Male Lymphoid Cells Research Articles

Purified donor T cells alone activate transplantation immunity to the male antigen but induce tolerance in combination with Mac-1+ donor cells.

In most experimental systems examined, "professional" antigen-presenting cells (APCs), such as dendritic cells, have been found to activate T cells, whereas "nonprofessional" antigen-bearing cells (nonAPC) may induce tolerance. Some recent studies have suggested that nonAPCs may under certain conditions prime a T-cell immune response. We have attempted to separate the roles of transplanted T cells and monocytic/dendritic cells in activating or tolerizing antigen-specific T cells in vivo, by examining the consequences of parenteral exposure to male antigen in anti-male TCR transgenic female mice. Qualitative and quantitative changes in the large population of male-reactive transgenic T cells to various male donor cell populations in transgenic female mice were followed after injections of highly purified male lymphoid cells. Changes in male-reactive T cells with time and the long-term outcome of male skin grafts were measured. When a nonAPC population consisting of highly purified male T cells alone was injected intravenously into H-Y antigen-specific TCR transgenic female mice, the number of host transgenic T cells was sustainably increased, and male graft rejection was accelerated. Injection of a combination of purified T cells and purified Mac-l+ cells induced massive and permanent deletion of the host male-reactive T-cell population and permanent graft tolerance. Mac-l+ cells alone gave no appreciable change in responsive T cells or graft rejection times. The data indicate that highly purified T cells engrafted alone induce rapid sensitization toward the male antigen. They also show that both male donor T cells and a population of male monocytic/ dendritic cells are required to induce peripheral tolerance toward this antigen and that this tolerance is related to permanent peripheral deletion of male-reactive T cells.

Role of infused CD8+ cells in the induction of peripheral tolerance.

The CD8 molecule when present on a cell recognized by a mature T cell has been shown to have an inhibitory effect on the development of an in vitro immune response by that T cell. To test whether CD8 has a role in the induction of peripheral tolerance in vivo, we have studied tolerance induction in B6 female mice carrying a transgenic TCR specific to male H-Y Ag. Previous studies have shown that male Ag reactive cells are inactivated in these mice after the i.v. injection of viable male lymphoid cells. Here, B6 female transgenic mice were injected with B6 male lymph node cells from CD4 or CD8 gene knockout mice, and the fate of male Ag-specific T cells was followed in vivo. Our results indicate that injection of male CD4 knockout (express CD8) lymphoid cells but not CD8 knockout (do not express CD8) lymphoid cells induced a significant reduction of male H-Y Ag reactive cells in the periphery, suggesting that CD8 plays an important role in the induction of peripheral tolerance of class I-restricted T cells. These results imply that peripheral tolerance can be induced not just by absence of signals required for activation but by the presence of additional signals.

Induction of long-term H-Y-specific tolerance in female mice given male lymphoid cells while transiently depleted of CD4+ or CD8+ T cells.

Rejection of H-Y-bearing primary skin grafts and generation of H-Y-specific cytolytic T cells by female mice requires the participation of both CD4+ and CD8+ T lymphocytes. Studies were conducted to investigate long-term tolerance of H-Y antigen induced in female mice by transiently depleting them of CD4+ and/or CD8+ T cells and, at the same time, giving them an injection of male lymphoid cells. We confirmed that after recovery of CD4+ to normal levels, female mice that had been transiently depleted of CD4+ cells and concurrently given an injection of male spleen cells were unable to generate H-Y-specific cytolytic T cells. Tolerance was also manifest by greatly extended survival (probably permanent in most cases) of male skin grafts. Further investigations revealed that female mice transiently depleted of CD8+ cells, and concurrently given an injection of male spleen cells, were similarly tolerant of H-Y antigen later when numbers of CD8+ T cells returned to normal. Moreover, small numbers of male cells were detectable in spleen and lymph nodes of tolerant females long after they had been given an injection of male cells and depleted of either CD4+ or CD8+ T cells, whereas no male cells were detected in (nontolerant) females given male cells and control antibodies. These findings show that tolerance of the relatively weak transplantation antigen, H-Y, can be achieved simply by giving male antigen-bearing spleen cells to the host while it is transiently depleted of a type of cell it needs in order to reject those cells, thus allowing the male cells to persist in the host. Furthermore, depletion of helper cells is not obligatory to achieve tolerance. It has been hypothesized that tolerance of H-Y antigen in females given male lymphoid cells while temporarily depleted of CD4+ lymphocytes results from unresponsiveness (anergy) induced in H-Y-specific CD8+ cells that are exposed to H-Y antigen in the absence of help from CD4+ cells. Interpretations of our findings are discussed in relation to this hypothesis.

Peripheral tolerance through clonal deletion of mature CD4-CD8+ T cells.

Transgenic mice bearing the alpha beta transgenes encoding a defined T cell receptor specific for the male (H-Y) antigen presented by the H-2Db class I MHC molecule were used to study mechanisms of peripheral tolerance. Female transgenic mice produce large numbers of functionally homogeneous CD8+ male antigen-reactive T cells in the thymus that subsequently accumulate in the peripheral lymphoid organs. We have used three experimental approaches to show that male reactive CD8+ T cells can be eliminated from peripheral lymphoid organs after exposure to male antigen. (i) In female transgenic mice that were neonatally tolerized with male spleen cells, male reactive CD8+ T cells continued to be produced in large numbers in the thymus but were virtually absent in the lymph nodes. (ii) Injection of thymocytes from female transgenic mice into female mice neonatally tolerized with the male antigen, or into normal male mice, led to the specific elimination of male-reactive CD8+ T cells in the lymph nodes. (iii) Four days after male lymphoid cells were injected intravenously into female transgenic mice, male antigen-reactive CD8+ T cells recovered from the lymph nodes of recipient mice were highly apoptotic when compared to CD4+ (non-male reactive) T cells. These data indicate that tolerance to extrathymic antigen can be achieved through elimination of mature T cells in the peripheral lymphoid organs.

MAJOR HISTOCOMPATIBILITY COMPLEX RESTRICTION AND CROSS-PRIMING OF H-Y ANTIGEN IN RATS

Although female rats can be sensitized to H-Y-incompatible male skin isografts following exposure to MHC-incompatible male lymphoid cells, these cells are not as effective as MHC-compatible male cells. Evidence is presented that the effectiveness of the MHC-incompatible cells is a consequence of crosspriming and that such crosspriming only occurs if these cells are rejected.

Expression of cell-defined H-Y antigen on mouse epidermal cells.

Cytotoxic T lymphocytes (CTL)5 of female origin that readily lysed syngeneic male lymphoid cells in specific, dose-dependent, H-2 restricted fashion had little or no activity against syngeneic male epidermal cells (EC) in short-term or long-term chromium-release assays. Moreover, although male EC were quite capable of priming syngeneic female lymphocytes in vivo for the accelerated rejection of male-specific skin grafts and for the subsequent generation of H-Y-specific CTL by exposure of primed female spleen cells (SC) to irradiated, syngeneic male SC in vitro, male EC themselves were incapable of stimulating the development of H-Y CTL when cocultured with primed female SC. Tests of EC from reciprocal male-female radiation chimeras revealed that keratinocytes, not marrow-derived EC (Langerhans cells), were responsible for the priming ability of EC in vivo. Moreover, H-Y antigen was serologically defined on EC that failed to express H-Y-specific CTL target-cell determinants. Alternative explanations of these findings are discussed, including the possibility that the inability of H-2-restricted T cells to lyse male EC results from the lack of association of H-Y antigen and H-2 restricting elements on the EC membrane.

Somatic cell hybrids of the Djungarian hamster: malignancy and evolution of the karyotype.

Djungarian hamster somatic cell hybrids were obtained by fusing malignant SV40-transformed fibroblasts (line DM15, HGPRT-), and normal male lymphoid cells. Tumorigenicity, growth in soft agar and karyotype changes of 10 independent hybrid clones were studied. All hybrids grew as tumors after injection of new-born hamsters with 1 x 10(6) cells. A total of 313 tumors occurred in 523 hamsters. The hybrids proliferated in soft agar as well. No correlation was noted between the ability of hybrids to grow in vivo and to form colonies in soft agar. The total chromosome number in hybrid cells was usually less than the expected sum of the parental chromosome sets. G-banding analysis showed that, in vitro, hybrids lost chromosomes of the normal parent, whereas marker chromosomes of the malignant parent were retained. In the majority of hybrid tumors the chromosome set was reduced to the diploid range. In tumors with a slightly reduced karyotype one or two homologues of chromosomes #4 and #8 were, as a rule, eliminated.

TOLERANCE TO NON-H-2 HISTOCOMPATIBILITY ANTIGENS

There have been several reports of observations which suggest that transplantation tolerance may be a result of positive immunoregulation rather than simply unresponsiveness attributable to a lack of competent effector cells. In particular, several investigators have reported that tolerance of the H-Y and H-1 histocompatibility antigens is mediated by a population of thymus-derived lymphocytes. In a companion report, we have presented evidence that supports the existence of a suppressor cell to the H-Y antigen. Furthermore, we have observed that female mice rendered tolerant of the H-Y antigens by neonatal exposure to male lymphoid cells or by multiparity accept male skin grafts indefinitely, but inactivate male peritoneal exudate cells (PEC) in a second-set fashion. This observation has led us to investigate whether tolerance of other non-H-2 antigens is controlled by a similar mechanism. Using mice congenic with C57BL/10 at the H-4 and H-7 loci, we have shown that mice rendered tolerant of the H-7a and H-4b antigens by neonatal exposure to histoincompatibe lymphoid cells are incapable of rejecting either skin or peritoneal cell allografts, suggesting that identical histocompatibility antigens are present on skin and peritoneal cells. Tolerance induced in neonatal mice to the H-4b and H-7a antigens could not be adoptively transferred to syngeneic recipients. These results suggest that tolerance involving the H-4 and H-7 antigens is most likely because of a clonal inactivation of alloantigen-reactive cells as a consequence of neonatal exposure to antigen.

AN INVESTIGATION OF ALLOGRAFT TOLERANCE

The extremely sensitive peritoneal exudate cell (PEC) transfer technique has been applied to an investigation of the immune response of female mice which are "tolerant" of the male antigen. Females rendered tolerant of male skin grafts by multiparity, neonatal inoculation of male spleen cells, or multiple inoculations of adult females with male lymphoid cells displayed second-set reactivity to male PEC while continuing to tolerate H-Y-incompatible skin grafts. Furthermore, this discordant response to male PEC and skin was adoptively transferrable to normal females by spleen cells from multiparous donors. Females rendered tolerant by irradiation and reconstitution with male cells were unresponsive to both H-Y-incompatible skin and PEC grafts. A model is proposed using two male-specific antigens, the response to which is controlled by independent genes.

Studies on the H-Y antigen in rats

The H-Y antigen has been studied under a variety of experimental conditions in BN and Lewis rats. The results indicate that 1. graft size is crucially important in determining the fate of male skin isografts on females; 2. H-Y incompatible ear skin grafts survive significantly better than those of trunk origin; 3. prior exposure of females to male lymphoid cells greatly increases their capacity to reject male skin isografts; 4. neonatal castration has no influence on the expression of H-Y; 5. multiparity can induce unresponsiveness to H-Y; and 6. although BN females respond better than do Lewis females to H-Y, the antigen is stronger in Lewis males. These findings are compared with the results of similar experiments conducted with mice.

Male specific antigen: homology in mice and rats.

Male lymphoid cells from some strains of rats can sensitize C57BL/6 female mice against C57BL/6 male skin grafts; this indicates that the male specific antigen of these two species is homologous.

Continued Expression of H-Y Antigen on Male Lymphoid Cells resident in Female (Chimaeric) Mice

REJECTION of male skin grafts by female mice of the same inbred strain is due to male-specific (H-Y) antigen1,2 (review3). As the only functions so far definitely traced to the Y chromosome are concerned with sex determination, a question arises as to whether the expression of H-Y antigen on male cells is secondary to development of the male phenotype, or whether a gene (or genes) on the Y chromosome specifies H-Y antigen (either directly or under regulation of an autosomal locus) independently of other sex-related characteristics. The fact that H-Y antigen is found on all male tissues that have been tested, rather than being restricted to sex-related tissues3,4, is perhaps an indication that it is not directly concerned with the attributes of male sexuality. Its appearance on male but not on female cells might instead be a fortuitous consequence of the lack of crossing over between the X and Y chromosomes; in this case, any change in cell surface antigenicity brought about by mutation of genes on the Y chromosome would automatically be transmitted only to males.

Influence of gonadal factors on skin test reactivity of CFW mice to Candida albicans.

Sex differences in the delayed skin test response to Candida albicans were studied in gonadectomized and control CFW mice sensitized either actively with sonically treated antigen or passively with lymphoid cells. In mice actively sensitized with antigen, the responses to candida skin testing were larger in the female. In both sexes, the skin test responses were larger in mice gonadectomized prior to sensitization than in controls of the same sex. In mice passively sensitized with male lymphoid cells, the responses to candida skin testing were essentially equal in the sexes. In both sexes, gonadectomy prior to passive sensitization with the male cells resulted in greater skin test reactivity. In mice passively sensitized with female lymphoid cells, the responses were larger in the males. In males, pre-sensitization gonadectomy resulted in smaller skin test responses, but in the females gonadectomy did not influence the responses. It is suggested that the greater skin test reactivity of the female to candida is due to a sex difference in the response of lymphoid cells to gonadal hormones. This difference may be important in the greater immune reactivity and resistance to infection which occur in the female.

A Serological Approach to the Study of the Male Isoantigen in Mice

Earlier tests for the H-Y (male) antigen in mice were limited to male graft rejection by syngeneic females. The experiments described in this report attest to the specificity of a new serological test for this antigen. Anti-H-Y sera and immune ascites fluid with statistically significant cytotoxic indices were produced in C57BL/6Ha females; a cytotoxicity test of sufficient sensitivity to detect and titrate cytotoxic activity, or, conversely, to detect the presence of the male antigen on C57BL/6Ha male lymphoid cells in vitro, was devised.

Studies on Tolerance of the Y Chromosome Antigen in Mice

Summary The principle of immunologic tolerance has been employed to study the isoantigen—usually referred to as the Y-factor—that is responsible for the rejection of male skin isografts by female mice of many different inbred strains. Various lines of evidence are presented that lend strong support to the view that this antigen is determined by a locus on the Y chromosome and that it behaves in all respects like the tissue transplantation antigens responsible for the destruction of tissue homografts. Tolerance of male skin isografts, like tolerance of skin homografts, can be abolished by transfer of normal or sensitized lymphoid cells from a genetically appropriate donor. Exactly the same Y antigen appears to be present in all male mice, irrespective of their strain of origin, as evidenced by the fact that C57 female mice could be made tolerant of subsequent male skin isografts by inoculation at birth with male cells derived from any one of four different and unrelated donor strains. The tolerance-responsive period of C57 female mice to this fairly weak antigen was found to be of the order of 17 days. Because of the very high degree of toleranceresponsiveness of C57 females to the Y antigen, this simple tolerance system has been employed to investigate several fundamental aspects of transplantation immunology. For example, it has been found possible to render female mice tolerant of male skin isografts by repeated inoculations with high dosages of a cell-free extract of male lymphoid cells. Highly suggestive evidence has also been obtained that persistence of tolerance of the Y antigen does not require persistence of the antigen.