Immunogenetics 13: 355-358, 1981 Im mullo= genetics

© Springer-Verlag 1981

H-Y Antigen in Sxr Mice Detected by H-2-Restricted Cytotoxic T Cells

Elizabeth Simpson ~, Peter Edwards 2, Stephen Wachtel z, Anne McLaren 3, and Phillip Chandler 1

1 Transplantation Biology Section, Clinical Research Centre, Warlord Road, Harrow, Middlesex, HA1 3UJ, England

2 Memorial Sloan Kettering Cancer Centre, New York 3 MRC Mammalian Development Unit, University College, London, England

Mice carrying the autosomal dominant mutation Sxr show some discrepancies between karyotypic and phenotypic sex. Sxr, XX individuals develop as males, although their testes are small and germ cells scarce or absent in the adult. Sxr, XY males have a slightly reduced testis size but they are of normal fertility (Cattanach et al. 1971). It has been proposed that Sxr may represent a small translocation from the Y chromosome to an autosome (Cattanach 1975). There is serological and graft- rejection evidence that Sxr, XX individuals have H-Y antigen on their epidermal cells (Bennett et al. 1977) and that Sxr, XY individuals may express an excess amount of H-Y antigen (Bennett et al. 1977).

This report describes the detection of H-Y antigen quantitatively and quali- tatively indistinguishable from normal in Sxr, XX males and Sxr, XY males. H-Y was detected (a) on peripheral blood lymphocytes (PBL) of normal and Sxr-carrier mice used as target cells in a 51Cr release assay of anti-H-Y cytotoxic T cells from normal H-Y-primed C57BL/10 mice, and (b) by attempting to sensitize Sxr, XX males to H-Y on normal C57BL/6 male cells. Since anti-H-Y cytotoxic T cells are H- 2-restricted and the response is under H-2-1inked, Ir-gene control with H-2 b mice as invariable responders (Simpson and Gordon 1977), we chose the H-2 b strain C57BL/10 to produce the H-2Db-restricted, H-Y-specific cytotoxic cells (Simpson and Gordon 1977) for (a) above, and the H-2 b strain C57BL/6 to make the F1 hybrids whose PBL were tested in (a) above, and who were in turn tested as anti-H-Y responders in (b) above. The other parent of the F1 hybrids was an Sxr, XY male. Two sets of such males were used, one from the stock maintained at the Sloan Kettering Institute, New York, and the other from stock maintained at University College, London, by Dr. Anne McLaren.

Table 1 shows the results of two separate experiments in which Con A blasts were cultured from peripheral blood lymphocytes of the same eight individuals on two occasions 1 month apart and then used as target cells in an anti-H-Y T-cell cytotoxicity assay. The levels of cytotoxicity in the second experiment are approximately twice those in the first, but in each experiment the level of kill in the

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Table 1. Presence of H-Y on PBL of Sxr-bearing mice

E. Simpson et al.

PBL Con A blast target cells* from Cytotoxicity at A : T 4 : 1 *

Mouse no. Description Exp. 1 Exp. 2

72 1 21.5-+0.6 37.8-+0.9 73 / (B6/J x Sxr/+ Ta~)F 1 27.2-+ 3.4 38.1 _+ 2.3 74 XY~ d 17.7 _+ 5.0 33.9 _+ 2.2 75 23.4 _+ 3.5 41.0 +_ 5.8

76 ] 20.1_+3.3 50.0_+1.7 77 t (B6/J x Sxr/+ Ta/Y)F 1 25.5_+0.9 49.8_+0.5 78 Ta/X XXc~ d r 27.8 _+ 1.8 40.9 _+ 1.8 79 30.7 _+ 0.5 41.6 -+ 2.0

C57BL/10 normal male 19.0 + 3.4 37.2 + 1.7 C57BL/10 normal female - 2.2 _+ 1.6 1.9 _+ 0.6

* The Sxr-carrying mice in this experiment were bred and identified in New York and sent to London for testing. Blood samples were taken from the tail vein and PBL purified and cultured with Con A for 24 h before labelling with s 1Cr and used as targets according to Chandler et al. (1979). When these mice were eventually killed, their testes were weighed. Nos. 76-79 had testes weights ranging from 19-30 mgm and histologically they had no germ cells. Of the group 72-75, two weighed over 100 mgm and were taken to be normal, non-Sxr-carrying, whilst the other two weighed 75 and 80 mgm and were probably Sxr carriers. Anti-H-Y-specific, H-2b-restricted cytotoxic T cells were prepared from C57BL/10 female mice primed in vivo and boosted in vitro with syngeneic male spleen cells according to Simpson and. These cytotoxic cells were tested at four attacker to target (A:T) ratios, starting from 10:1, on the PBL targets. The 4 : 1 cytotoxicity figure given is taken from regression analysis of the four experimentally determined points. For each regression curve, the r 2 value was between 0.9 and 1.0.

Sxr, XX group (nos. 76-79) is no different from that in the Sxr, XY group (nos. 72-75). This result is consistent with the finding that individuals from both groups

express similar amoun t s of H-Y antigen. Table 2 represents the results from three experiments using Sxr-carrying mice as

ant i -H-Y responders. Phenotypic male and female F 1 progeny of the cross (B6 x Sxr) which had been pr imed in vivo with no rma l male B6 spleen cells, were boosted in vitro in mixed lymphocyte culture with B6 male cells then tested for the generat ion of H-Y-specific cytotoxic cells. Testis weights were taken as an indicat ion of genotype. None of the eight XX Sxr/+ mice in these experiments gave a response, a l though their female l i t ter-mate controls did so. Neither did the XY Sxr/+ males respond. This result suggests that the normal B6 H-Y ant igen is not different from the H-Y ant igen expressed by the Sxr, XX male. The very low weights of the Sxr/+ XX testes make them easily identifiable, bu t the intermediate weights of the Sxr/+ XY make them more difficult to assign. In a further, similar experiment four more Sxr/+ XX males failed to respond to H-Y on B6 male cells (data not shown).

Taken together these data provide no evidence either for a quant i ta t ive abnormal i ty in the a m o u n t of H-Y ant igen expressed by Sxr, XX or Sxr, XY males or for an incomplete t rans loca t ion of H-Y-coding mater ial from the Y chromosome.

H-Y Antigen in Sxr Mice

Table 2. Inability of Sxr/+ mice to respond to semisyngeneic + / + male cells

357

Pheno- typic

Exp. Responder sex Testis weight Probable R/L genotype

CTx at 4 : 1

B10c~ B109

1 ~ 24/22 XX Sxr/+ - 0.8 _+ 1.9 0.4 _+ 0.8 2 o ~ 22/28 XX Sxr/+ - 1.7_+1.4 - 2.1_+0.8 3 d r 26/25 XX Sxr/+ - 2.0+_0.3 1.0_+0.4 4 ~3 13/13 XX Sxr/+ 0.0 _+ 2.9 0.3 +_ 1.0 5 c~ 61/71 XY Sxr/+ - 1.4 _+ 1.5 2.0 _+ 1.9 6 ~ 80/78 XY Sxr/+ - 4.0_+ 1.3 0.2_+ 1.2 7 d r 100/104 XY + / + 1.7_+ 1.4 3.1 _+2.6 8 d r 94/86 XY + / + 1.3+_'2.0 - 3.2_+1.2 9 d 95/95 XY + / + - 2.1_+1.2 1.9_+1.8

10 d r 87/91 XY + / + - 1.1+_1.7 - 1.2_+0.1 11 ~ - XX + / + 14.2_+1.5 0.4_+1.5 12 ~ - XX + / + 12.1_+3.2 - 2.8_+3.3 13 9 - XX + / + 10.2+_1.3 0.6-+0.9

1 d 21/21 XXSxr/+ - 0.6+_1.6 - 4.7+_1.1 2 c~ 26/25 XXSxr/+ - 1.8_+0.4 - 3.2_+2.2 3 c? 78/75 XY Sxr/+ - 4.6_+0.6 - 6.4+_2.2 4 <¢ 85/82 XY Sxr/+ - 4.8_+0.4 - 7.0_+1.4 5 ~ 97/96 XYSxr/+ - 5.6_+1.4 - 5.6+_1.4 6 c~ 117/122 XY + / + - 6.0+__0.5 - 7.0_+1.7 7 ~ 111/111 XY + / + - 9.3-+2.8 -10.5-+0.2 8 9 - XX + / + 48.0_+1.9 4.2_+2.2 9 ~ - XX + / + 37.2_+1.1 6.0+_1.0

1 ~ 26/27 XXSxr/+ - 5.4_+1.1 - 5.8_+1.7 2 ~ 25/27 XXSxr/+ - 3.2_+1.0 - 3.5_+0.7 3 ~ 110/112 XY + / + - 5.2+_0.5 - 4.1+_1.1 4 c~ 110/111 XY + / + - 4.1_+0.5 - 3.8_+1.0 5 c~ 111/112 XY + / + - 1.5_+0.6 - 2.4_+1.1 6 ~ - XX + / + 42.4_+1.7 1.9_+1.3

The Sxr-carrying mice in these experiments were bred and tested in London. They were used as anti-H-Y responders (cf. Sxr-carrying mice in Table 1, whose cells were used as target cells in the anti-H-Y cytotoxicity assay). Mice in these experiments were primed in vivo with B6 male cells and 4-12 weeks later boosted in vitro in mixed lymphocyte culture (MLC) with irradiated B6 male cells. At the end of the 5-day culture period they were assayed against B6 male and female, 24 h con A blasts as targets (see also second footnote to Table 1).

Acknowledgements. P. E. and S. W. supported by grants NIH : CA 08748; HD 00171; and HD 10065.

References

Bennett, D., Mathieson, B. J., Scheid, M., Yanagisawa, K., Boyse, E. A., Wachtel, S., and Cattanach, B. M.: Serological evidence for H-Y antigen in Sxr, XX sex-reversed phenotypic males. Nature 265 : 255-257, 1977

Cattanach, B. M., Pollard, C. E., and Hawkes, S. G.: Sex reversed mice: XX and XO males. Cytogenetics I0: 318-337, 1971

358 E. Simpson et al.

Cattanach, B. M.: Sex reversal in the mouse and other mammals. In M. Balls and A. Wild (eds.): The Early Development of Mammals. pp. 305-319, Cambridge University Press, Cambridge, 1975

Chandler, P. R., Matsunaga, T., Benjamin, D. C., and Simpson, E.: Use and functional properties of peripheral blood lymphocytes in mice. J. Immunol. Methods 31 : 341-350, 1979

Simpson, E. and Gordon, R. D.: Responsiveness to H-Y antigen: Ir gene complementation and target cell specificity, lmmunol. Rev. 35: 59-75, 1977

Received January 29, 1981