217

Immunologic comments

Elizabeth Simpson

Clinical Research Centre, Transplantation Biology Section, Watford Road, Harrow, Middlesex HA13UJ, UK

The presence or absence of an intrinsic antigen can be deter- mined immunologically in basically two ways: first, the im- mune system of a test animal not expressing the antigen may be induced to respond to it by antibody and/or by a T cell re- sponse, providing the individual has the appropriate immune response genes. For relatively weak transplantation antigens, like H-Y, brisker and more reliable responses are obtained if the animal has been immunised in vivo before the test chal- lenge with antigen. Secondly, T-lymphocytes or antibodies from known responder animals immunised with the antigen can be used to type cells from the animal under test for the presence of the antigen. In recent years the development of monoclonal antibodies and T cell clones in vitro have pro- vided specific and powerful reagents for this second way.

Monoclonal antibodies to a large number of cell-surface antigens have now been made. Their reactions and specifici- ties have in general confirmed and extended information ini- tially obtained using polyclonal antisera, and the use of mono- clonal antibodies exchanged between labs has given reproduc- ible and reliable results. This has not unfortunately been true of monoclonal antibodies to male-specific antigens (H-Ys), and tests for the serologically detected moiety have relied on polyclonal reagents and have been characterised by difficulties in reproducibility between and within different laboratories. These problems have been exacerbated by a failure to read coded samples in all cases and a failure to quantitate (e.g., by quantitative absorption). The renaissance in serological typing for male-specific antigens referred to in the final paragraph on H-Y immunology is surely needed if one is to build useful hypotheses on the basis of H-Ys typing.

It is characteristic of minor H antigens other than H-Y that serological responses to them cannot be obtained: like H-Y, in vivo graft rejection responses and in vitro MHC-restricted cell responses are readily obtained, especially after immunisation. This may mean that the T and B cell repertoires are different since there are converse examples of cell surface alloantigens detected by B cells (antibodies) but not T cells (Simpson 1986a). It could well be, therefore, that any male-specific anti- gens detected by antibodies are not the H-Y antigen, which characteristically excites T cell responses.

T cell responses to H-Y can be measured in vivo by graft rejection (using animals or cells being tested for H-Y as re- sponder or target of the response - see para 1 above) or by T cells in vitro: these latter can either be class I MHC restricted (Tc) or class II MHC restricted (Th). However, graft rejection to H-Y is predominantly controlled by class II restricted T cells (Th or Tdth), and it is this fact which produces the phe- nomenon referred to in the final paragraph in the section "H-Y of gonadal tissue". It is not therefore mysterious that some mouse strains can mount graft rejection responses to H-Y but not a cytotoxic response, and vice versa. In the first case such strains possess responder alleles in their class II

MHC genes but not in their class I (like B10.A(5R): H-2KbAbD d, A b is a class II responder allele, neither K b nor D d are, see Simpson 1982). In the second case, such strains possess responder alleles in their class I MHC genes (like CBA: H-2K k, AkEkDk: A k and E k can be readily used as class II restriction elements for T help for the in vitro cytotoxic T cell response, but not for the graft rejection response, and K k and D k are both class I responder alleles for H-Y). The H-Y antigen detected by Tc and Th is according to all the evidence the same antigen, but it is recognised by Tc and Th in the con- text of different self MHC molecules. The same evidence which argues for the identity of H-Y seen by Tc and Th argues for the identity of H-Y detected by graft rejection in vivo, namely, that each of these T cell systems detects H-Y coordi- nately, on tissues of mice carrying an entire Y chromosome (male or female) or of mice carrying the Sxr fragment of the Y (male or female), but not male or female mice carrying the mutant Sxr' fragment. In this latter case, a single mutational event has altered the antigen recognised by Tc, Th, and graft rejection. This does not amount to proof of identity at the molecular level of H-Y detected by Tc, Th, and graft rejec- tion, but there is no more (or less) reason to question the iden- tity of the H-Y antigen detected by Tc and graft rejection, than that by Tc and Th.

Cytotoxic T cells (Tc) specific for H-Y recognised in the context of human class I MHC antigens (HLA-A and HLA-B) can be used to type ceils from various types of patients. The most informative from the point of view of separating TDF and the gene for H-Y in man have been XX males carrying pa- ternal Yp sequences, who are H-Y negative, and XY females who carry an incomplete paternal Y chromosome (lacking part of Yp) who are H-Y positive (Simpson et al. 1987). It is the existence of these H-Y positive females which renders un- likely the proposal put forward in the section on "H-Y of non- gonadal tisue" that XX males are negative for H-Y detected by Tc only because they fail to modify a glycoprotein detected by anti-H-Ys antibodies: the XY females are H-Y positive and lack Yp-encoding TDF and the putative H-Ys controlling gene.

It is clear that in mouse as well as the wood lemming (point raised in discussion of hypothesis II) that the possession of Tdy and H-Yt is insufficient for testis determination. Female XY mice carrying the Y chromosome from wild mouse strains, M.poschiavinus, M. orbis, and others, as well as XY females carrying the chromosome 17 deletion Thp are H-Yt positive (Simpson et al. 1983). These findings have been argued per- suasively by Eicher as evidence for the presence of autosomal testis-determining genes in addition to and interacting with the Y-linked Tdy gene (Eicher et al. 1982; for review see Simpson 1986b).

Finally, the question of the H-Y status of testis tissue (penultimate paragraph of section "H-Y of gonadal tissue"), although not yet adequately tested, is readily amenable to in- vestigation. From the results of the Wiberg and Lattermann (1987) paper quoted it appears possible that the failure to im- munise for secondary H-Y responses normal C57BL/6 females with testis tissue from XY or XXSxr mice is not due to toler- ance induction, since the females reject male grafts in a first- set fashion after such procedures. From these data, there is also no evidence of immunosuppression. Tests for specificity, with positive controls, are needed before these findings can be interpreted, and these could be done using minor and/or major H antigens in a similar experimental system.

218

References

Eicher E, Washburn LL, Whitney JB III, Morrow KE (1982) Mus poschiavinus Y chromosome in the C57BL/6J murine genome causes sex reversal. Science 217: 535-537

Simpson E (1982) The role of H-Y as a minor transplantation antigen. Immunol Today 3 : 97-106

Simpson E (1986a) T and B lymphocytes: two repertoires or one? Immunol Lett 12: 185

Simpson E (1986b) The H-Y antigen and sex reversal. Cell 44:813 Simpson E, Chandler P, Washburn LL, Bunker HP, Eicher EM

(1983) H-Y typing of karyotypically abnormal mice. Differentia- tion 23 : Sl16

Simpson E, Chandler P, Goulmy E, Page DC, Disteche C, Ferguson- Smith MA (1987) Separation of the genetic loci for the H-Y anti- gen and testis determination on human Y chromosome. Nature 326 : 876-878

Wiberg UH, Lattermann U (1987) Syngeneic male graft rejection by B6 female mice primed with spleen and testes of Sxr and Sxr' mice. Exp Clin Immunogenet (in press)

Received March 16, 1987

Sxs antigen and the heterogametic gonad

Ulrich Wolf

Institut ftir Humangenetik und Anthropologie der Universit~t, Albertstrasse 11, D -7800 Freiburg i. Br., Federal Republic of Germany

I take the opportunity here to comment on the article by Wiberg considering sex-specific antigens, first of all because f welcome the introduction of the term "Sxs" antigen for what has been called H-Y antigen as detected by serological tech- niques. Furthermore, I should like to comment briefly on some genetic and functional aspects of Sxs antigen.

After, in certain cases mentioned in Wiberg's article, typ- ing for H-Y antigen gave discordant results depending on the method used for its detection, different terms referring to the respective methods were introduced. In contrast to former proposals, Wiberg's term Sxs also takes into consideration that an antigen cross-reacting with mammalian-derived anti- Sxs antiserum is commonly found in the heterogametic sex of species studied so far throughout the vertebrate subphylum. I will refer here only to the Sxs antigen as defined by Wiberg.

In nonmammalian vertebrates, the mechanism of sex de- termination can be either XX/XY or ZW/ZZ. In the ZW/ZZ system, Sxs antigen has been found to be present normally in the female, but (per definitionem) absent in the male. Exam- ples studied are some species of birds, reptiles, amphibians, and fish (for review see Wachtel 1983). In contrast, Sxs anti- gen is detected in the male if this is the heterogametic sex (XY), but not in the homogametic female, as shown in, e.g., some amphibian and fish species (for review see Wachtel 1983).

Interestingly, in nonmammalian vertebrates, Sxs antigen can be induced in the homogametic sex by the sexual steroid hormone of the opposite sex. Thus, Z Z males treated with es- tradiol during early ontogenetic development become positive

for Sxs antigen. This has been shown in the chicken (Mtiller et al. 1979), quail (Miiller et al. 1980), and Xenopus (Wachtel et al. 1980). In the protogynous hermaphroditic fish Coris julis, spontaneous sex change from original females (Sxs negative) to secondary males (Sxs positive) occurs. Administration of testosterone to the female also results in masculinization, and the treated animals again become Sxs positive (Reinboth et al. 1987).

From these experiments, it can be concluded that the gene(s) for Sxs antigen is shared by both sexes, at least in the species examined, and it is plausible to assume that this is true for nonmammalian species in general. Due to the cross-reac- tivity of mammalian-derived Sxs antisera with the hetero- gametic sex of nonmammalian species, it is to be assumed that Sxs has been conserved phylogenetically (Wachtel et al. 1975). Thus, the genes for Sxs antigen in mammals and non- mammalian vertebrates most probably share homology phylo- genetically. Both arguments, the homology of the gene(s) throughout vertebrates and the phylogenetic presence of it in both sexes of nonmammalian species, taken together favor the view that in mammals the Sxs gene is present in both sexes as well. I consider this as supporting evidence for the point made in Wiberg's article, that the Sxs gene is not Y-linked in mam- mals and is probably autosomal.

Considering the possible function of Sxs antigen, apart from the numerous in vitro experiments pointing to a role of Sxs antigen in morphogenesis of the heterogametic gonad (e.g., Miiller and Urban 1981; Zenzes et al. 1978, 1980), as pointed out in Wiberg's article, our recent studies on Sxs in chicken gonadal development are to be mentioned. We have shown that the chicken embryo at the indifferent stage of the gonadal anlage is negative for Sxs antigen not only in the male, but also in the heterogametic female. Between days 6 and 7, when gonad differentiation starts, the female becomes Sxs positive. This is true for the gonads as well as for non- gonadal tissues (Ebensperger et al., in preparation). This find- ing can be considered as circumstantial evidence for a func- tional role of Sxs antigen at this critical developmental stage.

Under the prospect of Sxs homology, the assumption of a corresponding role in mammals, i.e., in the organization of the testis from the indifferent gonadal anlage, is at hand.

References

Miiller U, Urban E (1981) Reaggregation of rat gonadal cells in vitro: experiments on the function of H-Y antigen. Cytogenet Cell Genet 31 : 104-107

Miiller U, Zenzes MT, Wolf U, Engel W, Weniger J-P (1979) Ap- pearance of H-W (H-Y) antigen in the gonads of oestradiol sex-re- versed male chicken embryos. Nature 280:142-144

Mfiller U, Guichard A, Reyss-Brion M, Scheib D (1980) Induction of H-Y antigen in the gonads of male quail embryos by diethylstil- bestrol. Differentiation 16: 129-133

Reinboth R, Mayerovfi A, Ebensperger C, Wolf U (1987) The occur- rence of serological H-Y antigen (Sxs antigen) in the diandric pro- togynous wrasse Coris julis (L.) (Labridae, Teleostei). Differenti- ation 34:13-17

Wachtel SS (1983) H-Y antigen and the biology of sex determination. Grune & Stratton, New York

Wachtel SS, Koo GC, Boyse EA (1975) Evolutionary conservation of H-Y ("male") antigen. Nature 254 : 270-272

Wachtel SS, Bresler PA, Koide SS (1980) Does H-Y antigen induce the heterogametic ovary? Cell 20 : 859-864

Zenzes MT, Wolf U, Engel W (1978) Organization in vitro of ovarian cells into testicular structures. Hum Genet 44 : 333-338