Concanavalin A-induced splenocyte supernatant causes a preferential increase in H-2K over H-2D and vice versa depending on culture conditions

Mouse embryo fibroblasts (MEF) from BALB/c mice were exposed to the culture supernatant (CS) from concanavalin A-stimulated splenocytes for 16 h. This caused an increase in the amount of Kd and Dd antigens expressed on the cell surface of the MEF. However, on MEF cultures at 37 degrees C and subcultured daily for the first 5 days after embryonic trypsinization, CS caused the K antigens to increase significantly more than the D antigens, while on MEF cultured for 24 days with a combination of 37 degrees C and room temperature, CS invariably caused the D antigens to increase more than the K antigens. In another protocol involving MEF culture at 37 degrees C and subculture every 4 days, either response was possible when the MEF were exposed to CS. We propose that cells may differentially control K and D antigen increase and discuss how this may be useful in the eradication of viral infection in the animal.     

MLR blast cells generated in mutant-standard strain combinations bind H-2K and H-2D antigens.

Strains CBA (M523) (= M523) and CBA differ by a mutation which has been mapped genetically into the K region of the H-2 complex. Similarly, strains B 10.D2 (M504 (= M504) and b 10.D2 differ in a mutation which occurred in the D region. The data presented in this study show that mixed lymphocyte culture in M523 anti-CBA and M504 anti-B 10.D2 strain combinations leads to the release of membrane fragments from the stimulating cells and binding of these fragments by blast cells. The fragments always carry H-2K (in the M523 anti-CBA combination) or H-2D (in the M504 anti-B 10.D2 combination) antigens present in the stimulating and absent in the responding cells (antigens H-2.60 and H-2.40, respectively). Although Ia antigens may occasionally be present on the CBA membrane fragments, these antigens do not participate in the M523 anti-CBA MLR stimulation. The data thus demonstrate that serologically detectable H-2K and H-2D antigens can induce a MLR and that a mutation can change properties of H-2K or H-2D molecules so that the alteration is detectable by both serological means and lymphocyte activation assays.     

Specific immune lysis of paramyxovirus-infected cells by H-2-compatible thymus-derived lymphocytes.

Mice exposed to paramyxovirus (Sendai) generate specifically sensitized thymus-derived lymphocytes (T cells) which, in an in vitro 51Cr release assay, interact only with virus-infected target cells sharing strong transplantation antigens. Reciprocal exclusion of cytotoxic T-cell activity is found for Sendai virus, lymphocytic choriomeningitis virus and ectromelia virus. Immune T cells are detected as early as 3 days after intraperitoneal inoculation with a large dose of Sendai virus, and cytotoxicity is generally maximal on days 5-7. Lysis is restricted to interactions where sensitized lymphocytes and virus-infected target cells (fibroblasts, tumour cells or macrophages) are compatible at the K or the D locus of one H-2 haplotype. Identity of immune response (Ir) genes is neither sufficient nor necessary. Levels of T-cell responsiveness show some variation with H-2 type. Cytotoxic T-cell activity associated with H-2b is less than that recognized for H-2k or H-2d. These differences are, however, not obviously related to Ir gene control.     

Age decreases the capacity of mouse embryo fibroblasts to increase surface H-2 when stimulated with concanavalin A-induced splenocyte culture supernatant

With increasing time in culture and increasing mitotic number there is a decreased sensitivity of BALB/c mouse embryo fibroblasts (MEF) to the effects of the culture supernatant from concanavalin A stimulated splenocytes. When exposed to culture supernatant in vitro for 16 h 'old' MEF (12 days in culture at 37 degrees C with three subcultures) showed less increase in class I major histocompatibility complex antigens (H-2Kd and H-2Dd) than 'young' MEF (up to 8 days in culture at 37 degrees C with two subcultures). The subculture number alone was not the cause of decreased sensitivity to culture supernatant, since MEF subcultured five times over 5 days in vitro did not exhibit it. We propose that the insensitivity to the major histocompatibility complex increasing effects of culture supernatant precedes cell senescence and discuss how it could contribute to depression of cell-mediated immunity in aged animals.     

Cytotoxic T cell lysis of H-2-negative murine sarcoma cells

The DOH cell line was established from C3H.OH (H-2Kd,Dk) embryonic fibroblasts transformed with Rous sarcoma virus (RSV) in vitro. When injected into syngeneic mice, DOH cells were very weakly tumorigenic and induced a cytotoxic immune response. Cytotoxic T lymphocytes (CTL) specifically lysed DOH cells but not other RSV-induced sarcoma cells, which shared the H-2Kd or H-2Dk antigen, respectively, with DOH. Serological and immunochemical analysis of H-2 antigens subsequently showed that DOH sarcoma cells did not express syngeneic H-2K and H-2D antigens. Surprisingly, a H-2Kk-specific monoclonal antibody (100-5) bound to DOH cells and was inhibitory for syngeneic CTL specific for DOH. In addition, DOH cells were lysed by alloreactive H-2Kk-specific CTL. The demonstration of immunogenic H-2-negative sarcoma cells suggests that either the H-2K antigens have been extensively altered or that hitherto unidentified major histocompatibility complex class I molecules are expressed on DOH sarcoma cells surfaces, acting as target antigens for tumor-specific CTL.     

The role of H-2 and H-2-like determinants in syngeneic and allogeneic cytostasis.

Specific sensitization against H-2 determinants was affected by immunizing allogeneic mice with spleen and lymph node cells in H-2 congenic combinations. Lymph nodes from the sensitized and non-sensitized mice were respectively cultured together with H-2 syngeneic tumour cell lines. The growth and viability of the tumour cells was subsequently measured by the amount of radiothymidine incorporation. If the tumour cells incorporated less isotope when cultured with the immune cells than with the normal cells this was termed 'cytostasis'. To identify the H-2 genes controlling the sensitization phase in the cytostasis assay, we studied the effect on different transplantable tumour target cells of lymphoid cells from mice sensitized against different congenic spleen cells. The results suggest that the cytostasis assay can can measure an in vitro specific response to H-2-incompatible sensitizing antigens, and that I--B incompatibility, together with K and/or D, is essential to produce effectors. Furthermore, H-2 allogeneic sensitization could induce cytostasis even against tumour cells syngeneic for the H-2 halotype of the responder strain. The implications of these findings are discussed.     

Differential effect of adenovirus 2 E3/19K glycoprotein on the expression of H-2Kb and H-2Db class I antigens and H-2Kb- and H-2Db-restricted SV40-specific CTL-mediated lysis

The E3/19-kDa glycoprotein (E3/19K) coded by adenovirus type 2 (Ad2) is known to inhibit the cell-surface expression of major histocompatibility complex (MHC) class I antigens by binding to the MHC antigens intracellularly, and thus reduces recognition of antigens by MHC-restricted cytotoxic T lymphocytes (CTL). We have studied the effect of the E3/19K expression in SV40-infected monkey cells, TC-7/H-2Kb and TC-7/H-2Db expressing transfected H-2Kb and H-2Db antigens, respectively, on the cell-surface H-2 class I antigens and on lysis of the cells by SV40 large tumor (T)-antigen-specific H-2Kb- and H-2Db-restricted CTL clones. H-2Db antigen expression on TC-7/H-2Db cells was drastically reduced by infection with Ad2 but not with an E3/19K-negative SV40-Ad2 hybrid virus, Ad2+ND1, as early as 12 hr postinfection. However, H-2Kb antigen expression on Ad2-infected TC7/H-2Kb cells remained unaltered, even at 24 hr postinfection. Specific lysis of SV40-infected TC-7/H-2Db cells by H-2Db-restricted SV40 T-antigen-specific CTL clones, Y-1 and Y-3, was strongly reduced by coinfection of the target cells with Ad2 but not with Ad2+ND1. Lysis of SV40-infected TC-7/H-2Kb cells by a H-2Kb-restricted SV40 T-antigen-specific CTL clone Y-4 was also reduced significantly by Ad2 infection, but not Ad2+ND1. These results indicate that the E3/19K protein affects cell-surface expression of H-2Db antigen but not H-2Kb antigen.     

Variation in H-2 antigen expression on lymphomyeloid cells in semi-allogeneic irradiation chimeras

Spleen cells from 30 individual murine irradiation chimeras of the type (P1 X P2)F1----P1 were compared in a rosetting assay for H-2K and H-2D cell surface antigen expression with normal (P1 X P2)F1 hybrid controls. Eleven out of the 30 chimeras were in the normal range, but the other 19 differed from F1 controls by 4- to 100-fold in endpoint titre for at least one H-2K or H-2D antigen. Every possible class of variation was found, i.e. up or down variation of H-2K or H-2D antigens of P1 or P2 type. This evidence, together with data from T6 chromosome marker experiments which also showed full reconstitution of lethally irradiated P1 recipients by (P1 X P2)F1 donor lymphomyeloid stem cells, suggested that incomplete reconstitution was not the cause of H-2 antigenic variation. Low expression of P2 H-2 antigens on spleen cells derived from (P1 X P2)F1----P1 chimeras was investigated further. Fifteen lethally irradiated (P1 X P2)F1 recipients of bone marrow cells from two such chimeras were all of normal F1 H-2 phenotype when tested 10-12 weeks after reconstitution, thus excluding stable, low P2 H-2-expressing variant F1 stem cells as a cause of the phenomenon. If P1 recipients were hyperimmunized against P2 cells before lethal irradiation and reconstitution with (P1 X P2)F1 stem cells, there were significantly fewer Till- McCulloch colonies in their spleens 10 days after reconstitution than in spleens of unimmunized controls. Also greater than 90% of immunized recipients died by 6 weeks after stem cell injection but two survivors both showed very low levels of P2 H-2K and H-2D antigens. These results together with previously published evidence of anti-P2 Tc cell activity and P2 skin graft rejection in (P1 X P2)F1----P1 chimeras suggested that residual anti-P2 immunological capability in lethally irradiated P1 recipients may be associated with low P2 H-2 expression on their F1-derived spleen cells, although the mechanism does not involve selection of stable, variant F1 stem cells. The mechanism(s) of other classes of variation in H-2 expression in (P1 X P2)F1----P1 chimeras were not investigated.     

THE REGULATORY ROLE OF I-J SUBREGION IN NEONATAL TOLERANCE INDUCTION TO H-2D ALLOANTIGENS

The mechanism of neonatally induced transplantation tolerance was studied in two mouse strain combinations involving differences at the D region of the H-2 complex only or at the same D region plus I-J subregion (including I-E, I-C, S and G regions). In the strain combination with the H-2D difference only, cells from tolerant mice proliferated markedly in the MLR assay when incubated with antigens tolerated in vivo, whereas the MLR reactions were negative in the combination with D plus I-J region disparities. In the latter combination cells from tolerant mice also did not respond to third-party antigens and their incubation with the tolerated antigens led to the suppression of cell proliferation. This non-specific suppression was absent in cells from tolerant mice in the strain combination, which differed in I-C, S, G and D alloantigens. Specific suppressor cells, which inhibited the development of cytotoxic cells, were demonstrated in tolerant mice of both strain combinations. The results show that, in addition to the specific suppressor cells induced by H-2K or H-2D alloantigens, non-specific suppressor cells induced by the I-J region disparity that may regulate the resultant activity against H-2D (and probably also H-2K) alloantigens are involved in transplantation tolerance.     

ABSORPTION ANALYSIS OF H-2D AND K ANTIGENS ON SPERMATOZOA

The presence of H-2D and K antigens on mouse spermatozoa has been investigated by absorption followed by testing on the proper target lymphocytes. It is concluded that, in addition to Ia antigens, H-2D and K antigens are indeed expressed on mouse sperm cells.     

Specificity of Cell-Mediated Lympholysis for Public and Private H-2 Determinants

The specificity of cell-mediated lympholysis in the mouse was investigated by studying the cross-reactivity of in vitro-generated effector cells against targets carrying H-2 haplotypes different from that of the stimulator. Congenic resistant partners of the C57BL/10 strain carrying ten different, independently derived haplotypes were tested in more than 600 combinations. Strong cytotoxicity was observed when stimulator and target cells shared a private determinant, but considerable cross-killing was also seen in combinations where stimulator and target cells shared only public determinants. Cross-killing is specific and can be blocked only by unlabeled cells of the stimulator or target strain. Although cross-killing does not correlate with the number of stimulating public determinants carried by the target individual determinants 5, 15. 25, and 27 + 28 + 29 are associated with increased cross-killing. Cross-killing can occur both at the K and the D end of H-2 and is not explained by antigens determined by the I region. We conclude that the serologically defined H-2K and H-2D molecules are the primary targets for cytotoxic T lymphocytes but that they may be perceived differently by T and B lymphocytes.     

Mechanisms of the H-2 Effect on Viral Leukemogenesis

Evidence has been gathered which supports the notion that two distinct but interacting mechanisms, controlled by loci mapping within the H-2 complex, influence Friend murine leukemia virus (FV) disease. One mechanism, controlled by a gene mapping in or close to H-2D, influences the capacity of the H-2D gene product to form molecular complexes with FV molecules in the plasma membrane of infected cells. Formation of a complex appears to provide a target antigen for syngeneic cytotoxic T-lymphocytes, to cause co-capping of FV and H-2D antigens, to permit the selective inclusion of H-2Db molecules into progeny Friend virions, to influence the long-term maintenance of virus production in vitro and, in conjunction with the second mechanism, to stimulate the generation of cytotoxic T-lymphocytes. This second mechanism is controlled by a gene in the H-2K or H-2I region, and, in the presence of an H-2/FV molecular complex immunogen, influences the generation of H-2 restricted cytotxic T-lymphocytes and the rate of rejection of syngeneic FV-induced tumor cells.     

Impaired H-2 expression in B16 melanoma variants

We studied the expression of H-2b alloantigens in three different B16 melanoma lines cultures in vitro. Cell lines were B16-F1 and two cell cultures (named B16-A and B16-B) newly derived from two different in vivo sublines of B16 melanoma. The assays used were in vivo tumour growth in allogeneic (BALB/c and B10.BR) as compared to syngeneic mice, in vitro cell-mediated cytotoxicity by anti-H-2b immune lymphocytes and absorption of anti-H-2b antisera activity. The B16-F1 line was able to efficiently kill allogeneic hosts, could not be lysed by anti-H-2b cytotoxic effectors and did not express any serologically detectable amount of H-2b alloantigens. The B16-A line was H-2 positive during the early in vitro passages, then, at the 8th-10th passages, it acquired the capacity to kill allogeneic hosts, lost the sensitivity to anti-H-2b cytotoxic effectors and the H-2Kb antigens became undetectable The expression of H-2Db was reduced, although at a lower degree. Similar data were obtained with B16-B cells, which after 10 in vitro passages grew and killed allogeneic hosts, showed a decreased sensitivity to cytotoxic anti-H-2b effectors and a very low expression of the K region antigens. The results indicate that H-2 expression is altered in B16 melanoma lines and this may influence the different metastatic capacity of such cells.     

Absorption analysis of H-2D and K antigens on spermatozoa.

The presence of H-2D and K antigens on mouse spermatozoa has been investigated by absorption followed by testing on the proper target lymphocytes. It is concluded that, in addition to Ia antigens, H-2D and K antigens are indeed expressed on mouse sperm cells.     

H-2 Antigen Frequencies Among Wild Mice From Chile

Thirty-five wild mice ( Mus musculus L. ) were captured at four different localities in Chile. The mice were typed for the presence of 15 K-, 11 D-, 14 A-, and two E-region H-2 antigens, using the complement-antibody microcytotoxicity assay. The mice from the sample representing the largest locality had a characteristic antigenic profile distinguishing them from other mouse populations thus far studied. With respect to class I H-2 antigens, the profile was characterized by the presence of antigens H-2K.16, 31, 103, 19, 108, 21, 33, 26, 115, and H-2D.4, 106, 30, 32, 111, 114, and 18 (in order of decreasing antigen frequency), and by the absence of antigens H-2K.15, 17, 20, 113, 116 and H-2D.2, 9, 110, and 112. The profile was, furthermore, characterized by the relatively high frequency of antigens H-2K.16, 31, and H-2D.4 and 106. The profile of class II antigens was also unique to the Chilean population but less conspicuously so than that of the class I antigens. Analysis of antigenic associations suggested that among the 34% blank H-2K alleles there were at least two coding for relatively frequent but as yet unidentified K antigens. Similarly, among the 57%blanks at the H-2D locus there were at least two frequent alleles encoding unidentified D antigens. Analysis of genetic distances suggested similarity between South American mice and mice from coastal regions of Europe.     

THE ROLE OF H-2 AND H-2-LIKE DETERMINANTS IN SYNGENEIC AND ALLOGENEIC CYTOSTASIS

Specific sensitization against H-2 determinants was effected by immunizing allogeneic mice with spleen and lymph node cells in H-2 congenic combinations. Lymph nodes from the sensitized and non-sensitized mice were respectively cultured together with H-2 syngeneic tumour cell lines. The growth and viability of the tumour cells was subsequently measured by the amount of radiothymidine incorporation. If the tumour cells incorporated less isotope when cultured with the immune cells than with the normal cells this was termed ‘cytostasis’. To identify the H-2 genes controlling the sensitization phase in the cytostasis assay, we studied the effect on different transplantable tumour target cells of lymphoid cells from mice sensitized against different congenic spleen cells. The results suggest that the cytostasis assay can measure an in vitro specific response to H-2-incompatible sensitizing antigens, and that I-B incompatibility, together with K and/or D, is essential to produce effectors. Furthermore, H-2 allogeneic sensitization could induce cytostasis even against tumour cells syngeneic for the H-2 halotype of the responder strain. The implications of these findings are discussed.     

H-2-RESTRICTED RECOGNITION OF MINOR HISTOCOMPATIBILITY ANTIGENS IN DELAYED TYPE HYPERSENSITIVITY

Subcutaneous (s.c.) immunization of mice with viable allogeneic H-2 compatible spleen cells can induce a persistent state of delayed type hypersensitivity (DTH) to minor histocompatibility (H) antigens which can be evaluated with a footpad swelling assay. The importance of H-2 compatibility of the injected spleen cells with the recipient for (1) the elicitation of the DTH-reaction, (2) the induction of DTH-related effector and memory T cells and (3) the activation of T memory cells was examined with congenic mouse strains. Spleen cells sharing either the K or D region of the H-2 complex with the recipient could elicit strong DTH-reactions to minor H antigens, though somewhat less than did fully H-2 compatible allogeneic spleen cells. H-2 incompatible cells or cells only sharing I-region coded antigens elicited relatively weak, though significant, DTH-reactivity to the minor H antigens. Similar H-2 requirements for recipient mice were demonstrated in the immune lymphocyte transfer assay. Optimal induction of primary DTH and DTH-related T memory cells for minor H antigens also required H-2 identity of the immunizing cells and the recipient. To some extent, H-2 incompatible cells were able to induce primary DTH-reactivity and memory for minor H-antigens. The secondary DTH-reactivity was not or slightly dependent on the H-2 haplotype of the cells used for booster injection. It is concluded that the DTH-related effector cells are restricted in their recongnition of minor H antigens by K- or D-region-coded antigens. Presumably, macrophage processing of the allogeneic spleen cells after primary and secondary immunization accounts for the capacity of the minor H antigens to activate unprimed T cells and memory T cells when these antigens are presented on H-2 incompatible cells.     

Lack of H-2Ld locus products on a BALB/c fibrosarcoma expressing H-2k-like alien antigens

The presence of H-2Ld antigens was evaluated in methylcholanthrene-induced BALB/c fibrosarcomas by a variety of approaches. Transplantation experiments showed that BALB/c-H-2dm2 mice, a mutant strain whose cells do not express H-2Ld antigens, after immunization with BALB/c normal tissues developed a resistance to the growth of two tumours (C-3 and GI-17), but not to a third neoplasm, C-1, which is known to have H-2d- as well as H-2k-like alien antigens. In vitro experiments with cytotoxic T lymphocytes generated against Ld antigens confirmed a loss of Ld antigens on C-1 but not on C-3 tumour cells. Serological experiments with an anti-Ld serum again revealed the presence of H-2Ld determinants on C-3 but not on C-1 cells. Biochemical analysis in SDS-PAGE of immunoprecipitates obtained by specific anti-H-2 sera with NP40 lysates of the tumours studied could detect H-2Kd, H-2Dd and H-2Ld antigens in C-3 fibrosarcoma cells whereas Kd and Dd were the only H-2d molecules found in C-1 lysate along with the H-2k-like specificities. The possible genetic mechanisms which may explain this apparent gain and loss modification of the H-2 profile of C-1 are discussed.     

Requirement of H-2-subregion differences for graft-versus-host autoimmunity in mice: superiority of the differences at class-II H-2 antigens (I-A/I-E)

The effect of various H-2-subregion differences on graft-versus-host (GVH) autoimmunity in mice was investigated by testing a variety of GVH combinations in which non-irradiated adult F1 hybrid mice were injected with parental strain lymphoid cells. As with previous results, the superiority of Class-II H-2 antigen (I-A/I-E) differences to other kinds of H-2 incompatibilities, such as Class-I H-2 antigen (H-2K/H-2D) differences, was largely confirmed. Anti-nuclear antibodies were produced significantly across Class-I as well as Class-II H-2 differences. However, the productions of anti-erythrocyte and anti-thymocyte autoantibodies were mainly confined to GVH reactions induced across Class-II H-2 antigens. Elevated proteinuria was elicited only in the GVH combinations that included the differences at Class-II H-2 antigens. GVH autoimmunity, however, did not always result in the significant occurrence of elevated proteinuria. The level of in-vitro IgG production by GVH spleen cells correlated closely with the degree of autoimmunity.