Fine specificity of alloimmune cytotoxic T lymphocytes directed against H-2K. A study with Kb mutants

The fine specificity of alloimmune cytotoxic T lymphocytes (CTL) was investigated in CTL responses across the smallest known H-2 differences, those based on mutation at a single H-2 locus. CTL were generated in all possible mixed lymphocyte culture (MLC) combinations among seven H-2Kb mutants and the mouse strain of origin, C57BL/6 (B6-H-2b). CTL were also generated in all F1 hybrid responder/homozygous stimulator-cell combinations among four Kb mutants and B6-H-2b. CTL activity was measured in cell-mediated lympholysis (CML) against target cells from all Kb mutants and B6-H-2b. Cross-reactivity against targets other than the MLC stimulator was extensive and led to the distinction of 64 CML target determinants. Two Kb mutants (B6-H-2bm6 and B6.C-H-2bm9) showed identical typing for all 64 CML determinants. CML reactions after MLC between these two haplotypes were mutually negative. The mutants B6-H-2bm3 and B6.C-H-2bm11 showed identical typing for 47 of the 64 determinants. Their close relationship is in agreement with the finding that H-2bm3 anti-H-2bm11 CTL were the only ones that exclusively lysed target cells of stimulator-cell genotype. On the basis of CML typing, the sequence of relatedness of the mutants with H-2b is as follows: bm6/bm9-bm10-bm3-bm1-bm11, bm6/bm9 being the closest to, and bm11 the most distant from H-2b. The extensive cross-reactivity of alloimmune CTL appears to reflect immunogenetic complexity rather than lack of specificity. Comparison with other reports supports the notion that the system of Kb CML determinants, recognized by alloimmune CTL, is at least partially overlapping with the H-2Kb specificity repertoire involved in the associative T cell recognition of virus-infected cells.     

Regulation of T-cell-mediated lympholysis by the murine major histocompatibility complex. I. Preferential in vitro responses to trinitrophenyl-modified self K- and D-coded gene products in parental and F1 hybrid mouse strains

Spleen cells from H-2b,k,d C57Bl/10 congenic mice were sensitized in vitro to trinitrobenzenesulfonate (TNBS)-modified autologous spleen cells. Cold target competition studies at the lytic phase demonstrated three distinct patterns of cytotoxic responsiveness: (a) H-2b spleen cells generated approximately equivalent CTL responses against Kb and Db modified self products, (b) H-2d spleen cells generated preferential responses against Dd modified self products, and (c) H-2k spleen cells generated cytotoxic responses which could only be detected against Kk self products in association with TNP. F1 spleen cells were sensitized against autologous TNBS-treated cells. The results showed that, although H-2b parental cells generated approximately equivalent Kb-TNP- and Db-TNP-specific CTL, the presence of the H-2b haplotype did not result in the generation of (a) Dk-TNP CTL response by (H-2b x H-2k) spleen cells, nor (b) a Db CTL response by (H-2b x H-2a) F1 spleen cells. Additionally, (H-2d x H-2k) F1 cells failed to generate detectable Dd-TNP-specific CTL, although H-2d parental cells generated D-regional-specific CTL. The findings demonstrated that these F1 response patterns paralleled those of the H-2k and H-2a parents, i.e. weak or no D-region TNP-specific CTL were induced. Because (H-2d x H-2a) F1 responders stimulated with H-2d TNBS-treated cells did generate good Dd TNP responses, the results illustrated that the presence of responder genes was not sufficient to result in a D-region TNP CML. It is suggested that the absence of Kk alleles on the stimulating population is necessary for the generation of D-region TNP CTL in these F1's. Mechanisms which could account for these response patterns in parental F1 mice are discussed including immunodominance, suppression, T-cell response , and Ir-gene defects.     

Ir-genes in H-2 regulate generation of anti-viral cytotoxic T cells. Mapping to K or D and dominance of unresponsiveness

H-2 dependent and virus-specific Ir genes regulate the generation of primary virus-specific K or D restricted cytotoxic T-cell responses in vivo. The following examples have been analyzed in some detail: first, Dk restricted responses to vaccinia in Sendai viruses are at least 30 times lower than the corresponding K-restricted responses irrespective of the H-2 haplotypes (k, b, d, dxs, dxq) of K and I regions; in contrast, LCMV infection generates high responses to Dk. These findings are consistent with but do not prove that this Ir gene maps to D. Second, Db restricted responses to vaccinia and Sendai viruses are high in strains possessing the Kq or KbIb, KbaIb haplotype, are very low in strains with Kk, and relatively low in mouse strains of the KdI-Ad haplotype; LCMV generates high Db restricted response in the presence of Kk. This Ir gene for the response to vaccinia and Sendai viruses maps to K since B10.BYR (KqIkdDb) is a responder and B10.A (2R) is a nonresponder (KkIkdDb). Third, virus and K or D allele specific nonresponsiveness is dominant with variable penetrance; in heterozygous mice the nonresponder Kk allele over-rides responsiveness normally found in KbDb or KqDb combinations. Fourth, when (responder X nonresponder)F1 lymphocytes are stimulated in an environment expressing vaccinia virus plus only a high responder Kb or Kq allelle and Db, response to vaccinia Db is high; in contrast when the same F1 cells are stimulated in an environment expressing the low responder allele Kk, response to vaccinia Db is low. Thus absence of Kk during immunization allows generation of high responsive Db restricted vaccinia specific cytotoxic T cells. The Dk dependent low response to vaccinia Dk can be explained by a preclusion rule or by failure of vaccinia to complex with Db; however the analysis of Kk dependent low response to vaccinia Db does not support these explanations or that self-tolerance is responsible for this Ir effect but is compatible with the interpretation that Kk vaccinia is immunodominant over Db vaccinia. These results are discussed with respect to (a) possible mechanisms of regulation by Ir genes and (b) H-2 polymorphism and HLA-disease association.     

Quantitative differences in the expression of parentally-derived H-2 antigens in F1 hybrid mice affect T-cell responses

Quantitative absorption with specific anti-H-2 sera has shown that the H-2Kb and H-2Dd antigens coded by the B10.A(5R) haplotype are expressed in about fourfold lower amount on the spleen cells of [B10.A(5R) X B10.A(2R)]F1 hybrids than on parental B10.A(5R) cells. In contrast, the H-2Kk and H-2Db antigens of B10.A(2R) are expressed equally on parental and F1 cells. These quantitative differences are reflected in cytotoxic T-cell (Tc-cell) function. Macrophage target cells from F1 mice are killed less efficiently than B10.A(5R) targets by alloreactive or H-2 restricted Tc cells specific for H-2Kb or H-2Dd, and spleen cells of F1 mice are less efficient stimulators of alloreactive Tc cells specific for B10.A(5R) H-2 antigens, whereas F1 and B10.A(2R) cells are equal as targets and stimulators for Tc cells recognizing B10.A(2R) H-2 antigens.     

Allospecific cytolytic T lymphocytes recognize conformational determinants on hybrid mouse transplantation antigens

Alloreactive cytolytic T cell (CTL) lines and clones have been used to identify the sites of polymorphism of antigens of the major histocompatibility complex (MHC). Specific CTL were generated against wild-type H-2b products by cells from H-2b mutant mice that had one or a few amino acid changes in either the alpha 1 or alpha 2 domains of the Kb or Db class I molecules. These CTL populations, which might be expected to react with determinants expressed on single MHC domains, were examined for lytic activity on L cells expressing newly constructed hybrid class I molecules. Transformed cell lines expressing native class I molecules or hybrid class I molecules in which the alpha 1 and alpha 2 domains of H-2Kb had been substituted by those domains of H-2Db were lysed by H-2Db-specific CTL. Similarly, all H-2Kb-specific CTL recognized hybrid molecules in which the alpha 1 and alpha 2 domains of H-2Kb were inserted into the H-2Db molecule. In contrast, exchange of the alpha 1 domains of H-2Kb and H-2Db resulted in a total loss of recognition by Kb and Db-specific CTL. These results suggest that the allodeterminants recognized by H-2 mutant CTL are influenced by interactions between the alpha 1 and alpha 2 domains, findings similar to those seen using conventional alloreactive T cells (11). These results were compared to the binding of alloreactive mAbs, including 5 new mAbs specific for the Kb molecules. Finally, it was shown that primary and secondary CTL responses could be generated by direct sensitization against hybrid class I molecules, demonstrating that these molecules express neoantigenic determinants recognized by alloreactive CTL.     

Neonatal tolerance of major histocompatibility complex antigens alters Ir gene control of the cytotoxic T cell response to vaccinia virus

The K region of H-2 controls the Tc cell response to vaccinia-Db. The Kb, Kd, and Kq alleles allow good Tc cell responses against vaccinia- Db. In contrast, the presence of Kk in H-2 recombinants 2R (Kk,Db) and 4R (Kk,Db) or in F1 hybrids greatly reduces the anti-vaccinia-Db response. The defect does not lie in antigen presentation, as infected 4R cells can stimulate anti-vaccinia-Db Tc cells in vitro. Furthermore, nonresponder animals possess Tc cell precursors for vaccinia-Db, as transfer of F1 nonresponder spleen cells into infected, lethally irradiated responder recipients allowed generation of anti-vaccinia-Db effector Tc cells. Secondary responses to vaccinia-Db can also be obtained in vitro from T cells of 4R animals. Feedback inhibition was excluded in experiments with mixed chimeras in which Kk and Db were expressed on separate cell populations. Neonatal tolerance of B10 animals to Kk suppressed the anti-vaccinia-Db response but did not affect anti-vaccinia-Kb, anti-lymphocytic choriomeningitis virus, or anti-H-2d responses. In cold target competition experiments, H-2k competitors inhibited vaccinia-Db-specific target cell lysis by Tc cells, which suggests that anti-vaccinia-Db and anti-H-2Kk Tc cells may cross-react. Therefore, we propose that the suppressive influence of Kk on anti-vaccinia-Db Tc cell responses is a consequence of self- tolerance and that suppression of anti-Kk Tc cells results in cross- reactive suppression of anti-vaccinia-Db Tc cells.     

Thymus dictates major histocompatibility complex (MHC) specificity and immune response gene phenotype of class II MHC-restricted T cells but not of class I MHC-restricted T cells

Athymic H-2b nude mice received grafts from C57BL/6 (Sendai virus and H-Y antigen cytotoxic T lymphocyte [CTL] responder type), bm1 (H-2Kb mutant, Sendai CTL nonresponder type), or bm12 (H-21-A mutant, H-Y CTL nonresponder type) neonates. In observations of the CTL response to H-Y, both recipients and thymus donors were female. All types of thymus engraftment resulted in mature H-2b splenic T lymphocyte surface phenotype in nude hosts. T cell immunocompetence (as measured by major histocompatibility complex [MHC] CTL responses to allogeneic cells) was restored, and induced nonresponsiveness to the MHC determinants of the engrafted thymus in the nude host. The CTL reaction to Sendai virus in both responder type C57BL/6 and nonresponder type bm1 neonatal thymuses allowed maturation of Sendai-specific, H-2Kb-restricted CTL. For the CTL reaction to H-Y, only responder type C57BL/6 thymuses restored the CTL response, whereas this was not achieved with thymuses from nonresponder type bm12 neonatal females. Results of double thymus (B6 and bm12) engraftment excluded the possibility that this latter effect was caused by suppression. In addition, athymic bm1 mice were engrafted with thymuses from either B6 (Sendai CTL responder type) or syngeneic bm1 neonates (Sendai CTL nonresponder type). Again, both types of neonate thymuses restored T cell competence as measured by MHC/CTL responses to allogeneic cells. However, neither responder B6 nor nonresponder bm1 neonate thymus grafts allowed maturation of Sendai-specific CTL. In conclusion, the thymus dictates MHC specificity and immune response gene phenotype of T cells restricted to class II MHC molecules but not of T cells restricted to class I MHC molecules.     

Haplotype-specific suppression of cytotoxic T cell induction by antigen inappropriately presented on T cells

To detect a strong cytotoxic T lymphocyte (CTL) response to minor histocompatibility (H) antigens in a 5-d mixed lymphocyte culture, it is necessary to use a responder that has been primed in vivo with antigen-bearing cells. It has previously been shown that minor-H- specific CTL can be primed in vivo both directly by foreign spleen cells and by presentation of foreign minor H antigens on host antigen- presenting cells. This latter route is evident in the phenomenon of cross-priming, in which H-2 heterozygous (A x B)F1 mice injected 2 wk previously with minor H-different H-2A (A') spleen cells generate both H-2A- and H-2B-restricted minor-H-specific CTL. In a study of the kinetics of direct- vs. cross-priming to minors in F1 mice, we have found that minor H-different T cells actually suppress the induction of virgin CTL capable of recognizing them. CTL activity measured from F1 mice 3-6 d after injection with viable A' spleen cells is largely H-2B restricted. The H-2A-restricted response recovers such that roughly equal A- and B-restricted activity is detected in mice as early as 8-10 d postinjection. This temporary hyporeactivity does not result from generalized immunosuppression--it is specific for those CTL that recognize the foreign minor H antigen in the context of the H-2 antigens on the injected spleen cells. The injected spleen cells that mediate this suppression are radiosensitive T cells; Lyt-2+ T cells are highly efficient at suppressing the induction of CTL in vivo. No graft vs. host reaction by the injected T cells appears to be required, as suppression of direct primed CTL can be mediated by spleen cells that are wholly tolerant of both host H-2 and minor H antigens. Suppression cannot be demonstrated by in vitro mixing experiments. Several possible mechanisms for haplotype-specific suppression are discussed, including inactivation of responding CTL by veto cells and in vivo sequestration of responding CTL by the injected spleen cells.     

Private specificities of H-2K and H-2D loci as possible selective targets for effector lymphocytes in cell-mediated immunity

Receptors of effector T lymphocytes of congeneic strains of mice do not recognize public H-2 specificities and react to private H-2 specificities only. This has been established with the use of three tests: direct cytotoxicity assay of immune lymphocytes upon target cells, specific absorption of the lymphocytes on the target cells, and rejection of skin grafts at an accelerated fashion. Immunization with two private H-2 specificities in the system C57BL/10ScSn leads to B10.D2 induces formation of two corresponding populations of effector lymphocytes in unequal proportion: a greater part of them is directed against the private specificity H-2.33 (Kb), while the smaller part is towards H-2.2 (Db) private specificity. These two populations of effector lymphocytes do not overlap, as demonstrated by experiments on their cross-absorption on B10.D2 (R107), B10.D2 (R101), B10.A(2R), and B10.A(5R) target cells, as well as on mixtures of R107 and R101 targets. Following removal of lymphocytes reacting with one of the private H-2 specificities, lymphocytes specific to the other specificity are fully maintained. A mixture of target cells, each bearing one of the two immunizing private specificities, absorbs 100% of the immune lymphocytes and is totally destroyed by them. It is suggested that H-2 antigens are natural complexes of hapten-carrier type, in which the role of hapten is played by public H-2 specifities and that of the carrier determinant by either private H-2 specificities or structures closely linked to them. Various models of steric arrangement of MHC determinants recognized by receptors of effector T lymphocytes are discussed.     

Genetic regulation of the antibody response to H-2Db alloantigens in mice. II. Tolerance to non-H-2 determinants abolishes the antibody response to H-2Db in B10.A(5R) mice

B10.A(5R) mice (H-2i5), immunized with spleen cells from congenic B10 mice (H-2b), responded to alloantigens of the H-2Db region by producing antibodies of only IgM type. In contrast, they produced both IgM and IgG antibodies when immunized with noncongenic H-2b cells that carry other foreign cell surface antigens (non-H-2) in addition to H-2Db. A hypothesis was proposed comparing the H-2Db antigen on a congenic cell to a hapten on a nonimmunogenic carrier which fails to induce T-cell helper function responsible for the switch from IgM to IgG secretion in B cells. Data presented here confirmed this hypothesis. 5R mice rendered tolerant to the relevant non-H-2 antigens were unable to mount the anti-H-2Db IgG response in a noncongenic immunization. Tolerance induction did not lead to abrogation of the T-cell mediated cytotoxicity.     

Genetic control of immune response to myoglobin. Ir gene function in genetic restriction between T and B lymphocytes

We studied the genetic restrictions on the interaction between T cells, B cells, and antigen-presenting cells (APC) involved in the H-2-linked Ir gene control of the in vitro secondary antibody response to sperm whale myoglobin (Mb) in mice. The B cells in this study were specific for Mb itself, rather than for a hapten unrelated to the Ir gene control, as in many previous studies. Low responder mice immunized in vivo with Mb bound to an immunogenic carrier, fowl gamma globulin (F gamma G), produced B cells competent to secrete anti-Mb antibodies in vitro if they received F gamma G-specific T cell help. However, (high- responder X low responder) F1 T cells from Mb-immune mice did not help these primed low responder (H-2k or H-2b) B cells in vitro, even in the presence of various numbers of F1 APC that were demonstrated to be component to reconstitute the response of spleen cells depleted by APC. Similar results were obtained with B6 leads to B6D2F1 radiation bone marrow chimeras. Genotypic low responder (H-2b) T cells from these mice helped Mb-primed B6D2F1B cells plus APC, but did not help syngeneic chimeric H-2b B cells, even in the presence of F1 APC. In contrast, we could not detect any Ir restriction on APC function during these in vitro secondary responses. Moreover, in the preceding paper, we found that low responder mice neonatally tolerized to higher responder H-2 had competent Mb-specific helper T cells capable of helping high responder but not low responder B cells and APC. Therefore, although function Mb-specific T cells and B cells both exist in low responder mice, the Ir gene defect is a manifestation of the failure of syngeneic collaboration between these two cell types. This genetic restriction on the interaction between T cells and B cells is consistent with the additional new finding that Lyb-5-negative B cells are a major participant in ths vitro secondary response because it is this Lyb-5- negative subpopulation of B cells that have recently been shown to require genetically restricted help. The Ir gene defect behaves operationally as a failure of low responder B cells to receive help from any source of Mb-specific T cells either high responder, low responder, or F1. The possible additional role of T cell-APC interactions, either during primary immunization in vivo or in the secondary culture is discussed.     

Primary cell-mediated lympholysis response to a maternally transmitted antigen

Mta-specific cytotoxic T lymphocyte (CTL) can be generated in primary cultures of (NZB X B10.D2)F1 spleen cells with H-2-compatible BALB/c stimulator cells. The CTL lyse reciprocal Mta+ (B10.D2 X NZB)F1 as well as H-2-disparate targets, such as B10, B6, and B6-Tlaa; they do not lyse targets from NZB or any F1 hybrid of an NZB mother. The lysis of 51Cr-labeled B10 targets is completely inhibited by unlabeled targets from Mta+ (B10.D2 X NZB)F1, but not from the reciprocal Mta- F1, thus demonstrating H-2-unrestricted lysis of Mta.     

Distinct Ir genes for helper and killer cells in the cytotoxic response to H-Y antigen.

The H-Y-specific cytotoxic T-cell response requires helper cells: cells from bone marrow chimeras B6 X CBA leads to B6, B6 X CBA leads to B10.A (5R), or B6 X CBA leads to CBA are each unable to respond to H-2k male cells. If, however, cells from B6 X CBA leads to B6 or B6 X CBA leads to B10.A (5R) chimeras are adoptively transferred together with cells from B6 X CBA leads to CBA chimeras, H-Y-specific CTL restricted to H-2k can be obtained. Thus, cells from B6 X CBA leads to B6 or B6 X CBA leads to B10.,A (5R) chimeras (restricted to the left end of the H-2b haplotype) can help CTL precursors from B6 X CBA leads to CBA chimeras (restricted to H-2k). The two classes of T cells required for the CTL response to H-Y antigen are controlled by different IR genes. All H-Y-specific CTL obtained from chimeras B6 + CBA leads to B6 X CBA were found to be of B6 origin. This suggests that CTL or their precursors must express antigens encoded in the left end of the H-2b haplotype for interaction with helper cells.     

Genetic regulation of the antibody response to H-2Db alloantigens in mice. III. Inhibition of the IgG Response to noncongenic cells by preimmunization with congenic cells

When B10.A (5R) mice (H-12i5) are immunized with spleen cells from congenic B10 mice (H-12b), they respond to alloantigens of the H-2Db region by producing antibodies of only IgM type. In contrast, they produce both IgM and IgG antibodies when immunized with A.BY cells (H- 2b) that carry other foreign cell surface antigens (non-H-2) in addition to H-2Db. Preimmunization of 5R mice with two injections of congenic cells leads to an H-2Db specific inhibition of the IgG response to a subsequent immunization with A.BY cells. It is concluded that congenic B10 cells fail to activate helper T cells which are necessary to induce the switch from IgM to IgG production. Instead T- or B-cell tolerance may be induced with prohibits the subsequent IgG response to A.BY cells, possibly by way of suppressor T cells which may act either on B cells directly or via helper T cells.     

Genetic regulation of the antibody response to H-2Db alloantigens in mice. I. Differences in activation of helper T cells in C57BL/10 and BALB/c congenic strains

B10.A(5R) mice immunized with C57BL/10 spleen cells demonstrate a normal T-cell-mediated cytotoxicity to H-2Db tumor cells but they do not mount any IgG antibody response to H-2Db alloantigens. B10.A(5R) mice do show a high titered IgG response when immunized with A.BY cells, which differ at H-2Db plus non-H-2 cell surface antigens, or with B10.A(2R) cells, which differ at H-2Db, H-2Kk, and H-2Ik cell surface antigens. These findings indicate a failure of the T-helper cells to induce the switch from IgM to IgG when the H-2Db alloantigens are the only difference on the immunizing cell. In immunizing H-2d mice with congenic H-g2 cells which differ only in the H-2Db region, mice of the C57BL/10 background made only IgM antibodies whereas mice of the BALB/c background made IgG antibodies. This comparison confirms that genes separate from H-2 regulate the T-cell helper function. The genes that influence the T-cell helper function do not regulate the T-cell- mediated cytotoxicity.     

Genetic control of the immune response to myoglobins. Both low and high responder T cells tolerant to the other major histocompatibility complex help high but not low responder B cells

We sought to examine the role of immune response (Ir) genes in helper T cells. To eliminate allogeneic effects, we used neonatally tolerized mice. The results bear not only on the mechanism of Ir genes, but also on the development of the T cell repertoire. B 10.BR (H-2(k)) or C57BL/10 (H-2(b)) mice, which were low responders to myoglobin (Mb), were neonatally tolerized to high responder H-2(d) alloantigens, and B10.D2 mice, which were high responders to Mb, were neonatally tolerized to low responder H-2(k) or H-2(b) alloantigens. Spleen cells from these tolerized mice did not show any reactivity in mixed-lymphocyte reaction or cell-mediated lympholysis against alloantigens used in tolerization. Mb-immune F(1) B cells were helped comparably by Mb-immune tolerized low or high responder T cells. Thus, low responder T cells functioned equivalently to high responder T cells. The failure of nonimmune T cells from tolerized low responder mice to help F(1) B cells and antigen-presenting cells (APC) indicated that collaboration between B10.BR or C57BL/10 T cells and F(1) B cells was not caused by a positive allogeneic effect. Spleen cells from tolerized mice were contaminated with 2-4 percent chimeric F(1) cells, as judged by fluorescence-activated cell sorter analysis, and no F(1) alloantigens were detectable in the thymus. However, removal of chimeric F(1) T cells from the tolerized cell population by treatment with anti-H-2 and complement did not change the helper activity of tolerized low responder T cells. These data indicated that helper activity in the T cell population from low responder mice was not due to F(1) cells. Also, the level of contamination was not sufficient to quantitatively account for the help. In examining the genetic restriction of these tolerized T cells, we found that T cells from tolerized low responder B10.BR or C57BL/10 mice helped F(1) or high responder B10.D2 B cells and APC but not syngeneic B10.BR or C57BL/10 B cells and APC, which were immunized with Mb-coupled fowl gamma globulin instead of Mb to prime low responder B cells with Mb. On the other hand, high responder B 10.D2 tolerized T cells helped syngeneic B 10.D2 B cells but not allogeneic low responder B10.BR B cells. These data indicated that clones of helper T cells specific for Mb exist in low responder mice, and these are not phenotypically different from those in high responder mice, in that both help high responder and F(1) but not low responder B cells and APC. These data are discussed in terms of the mechanism for Ir gene control, and the mechanism of T cell repertoire development- whether intra- or extrathymically-in neonatally tolerized mice.     

Viral specificity of H-2-restricted T killer cells directed against syngeneic tumors induced by Gross, Friend, or Rauscher leukemia virus.

Cytolytic T lymphocytes (CTL) were generated against murine tumors induced by Gross, Friend, or Rauscher leukemia virus (LV) in syngeneic mixed leukocyte-tumor cell cultures. Analogous to the patterns of specificity observed with antibodies to LV-induced cell surface antigens, CTL could be classified into two major groups of specificity. Tumor cells induced by Friend, Moloney, or Rauscher virus and positive for the FMR antigen were killed by syngeneic CTL immune to any one of these three LV; the same CTL, however, were incapable of killing syngeneic tumor cells induced by Gross LV. The converse was true for Gross LV-specific CTL: these CTL were specific for syngeneic tumor cells expressing the Gross virus-associated cell-surface antigen (GCSA), and not the FMR antigen. The H-2 specificities of the two groups of LV-immune CTL were also compared, because in both cases, CTL were restricted in their killing activity to H-2-identical tumor target cells. When CTL from single strains of mice were generated against syngeneic FMR- or GCSA-positive tumor cells, differences were observed with respect both to the requirement for the expression of compatible H-2K or H-2D specificities, and to the intensity of the CTL response in congenic mice of the H-2b, H-2d, and H-2k haplotypes.     

T-cell-mediated cytotoxic immune responses to F9 teratocarcinoma cells: cytolytic effector T cells lyse H-2-negative F9 cells and syngeneic spermatogonia

Murine thymus derived (T) lymphocytes primed in vivo to mouse 129 (H-2bc) derived H-2-negative F9 embryonal carcinoma cells and rechallenged in vitro with X-irradiated F9 stimulator cells differentiated into anti-F9 cell immune cytotoxic T lymphocytes (CTL). Using CBA mouse derived splenic responder T cells, F9 stimulator cells triggered a primary cytotoxic anti-F9 response. The CTL generated lysed the F9 antigen-positive target cells F9. PCC3 and PCC4, but not the F9 antigen-negative mouse 129 derived PYS tumor cells, nor LPS induced H-2bc blast cells. Mouse 129 anti-F9 cell antisera but not H-2k anti-H-2bc antisera blocked the lytic interaction with F9 target cells. Similarily unlabeled F9 cells but not H-2bc blast cells inhibited the anti-F9 cell cytotoxicity H-2k anti-F9 cell immune CTL were found to be cytotoxic for syngeneic spermatogonia, known to express the F9 antigen. The results suggest not only that CTL can recognize and lyse H-2-negative target cells, but also that CTL precursors can be sensitized against H-2-negative stimulator cells. From the data available it may be inferred that anti-F9 Cell immune CTL recognize the F9 antigen, known to be linked with the T/t locus. Since anti-F9 cell immune CTL lyse syngeneic spermatogonia, the system may be useful to analyze in vitro the induction and effector phase of a T-cell-mediated cytotoxic autoimmune orchitis.     

Direct demonstration that cytotoxic T lymphocytes recognize conformational determinants and not primary amino acid sequences

The bm 1 H-2Kb mutant differs from the parental strain C57BL/6 (B6) only at amino acid (AA) positions 152, 155, and 156 of the H-2K molecule. The H-2Ld molecule is structurally identical with the H-2 Kbm1 molecule from positions 146-162, thus including all three AA substitutions in Kbm1. In direct lysis and monolayer adsorption studies, B6 anti-bml cytotoxic T lymphocytes (CTL) were shown to include at least five distinct CTL subsets of the following specificities. (a) Uniquely reactive with Kbm1; (b) cross-reactive with Kk; (c) cross-reactive with Dk; (d) cross-reactive with H-2d minus Ld, and (e) cross-reactive with Ld. If B6 anti-bm1 CTL were directed against the primary AA-sequence difference, then all five subsets are expected to react with Ld. However, four out of five CTL subsets including a major population uniquely directed against Kbm1 failed to react with Ld. These findings strongly strengthen the notion that CTL recognize conformational determinants and not primary AA sequences.     

The requirement for two complementing Ir-GLphi immune response genes in the T-lymphocyte proliferative response to poly-(Glu53Lys36Phe11)

The antibody response to poly-(Glu53Lys36Phe11) (GLphi) has been shown to be under the control of two independent, major histocompatibility-linked immune response genes, designated alpha and beta. In the present work we demonstrate that the T-lymphocyte proliferative response is also under the control of these two immune response genes. Thus, mice of the H-2a, H-2b, H-2k, and H-2s haplotypes were all nonresponders to GLphi. In contrast F1 hybrids between these strains, such as (B10 X B10.A)F1 and (C3H X SJL)F1, as well as several recombinant mice derived from the nonresponder haplotypes, such as B10.1(5R), B10.HTT, and B10.S(9R), were all responders to GLphi. The complementation between nonresponder genomes appeared to be stronger in the cis position than in the trans position for some strain combinations. The failure of strains bearing only one of the two responder alleles to show a T-lymphocyte proliferative response to GLphi, argues strongly that neither gene can be expressed exclusively in B lymphocytes. This conclusion is discussed in relation to another two gene model which has recently been proposed.