Eur. J. ImmunoL 1978.8: 685-687 Anti-H-Y responses of H-2b mutant mice 685

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15 Luft, J . H.,J. Biophys. Biochem. Cytol. 1961. 9: 409. 16 Metcalf, D., J. Zmmunol. 1976. 116: 635. 17 Andersson, J. , Coutinho, A., Lernhardt, W. and Melchers, F., Cell

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11 Rozenszajn, L. A., Michlin, H., Kalechman, I. and Sredni, B., 20 Jacobson, H. and Blomgren, H., J. Zmmunol. 1976.114: 1631. Immunology 1977. 32: 319.

Elizabeth Simpson4 R.D. Gordon’, P.R. Chandlernand D. Baileyo

Transplantation Biology Section, Division of Surgical Sciences, Clinical Research Centre, HarrowA, and The Jackson Labora- tory, Bar Harbor

Anti-H-Y responses of H-2b mutant mice

Two strains of H-2b mutant mice, H-2ba and H-2bf, in which the mutational event took place at H-2K, make anti-H-Y cytotoxic T cell responses which are H-2-restricted, Db-associated and indistinguishable in target cell specificity from those of H-2b mice. Thus, alteration of the H-2K molecule affects neither the Ir gene controlling the response, nor the associative antigen. On the other hand, one H-2Db mutant strain, H-2bo, although it makes a good anti-H-Y cytotoxic response, shows target cell specificity restricted t o its own Dbo anti- gen($, and neither H-2b, H-2ba o r H-2bf anti-H-Y cytotoxic cells kill H-2bo male target cells. Thus, the alteration of the H-2Db molecule does not affect the Ir gene of H-2b mice, but it does alter the H-2Db-associative antigen.

1 Introduction

Female mice of the H-2b haplotype reject syngeneic male skin grafts and develop H-2-restricted cytotoxic T cell responses to the male-specific antigen, H-Y, encoded or controlled b y gene(s) situated on the Y chromosome [l-31. Mouse strains of all the other independent haplotypes so far examined are nonresponders t o H-Y, although certain F1 hybrids of two nonresponding parental strains are responders with respect to the generation of cytotoxic responses indicating Ir gene complementation for this response [3,4] . Ir gene(s) con- trolling the response, both in H-2b strains and in the various F1 hybrids, appear t o lie within the H-2 complex (MHC), and at least one of the complementary genes in both H-2k/H-2S F1 hybrids and H-2k/H-2q F1 hybrids maps in t h e IC region [4,51.

In this study, we provide evidence (a) that the Ir gene(s) controlling anti-H-Y cytotoxic responses of H-2b mice are unlikely t o map a t either K or D, with the implication that they probably map in the I region; and that (b) the target cell specificity of the responses of H-2b mutant strains shows

[I 20781

+ Present address: Department of Surgery, Massachusetts General

Correspondence: Elizabeth Simpson, Transplantation Biology Section, Division of Surgical Sciences, Clinical Research Centre, Watford Road, Harrow, HA1 3UJ, Middlesex, GB

Abbreviations: MLC: Mixed lymphocyte culture A:T: Attacker-to- target cell ratio MHC: Major histocompatibility complex

Hospital, Boston, MA 02114, USA.

that the mutational event which alters the H-2Db-coded mole- cule restricts its ability t o act as part of the H-2Db-H-Y asso- ciation which serves as a target for sensitized H-2b-type cells.

2 Materials and methods

2.1 Mice and immunizations

C57BL/6 (H-2b), HZI (H-2ba), HZ170 (H-2bf) [6] and HZW42 (H-2bo) [7] mice were bred at the Jackson Labora- tory, Bar Harbor, ME. H-2ba and H-2bf are H-2Kb mutants H-2b0 is anH-2Db mutant, C57BL/10, BlO.A(SR) and BlO.A(2R) mice were bred a t the Clinical Research Centre. Two t o 4-month-old female mice were primed by intraperi- toneal injection of 1 x lo7 male spleen cells three weeks to five months before use.

2.2 In vitro sensitization and cytotoxic assay

The methods used were those previously published in detail [8]. In brief, mixed lymphocyte cultures (MLC) were set up between spleen cells f rom previously immunized mice and irradiated male spleen cells as antigen. After five days of in- cubation, these cells were harvested and assayed against ap- propriate SICr-labeled target cells at four attacker-to-target (A:T) cellratios, 10 :1 ,5 :1 ,2 .5 :1 and 1.25: l . Regressionana- lysis of t h e percent corrected lysis plotted against the A:T ratio was performed, and the results were expressed as the percent corrected lysis a t 4: 1 from t h e regression curve. Posi- tive results were only accepted when the r2 value for the re- gression curve lay between 0.90 and 1 .OO. All experiments reported were repeated at least three times.

686 E. Simpson, R.D. Gordon, P.R. Chandler and D. Bailey Eur. J. Immunol. 1978.8: 685-687

3 Results

C57BL/10 (B10) and C57BL/6 (B6) mice differ only at a few minor (non-H-2) loci. They make anti-H-Y responses of indistinguishable H-2-restricted specificity. The H-2 mu- tants used were derived from the H-2b haplotype in the B6 strain, and whenever they were cross-immunized with H-2b cells, either in vitro or in vivo, an attempt was made t o use B6 rather than B10 male cells. However, although in some experiments B 10 male cells were used instead of B6 male cells for immunizing the mutants, and vice versa, the results obtained did not differ.

Tables 1 and 2 show that not only the H-2b mice make a good anti-H-Y cytotoxic response after homologous priming and boosting, but so d o mice with both the K end and the D end mutant haplotypes, H-2ba, H-2bf and H-2bo. This sug-

Table 1. Anti-H-Y cytotoxicity of homologously primed and boosted H-2b mice and H-2b mutant mice

Female Priming and Cytotoxicity: 51Cr-labeled target cells”)

maleantigen 6 Q d 0 d 0

H-2b (B6) H-2b (B6) 26.2 0 20.7 2.7 6.6 0 ~ - 2 b a 14-2ha 55.2 4.0 42.0 4.0 5.4 0 H-2bf H.2bf 37.1 7.0 34.9 4.4 6.2 5.0 H-2bo H-2ho 17.5 3.9 19.1 7.5 3 1 . 3 0

responder boosting H-2b (B10) H-2ba H-2bo

a) Percent corrected lysis at A:T = 4: 1, from a regression analysis (see Sect. 2.2).

Table 2. Target cell specificity of anti-H-Y cytotoxicity of homologously primed and boosted B6 (H-2b) mice and two H-2Kb end mutant mouse strains

Cytotoxicitya) on male target cells Female Priming and H-2b H-2ba H-2bf BIO.A(SR) BIO.A(ZR) respondcr booTting (B 10) (bbbddd) (kkkddb)

male antigen

H-2b (BIO) H-2b(BlO) 14.1 22.4 N.D. 3.4 15.7 H-2bS H-2ha 16.9 26.3 N.D. 0 20.5 H-2bf H-2bf 28.0 N.D. 31.1 5.6 31.4

a) See legend to Table 1. N.D.: Not determined.

gests that the mutational event involving either the H-2K or the H-2D end does not affect any Ir gene controlling the re- sponse in H-2b mice. Tables 1 and 2 show that the target cell specificity of H-Y cytotoxicity of the two K end mutant mice is indistinguishable from that of the H-2b mice, and it is H-2Db- associated.,However, anti-H-Y cytotoxic cells from both the H-2K end mutants and H-2b mice kill male cells from the H-2D end mutant, H-2bo, only poorly, and anti-H-Y cyto- toxic cells from H-2bo females kill homologous H-2bo male cells twice as well as male cells of H-2b or the H-2K end mutant (Table 1). This suggests that the mutational event at the H-2D end has altered the H-2Db molecule to an extent that it cannot act as a recognizable target for H-Y associa- tion for wild strain-primed cells and vice versa, that anti-H-Y- primed, H-2D mutant cells can only “see” H-Y in association with their own unique mutated H-2Db molecule. The differ- ences observed between H-2b and H-2ba and H-2bf in the level of the cytotoxic response (shown in Table 1) is a result of mouse t o mouse variation, and none of the three strains was consistently a better or worse responder than the others (com- pare the responses of H-2b and H-2ba mice in Tables 1 and 2 which represent two experiments performed on different oc- casions).

The ability of H-2b mice t o recognize t h e mutated K and D antigens and of the mutant strains t o recognize K and D anti- gens of H-2b is shown in Table 3. In fact, the cytotoxic re- sponse t o these antigens “swamps” the anti-H-Y response, a result similar t o that which has been seen whenever H-2-in- compatible male cells are used for the in vitro challenge of homologously primed female cells [8]. Thus, spleen cells of anti-H-Y-primed H-2b mice boosted in vitro with male cells of the mutant strains make an anti-mutant cytotoxic response and virtually no H-Y response. This is shown by the killing of bo th male and female cells of the mutant strain used for in vitro boosting, and little, if any, killing of H-2b male cells. This result is obtained whether the in vivo priming was done homologously with H-2b male cells, or with male cells of the mutant strains subsequently used for in vitro boosting. There is some contrast in the results found with the K end mutant strains primed homologously and then boosted with B6 male cells, in that although the predominant cytotoxic response is directed at H-2b target cells regardless of sex, there is a smaller but substantial component directed against self male cells, i.e. an anti-H-Y response. This is not , however, found with the H-2Db mutant, H-2bo, which makes a pure anti-H-2b response (demonstrated against target cells of B10 male and B10 female),

Table 3. Recognition of K and D end antigens between H-2b and H-2b mutant strains

Cytotoxicitya) on targets H-2b3 H.2hf H.2ho Female Male antigen used Male antigen used H-2h

responder for priming for boosting (BI0) in vivo in iiitro d O d O S Q d Q

H-2b H-2b H-2” ~ - 2 h a H-2bf H-2bo H-2b H-2h

(B10) H-2h (B10) H-2h (B10) H-2b

H-2ba H-2bf H-2bo

(B6) H-2”” (86) H-2b0

H-2bd 7.1 (B10) H.2bf 3.1 (B10) H-2bo 0

H-2h (B6) 39.7 H-2b (B6) 35.0 H-2b (B6) 36.4 H-2ba 4.6 w 2 b o 9.9

u 60.0 0 N.D. 0 N.D.

45.3 26.4 27.9 N.D. 31.9 N.D.

1.8 28.2 2.0 N.D.

47.7 N.D. N.D. 0 N.D. N.D. 29.3 N.D.

N.D. N.D. N.D. 34.0 47.9 N.D. N.D. N.D. 31.9 N.D. N.D. N.D. 22.5 0 N.D. N.D. N.D. 0 N.D. N.D. N.D. N.D. N.D. 48.8

N.D N.D 24.5 N.D. N.D. 0 N.D. 47.0 a) See legend to Table 1.

N.D.: Not determined.

Eur. J. Immunol. 1 9 7 8 . 8 : 685-687 Anti-H-Y responses of H-2b mutant mice 687

and none against H-2bo male cells. This finding suggests that H-Y presentation with the appropriate H-2K or D molecule, H-2Db in this case, is necessary not only for target cell killing but also for induction of the response (Tables 1 and 3).

4 Discussion

Each of the three mutant strains used in this study are con- sidered t o have undergone a gain plus loss type of mutational event involving either the K end (H-2ba, H-2bf) or the D end (H-2bo) of the H-2 complex. This is evidenced by mutual skin graft rejection between each mutant strain and t h e B6 strain [6, 71 and by the mutual development of cytotoxicity during an MLC between each mutant strain and B6 strain (Table 3). In the case of t h e two K end mutants, this mutational event has altered neither their ability to make a secondary anti-H-Y cytotoxic response, nor t h e target cell specificity of that re- sponse, which is Db-associated (Tables 1 and 2). This result contrasts with the H-2-restricted cytotoxic responses of these same H-2 K end mutant strains to viruses [9] only because the virus antigens are both H-2K and H-2D-associated. Thus, H-2ba and H-2bf mice do not make H-2Kb-associated anti-viral re- sponses (as detected on the recombinant BlO.A(SR), which is KbDd), although they d o make H-2Db-associated responses, detectable on H-2b virus-infected targets. Since anti-H-Y re- sponses of H-2b mice are only H-2Db-associated, one would expect t o obtain a comparable finding only with an H-2D mutant, and this we are reporting. Thus, H-2bo females make a good homologous anti-H-Y response, indicating that the Ir gene(s) controlling this response is not affected by t h e muta- tion, bu t the target cell specificity is affected: male cells of the H-2Db mutant cannot act as target cells for B 6 anti-H-Y cytotoxicity, and t h e anti-H-Y response of the H-2bo cross- kills H-2b and H-2ba male cells much less efficiently than the homologous male target H-2bo.

Our findings therefore confirm those of Zinkernagel [9] and others working with H-2-restricted responses t o viruses in that mutational events of K/D molecules d o affect target cell speci- ficity of these responses, presumably b y altering the self H-2 component of the target antigen. In addition, t h e data pre-

sented in Table 3 argue that the mutational event alters H-Y presentation during induction of the response, a t least at the level of the in vitro boost, so that the H-2bo female mice, homologously primed with male cells, cannot be boosted with H-2b male cells for an anti-H-Y response, in contrast t o the two K end mutant strains, which can. This suggests that H-Y is Db-associated for induction as well as for cytotoxicity.

The finding that the ability t o make anti-H-Y responses p e r se is not affected by either K or D end mutational events sug- gests that the Ir gene(s) controlling the response, known to be situated in the H-2 complex [ 1-31, are not at the K or D end. By analogy with Ir genes controlling antibody re- sponses t o a variety of antigens, it would seem likely that they should be in the I region. Data from von Boehmer [ 101 suggest that the dominant H-2b gene controlling H-Y responses maps in IA o r IB, and we have recently localized this gene t o the IA subregion [5]. Mapping data o n anti-H-Y responses in F, mice from complementary haplotypes (other than H-2b) suggest that at least one of the complementary genes maps in IC [4, 51.

Received April 3, 1978; in revised form June 22, 1978.

5 References

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5 Hurme, M., Hetherington, C.M., Chandler, P.R. and Simpson, E.,

6 Bailey, D., The Jackson Laboratory Annual Report 1975. 46: 73. 7 Bailey, D., The Jackson Laboratory Annual Report 1 9 7 6 . 4 7 73. 8 Gordon, R.D., Simpson, E. and Samelson, L., J. Exp. Med. 1975.

9 Zinkernagel, R.M., J. Exp. Med. 1976. 143: 437.

and Simpson, E., Immunogenetics 1 9 7 7 . 5 : 453.

J. Exp. Med. 1 9 7 8 . 1 4 7 758.

142: 1108.

10 von Boehmer, H., Fathman, C.G. and Haas, W., Eur. J. Immunol. 1977. 7: 443.