropriate immunization and the correlation in CBA mice of positive secondary IlVCi responses and the ability to generate H-Y-specific cytotoxic I cells, secondary skin graft rejection and DTH only after fp priming. The singular inability of BALB/c (//-i'l) mice to generate //-i 'l-rcstricted cytotoxic T-ccll responses to H- \ ' correlates with sup|)ression of secondary H\ ' ( i . In contrast, the case with which C^.S7BL/K) (11-2'') mice can make primary and secondary anti-H-Y skin graft rejection res[)onscs and cytotoxic T-cell responses after immunization by any route correlates with their primary and secondary HVX; responses.

Anti-H-Y responses in man The influence of H-Y on allograft rejection was first

noted t)\' Oliver ' from a scries of kidney transplant patients in which the presence of II-Y on the graft (i.e. male donors) seemed to aflord the graft some protec­tion ([)erhaps enhancement) in female recipients. However, since then it has become apparent that the presence of H-Y on grafts can also be associated with more rapid graft rejection, both of bone-marrow grafts- and kidney grafts ' \ foifowing rapid rejec­tion by females of HLA-matched male grafts it has been possibie lo elicil in .\II,C HL.\-restricted H-Y-specific cytotoxic 'I'-cell responses from the [)eri[)heral blood lymphocytes of such patients - '.

The interesting series of patients reported by London ' and another similar series of Thomas

]:)rimint^; ,Ni': not lested

(personal communication) both provide evidence that hepatitis virus (HBs antigen) and H-Y antigen cross-react. Thus, female recipients who, after hepatitis eliminated the virus and had anti-HBs antibody, also had an increased tendency to reject mafe kidney grafts. In contrast, in female recipients who had become chronic carriers of the virus following hepati­tis, and had circulating HBs antigen but no antibody, male grafts were relatively protected from rejection.

Summary H-Y elicits many types of immune response. Ihe T-

cell responses, i.e. graft rejection, cytotoxic T-cell, D'LH and IlVCi responses are generally strong, whereas the B-cell, antibody response is weak. The T-cell responses involve different T-cell sub-populations'- and are controlled by Ir genes both in the h'/D regions"''-'^''^'-^^ and in the lA, //^ and E/C sub-regions ' - ' " ' - ' " "•i".^"'-''-''^-^'^.'^* of the II-2 complex as well as the so-far unmapped non-//-2 Ir gene(s) ' ' (see I'able IV). The mode of action of these Ir genes is not altogether understood. However, K/ I ) and I (class I and class II) antigens which are capable of'associat­ing' with II-Y in order for it to be recognized by Tc and Th respectively are necessary and some alleles may be non-assoeiative and thus may cause non-responsiveness e.g. A7; tint! Ih! in BIO./i('>Ri ihhbildil) mice' ' . Possession of Ir genes mapping to lAh'"''-'-'"'-*'^ and IBh'"-'^'-' confer strong and readily elicited responsiveness respectively for the helper component (Th) of the cytotoxic T-eell (Tc) response and for the skin graft rejection and DTH responses and these are dominant t r a i t s " ' - ' ' " . However, I region antigens of other haplotypes can be permissive for Th function"" although their activity can be modified by non-//-2 genes". The involvement of T suppressor cells (Ts) in producing nonresponsiveness is not yet clear, although there is good evidence that Ts can be elicited both in the res[)onder strain C.S7BL---'-" where they are associated with tolerance to H-Y and probably also in non-responder strains, e.g. C.BA and B/\LB/e (see

106 Immunology Today, vol. 3, J^o. 4, 1982

sections on D T H and HVG). Failure to make a response may in some instances be due to natural tolerance towards H-Y plus self" '"^ This is the most reasonable explanat ion for the phenomenon of parental preference*'"^"''.

Unravelling the complexities of the immune re­sponse to H-Y may give us better insight into the control of immune responses to other cell surface determinants acting as weak transplantation antigens. Tumour antigens also fall into this category.

I thank Drs D. Bailey, E. Eicher, L.Johnson, W. Fierz, \ . Mullbacher, G. .Stockinger and P. Chandler for discussion on this manuscript. Sir Peter Medawar has constantly helped and encouraged me in this work.

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185 ,963 4 Simpson, E., Brunner , G., He the r ing ton , C., Chand le r , P.,

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5 Silvers, W. K. a n d Bil l ingham, R. E. {\967) .Sneme, 158, 118 6 J o h n s o n , L. L. (1979) Immunagmflic.s S, 373 7 Kralova, J . and Lengerova, A. (1979) J. Immunogvnvlus 6, 429 8 Bailey, D. W. (1971) Transplanlalum 11, 426 9 Gasser , D. L. a n d Schreffler, D. C. (1974) Immunogmetus 1, 133

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26 Goldberg , E. H. , Boyse, E. A., Schied, M . and Bennet, D. (\912) .Nature .New Biol. 2^9., ii

27 Wach te l , S. S. (1977) Immunol. Rev. 33, 3

28 van Leeuwen, A., Goulmy, E. and van Rood, J . J . (1979) J. Exp.Med.\iO,\01i

29 Melvold, R. W. , Kohn , H. 1., Yerganian , G. a n d Fawcet t ,

D. W. (1977) Immunogenetics 5, 33 30 Wolf, U., Fraccoro, M. , Maye roue , A., Hecht , T. , Zuffardi, O .

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