Clonal analysis of H-2Kb + TNP recognition by T cells with the use of H-2Kbm mutants and H-2Kb-specific monoclonal antibodies

Eleven long-term cytotoxic T lymphocyte (CTL) clones derived from C57BL/10 T cells sensitized in vivo and in vitro with trinitrobenzene sulfonate- (TNBS) treated syngeneic cells were all restricted to the K end of H-2b. The fine specificity of these CTL clones was analyzed by using H-2Kbm mutant target cells and H-2Kb-specific monoclonal antibodies (mAb). Seven distinct patterns of reactivity of the T cell clones could be observed with the use of six H-2Kbm mutant target cells. Further heterogeneity could be detected in terms of the ability of anti-Lyt-2 mAb to inhibit CTL activity. Cross-reactivity between H-2Kb + TNP and H-2Kbm + TNP was observed for all clones tested for bm5 and bm6, but less frequently for bm3 (8/11), bm8 (7/10), bm4 (4/11), and bm1 (3/11). It was further observed that amino acid substitutions located in the first domain only (one clone), or in the second domain only (six clones), or in either the first or the second domain (three clones) of the H-2Kb molecule could affect target cell recognition by a given T cell clone. the latter type of reactivity suggested that some clones recognized "conformational" determinants of the H-2 molecule, or that amino acid substitutions in one domain might influence the structure of the next domain. One H-2Kb + TNP-reactive clone exhibited a heteroclitic behavior with decreasing avidities for target cells expressing H-2Kbm8 + TNP, H-2Kb + TNP, and H-2Kbm8, which further extends the various patterns of T cell cross-reactions observed within a given class of MHC products. The use of H-2Kb-specific mAb in blocking studies as an attempt to define further the H-2Kb epitopes recognized by CTL clones indicated that: a) TNBS treatment may affect the antigenicity of the H-2Kb molecule as assessed by some mAb; and b) that the T cell clone-target cell interaction may or may not be inhibited by a given mAb, depending on structural variations of the H-2Kb molecule (use of H-2Kbm mutants) that do not affect the interaction itself. These results indicate that this type of analysis does not permit correlation of serologic- and T cell-defined epitopes.     

H-2Kb mutations limit the CTL response to SV40 TASA

The cytotoxic T lymphocyte (CTL) responses directed towards SV40 tumor-associated specific antigen (TASA) in nine strains of spontaneously arising Kb mutant mice were analyzed. All nine mutants generated normal levels of H-2Db-restricted response, but the K-end-restricted CTL response varied. B6.C-H-2bm1 (bm1) did not produce K-end-restricted SV40 TASA-specific CTL upon immunization, and SV40-transformed bm1 cells were not lysed by intra-H-2 recombinant Kb [B10.A(5R)] CTL. Nonreciprocal cross-reactive lysis was seen between B6-H-2bm8 (bm8) and B10.A(5R). Strain B6-H-2bm8 mice produce highly specific Kbm8-restricted CTL that lyse SV40-transformed bm8 cells (Kbm8SV) but not B10.A(5R) target cells (K5RSV), although Kbm8SV targets can be partially lysed by B10.A(5R) CTL. The other seven Kb mutants cross-react with B10.A(5R). These experiments definitively show that genes mapping to the K and/or D region directly control the H-2-restricted CTL response to SV40 TASA.     

Characterization of dual-reactive H-2Kb-restricted anti-vesicular stomatitus virus and alloreactive cytotoxic T cells

Cross-reactive recognition of alloantigen by "self + X"-reactive cytotoxic T lymphocytes (CTL) has been documented in a variety of systems. It has been shown previously that the H-2Kb-restricted CTL response of C57BL/6 (B6) mice to vesicular stomatitis virus (VSV) infection is partially cross-reactive on uninfected target cells expressing the H-2Kbm8 mutation. In this report, we describe the isolation and detailed characterization of such dual-reactive CTL. By employing EL4 tumor lines transfected with genes encoding various VSV proteins, we demonstrated that the majority of dual-reactive CTL recognize the internal N protein of VSV and are also reactive against uninfected bm8 targets. Although the response of normal B6 mice to bm8 stimulators shows no measurable cross-reactivity on VSV-infected targets, the response of VSV-primed B6 mice to bm8 stimulation is almost entirely cross-reactive, lysing VSV-B6 targets and uninfected bm8 targets roughly equally. Furthermore, about 70% of CTL clones isolated from such mice by bm8 stimulation are dual-reactive with respect to effector function. Analysis at the population and clonal levels with cold target competition and antibody blocking suggests that the bulk of dual-reactive CTL have a higher avidity for VSV-B6 targets than for bm8 targets. The extreme case of this is illustrated by a fraction of CTL clones, isolated and maintained on bm8 stimulators, which lyse VSV-B6 targets but do not lyse bm8 targets. One such CTL clone is shown to be specific for the bm8 antigen in proliferation assays. These results demonstrate that: the specificity of an alloreactive CTL response may be dramatically altered by previous antigenic encounters; and dual-reactive CTL display a significant difference in affinity of the CTL receptor-determinant interaction, depending on the target which is recognized.     

Recognition of H-2Kb mutant target cells by Moloney virus-specific cytotoxic T lymphocytes from bm13 (H-2Db mutant) mice. I. Full recognition of Kbm11 by Kb-restricted CTL

In C57BL/6 (B6, H-2b) mice, the secondary in vitro CTL response against Moloney leukemia virus is restricted and regulated by the H-2Db locus. B6.C-H- 2bm13 ( bm13 ) mice, however, carrying a mutation at the Db locus, show an increased H-2Kb-restricted CTL response without a demonstrable CTL component restricted by the mutant Dbm13 molecule (D----K shift). These purely Kb-restricted bm13 virus-specific CTL were incubated with a series of Kb mutant virus-infected target cells to study the effect of the mutations at the target cell level. Of six Kb-mutant virus-infected target cells tested, bm1 cells were not recognized and bm8 cells were recognized only marginally by bm13 virus-specific CTL, whereas bm3 , bm5 , bm6 , and bm11 cells were fully recognized. Thus, the bm3 , bm5 , bm6 , and bm11 Kb mutants fully share the relevant H-2K restriction specificities with H-2Kb, whereas the bm1 mutant totally and the bm8 mutant almost completely lack these specificities. This result differs markedly from the restriction site relationships among B6 and these Kb mutants in other antigenic systems. The most striking example concerns the bm11 mutant, which is fully recognized by Moloney-specific CTL, but not at all by Sendai, minor H (H-3.1, H-4.2), and sulfhydryl hapten-specific CTL. Monoclonal anti-H-2Kb antibody B8-3-24 inhibited virus-specific lysis by bm13 CTL of all Kb virus-infected mutant target cells to which this antibody binds. Lysis of bm5 and bm11 but not of bm3 target cells was inhibited, in line with the fact that B8-3-24 antibody does not bind bm3 . On the other hand, not only bm5 and bm11 but also bm3 virus-infected target cells blocked virus-specific lysis to the same extent as syngeneic bm13 target cells. Therefore, bm13 virus-specific CTL populations do not recognize the discrete cluster alteration in the Kbm3 molecule, as identified by antibody B8-3-24. The bm1 and the bm8 mutations, which have structural alterations in completely different sites of the Kb molecule, show complete or almost complete loss, respectively, of Kb-Moloney restriction sites. This finding supports the notion that these virus-specific CTL recognize conformational determinants rather than linear amino acid sequences.     

In vivo induction of cytotoxic T lymphocytes in H-2Kbm mutant mice and cross-reactivity of narrowly specific killer clones

Anti-wild-type (B6) H-2Kbm mutant (bm) CTL were induced in the regional lymph nodes by 2 injections (with 2 week interval) of bm mice into foot-pads with B6 irradiated splenocytes. CTL were tested 7 days after the boost, including 3 days precultivation in monoculture (required for high CTL activity in bm). Active bm4 CTL inducible in vivo but not in the mixed lymphocyte culture (MLC), while bm1, bm3 and their F1 hybrids with BALB/c were equally active in both models. In vivo induced bm3 CTL were cloned with B6 irradiated splenocytes stimulators in the presence of rat interleukine-2. Of 9 Thy1.2 positive narrow-specific CTL clones 2 displayed cross-reactivity to allogeneic target cells (TC): the 1st lysed H-2Kk [TC B10.A(2R)] and the 2nd H-2Kd [TC B10.D2(R101)]. The results witness for non-identity of the in vivo and in vitro induced CTL. The variable cross-reactivity of the narrow-specific CTL clones possibly occur because of receptors' affinity difference.     

Dissection of H-2Db-restricted cytotoxic T-lymphocyte epitopes on simian virus 40 T antigen by the use of synthetic peptides and H-2Dbm mutants.

Five distinct cytotoxic T-lymphocyte (CTL) recognition sites were identified in the simian virus 40 (SV40) T antigen by using H-2b cells that express the truncated T antigen or antigens carrying internal deletions of various sizes. Four of the CTL recognition determinants, designated sites I, II, III, and V, are H-2Db restricted, while site IV is H-2Kb restricted. The boundaries of CTL recognition sites I, II, and III, clustered in the amino-terminal half of the T antigen, were further defined by use of overlapping synthetic peptides containing amino acid sequences previously determined to be required for recognition by T-antigen site-specific CTL clones by using SV40 deletion mutants. CTL clone Y-1, which recognizes epitope I and whose reactivity is affected by deletion of residues 193 to 211 of the T antigen, responded positively to B6/PY cells preincubated with a synthetic peptide corresponding to T-antigen amino acids 205 to 219. CTL clones Y-2 and Y-3 lysed B6/PY cells preincubated with large-T peptide LT220-233. To distinguish further between epitopes II and III, Y-2 and Y-3 CTL clones were reacted with SV40-transformed cells bearing mutations in the major histocompatibility complex class I antigen. Y-2 CTL clones lysed SV40-transformed H-2Dbm13 cells (bm13SV) which carry several amino acid substitutions in the putative antigen-binding site in the alpha 2 domain of the H-2Db antigen but not bm14SV cells, which contain a single amino acid substitution in the alpha 1 domain. Y-3 CTL clones lysed both mutant transformants. Y-1 and Y-5 CTL clones failed to lyse bm13SV and bm14SV cells; however, these cells could present synthetic peptide LT205-219 to CTL clone Y-1 and peptide SV26(489-503) to CTL clone Y-5, suggesting that the endogenously processed T antigen yields fragments of sizes or sequences different from those of synthetic peptides LT205-219 and SV26(489-503).     

Anti H-2Dd alloreactivity mediated by herpes-simplex-virus specific cytotoxic H-2k T lymphocytes is associated with H-2Dk

Herpes-simplex-virus (HSV) specific, H-2k-restricted, immune cytotoxic T lymphocytes also lyse noninfected H-2d target cells. Genetic mapping studies revealed that HSV-specific Dk-restricted CTL cross-react with allogeneic targets expressing Dd alloantigens. Cold target inhibition experiments indicate that only a minority of HSV-specific CTL mediate cross-reactive cytolysis. The data give an example of where the phenomenon of H-2-restricted versus nonrestricted responsiveness is not due to distinct subsets of T cells but solely depends on the antigenic determinants recognized.     

Induction in vivo of specific T suppressors in mice of mutant lines H-2KbT

The differences in the generation of specific suppressor T cells (SSTC) against H-2Kb wild type were investigated in H-2Kbm1, H-2Kbm3 and H-2Kbm4 mutants. Anti-Kb SSTC were produced only by bm3 mutant and F1(BALB/c X bm3) hybrid. T-cell nature of SSTC of bm3 mutant was confirmed by anti-Thy 1.2 monoclonal antibodies described in the same study.     

Anti H-2Dd alloreactivity mediated by herpes-simplex-virus specific cytotoxic H-2k T lymphocytes is associated with H-2Dk

Herpes-simplex-virus (HSV) specific, H-2^k-restricted, immune cytotoxic T lymphocytes also lyse noninfected H-2^d target cells. Genetic mapping studies revealed that HSV-specific D^k-restricted CTL cross-react with allogeneic targets expressing D^d alloantigens. Cold target inhibition experiments indicate that only a minority of HSV-specific CTL mediate cross-reactive cytolysis. The data give an example of where the phenomenon of H-2-restricted versus nonrestricted responsiveness is not due to distinct subsets of T cells but solely depends on the antigenic determinants recognized.This work was supported by the SFB 107 and the Stiftung Volkswagenwerk.     

Cross-reactivity patterns of influenza-specific cytotoxic T lymphocytes from H-2Kb mutant mice

Limit dilution cultures were used to test for influenza immune T cell populations from bm1 and bm3 mutant mice that were not lytic for virus-infected targets expressing the Kb and Db major histocompatibility complex glycoproteins. Both Kbm3- and Kbm1-restricted cytotoxic T cells were detected. Such effectors showed minimal cross-recognition of influenza on other mutant targets, except for the case of bm1 and bm10 targets. This is dissimilar to previous findings concerning vaccinia presentation in which bm3+bm11, bm1+bm9, and bm3+bm9 pairs each showed high cross-reactivity. These differences illustrate the role of the H-2K glycoprotein in immune responsiveness. Not only are multiple determinants on each H-2K glycoprotein involved in antigen presentation, they appear to play differential roles in the presentation of different viral antigens.     

H-2 class I mutants utilize novel restriction specificities in the trinitrophenyl-specific cytotoxic T-lymphocyte response

In antigen-specific cytotoxic T-lymphocyte (CTL) responses H-2 class I mutations usually result in a decreased recognition of the antigen in association with the mutant molecule by CTL from the strain of origin. However, the influence of class I mutations on the magnitude and specificity of CTL responses in the mutants has been studied in only a few instances, in which usually a partial or complete loss of responsiveness was found. We now report that class I mutants extensively use gained (novel) CTL restriction sites, generated by the mutations in the CTL response against the hapten trinitrophenyl (TNP), demonstrated both at the population level and in limiting dilution. TNP-specific CTL clones, restricted by mutant-specific determinants, were detected in all mutants. The percentages mutant-specific CTL clones in limiting dilution experiments were 43, 40, 35, and 13 in the Kb mutants bm1, bm8, bm3 and bm5, respectively, and 35 in the Db mutant bm 14. It is concluded that H-2 class I mutations led to changes in the TNP-specific CTL repertoire resulting in gain of CTLs uniquely restricted to the mutant molecule.     

Cell-mediated lympholysis responses against autologous cells modified with haptenic sulfhydryl reagents. IV. Self-determinants recognized by wild-type anti-H-2Kb and H-2Db-restricted cytotoxic T cells specific for sulfhydryl and amino-reactive haptens are absent in certain H-2 mutant strains

Virus-specific H-2-restricted cytotoxic T cells (CTL) have been found to discriminate between wild-type and mutant class I molecules. The only results reported concerning a hapten-self model, however, indicate that TNP-specific CTL do not discriminate between wild-type and mutant self determinants (7). In the present study, hapten-specific CTL generated against N-iodoacetyl-N'-(5-sulfonic-1-naphthyl) ethylene diamine-modified syngeneic cells (AED-self) were used to determine whether a hapten that is known to react with different cell surface sites than TNP can induce CTL that distinguish mutant H-2K and D molecules from those of wild type. The findings of this study indicate that H-2Kb-AED-self cytotoxic effector cells can discriminate between self-determinants of H-2Kb wild-type and the H-2bm1 and H-2bm11 mutants, but not between wild-type and the H-2bm6 and H-2bm9 mutants. H-2Db-AED-self effector cells were also found to discriminate between self-determinants of H-2Db wild-type and the H-2bm13 and H-2bm14 mutants. Furthermore, cold target competition experiments indicated that the bm1 and bm11 Kb products also lack some determinants recognized by anti-wild-type Kb TNP-specific CTL. These findings provide the first demonstration that hapten-self-specific effectors can detect alterations in H-2 mutant class I molecules. The results in the present report also support the hypothesis that haptens do not have to derivatize H-2 molecules in order to form antigens recognized by H-2-restricted CTL. These findings are discussed with respect to the involvement of self-determinants on MHC and non-MHC cell surface molecules.     

Demonstration of multiple antigenic sites of the SV40 transplantation rejection antigen by using cytotoxic T lymphocyte clones

Multiple antigenic sites on the simian virus 40 (SV40) tumor-specific transplantation antigen (TSTA) were detected by the use of cytotoxic T lymphocyte (CTL) clones isolated from continuous cultures of SV40-specific CTL (H-2b). Two independently derived clones, K11 and K19, specific for the SV40 TSTA in association with H-2Db, each recognized a different antigenic determinant of the SV40 TSTA. This conclusion was based on the observation that a human papovavirus BK virus (BKV) transformed cell line, which possesses a T antigen serologically cross-reactive with that of SV40, was lysed by a heterogeneous population of SV40-immune lymphocytes and by clone K19 but not by K11. Therefore, these CTL clones must recognize two different antigenic determinants of the SV40 TSTA:K19 recognizes a cross-reactive determinant of the SV40 and BKV TSTA, whereas K11 is reactive against an SV40-specific determinant.     

Structural basis of Kbm8 alloreactivity. Amino acid substitutions on the beta-pleated floor of the antigen recognition site

We have analyzed the functional significance of the four amino acid differences between the parental H-2Kb and mutant H-2Kbm8 glycoproteins. Six bm8 variants including single substitutions at residues 22, 23, 24, and 30 as well as paired substitutions at residues 23, 30 and 22, 24 were generated and transfected into L cells. Surface expression of these H-2Kb variants was analyzed using monoclonal antibodies which bind to well-defined H-2Kb epitopes. No alterations introduced into the conformational structure of H-2Kb by the amino acid substitutions were detected. The effect of these substitutions on CTL recognition was initially analyzed using the following bulk CTL: either H-2Kb anti-H-2Kbm8, H-2Kbm8 anti-H-2Kb, or third party anti-H-2Kb. The alloreactivity between H-2Kb and H-2Kbm8 is dominated by the amino acid substitution at residue 24 (Glu----Ser). The complete bm8 phenotype, however, also requires the additional substitution at residue 22 (Tyr----Phe). The H-2Kbm8 anti-Kb bulk CTL reacted with both variant H-2Kbm8 molecules containing single substitutions at amino acid positions 22 or 24 but not the variant molecule containing both substitutions. Further analysis using three individual H-2Kbm8 anti-Kb CTL clones indicated the complexity of the self Kbm8 phenotype. Clone 8B1.20 did not react to changes in residues 22 or 24. The 8B1.32 clone reacted with the change at residue 22 but not with the change at residue 24, although the 8B1.54 clone reacted with the change at residue 24 but not with the change at residue 22. The changes in residues 23 (Met----Ile) and/or 30 (Asp----Asn) did not impact significantly on the alloantigenic properties of Kbm8 as determined by both the bulk and cloned CTL populations. According to the three-dimensional class I structure the substitution at amino acid 24 is inaccessible to the TCR. The location of this substitution within the Ag recognition site implies that altered peptide binding, and not a disruption of MHC residues that interact with the TCR, is responsible for the alloreactivity between H-2Kb and H-2Kbm8.     

Studies of two H-2Db mutants: B6. C-H-2bm13 and B6.C-H-2bm14

Two new C57BL/6 H-2 mutants, B6.C-H-2bm13 and B6.C-H-2bm14 are described. They arose independently in C57BL/6 as spontaneous mutations of the gain and loss type. Complementation studies map the mutations in both bm13 and bm14 to the H-2Db gene. However, these two mutant strains are not identical, but occurred as independent mutations at the same locus, as shown by reciprocal graft rejection and by the inability of the (bm13 X bm14)F1 hybrid to accept C57BL/6 grafts. Serological studies by direct testing (cytotoxicity and hemagglutination) and by quantitative absorption demonstrated a decrease in the H-2Db private specificity H-2.2 in both bm13 and bm14 when compared to C57BL/6. This was confirmed by SDS-PAGE analysis using antisera detecting the H-2.2 specificity. Attempts to produce antibodies to either the gained or lost specificities of the two mutant strains failed.     

H-2 Effects on cell-cell interactions in the response to single non-H-2 alloantigens

Mice expressing mutant H-2Kb alleles were tested for their ability to generate cytotoxic effector T-cells specific for the non-H-2 histocompatibility alloantigen H-4.2. Cytotoxic effectors specific for H-4.2 are preferentially restricted by the Kb allele. Mutant Kb alleles were observed to differentially regulate the magnitude of the H-4.2-specific cytotoxic effector response. Mice expressing the Kbm5, Kbm6, Kbm7, and Kbm9 alleles generated cytotoxic T-cells to the same level as mice expressing the wild-type Kb allele. Kbm8 and Kbm11 responders generated intermediate levels of effectors, whereas Kbm1, Kbm3, and Kbm10 responders did not generate detectable levels of cytotoxic effectors. Kbm4 responders produced high levels of H-4.2-specific cytotoxic effectors that were variably reactive with wild-type Kb antigens with no H-4.2. The ability to generate H-4.2-specific effectors generally correlated with (1) the ability of mutant Kb molecules to present H-4.2 to wild-type Kb-restricted effectors, and (2) the position of the respective amino acid interchanges on the Kb molecule. Mutations that altered the amino acid sequence in the vicinity of the disulfide bond in the C1 domain had the greatest deleterious effects on Kb-controlled responsiveness to H-4.2. The only exception was the Kbm11 intermediate responder, which differs from Kbm3 in both responsiveness and in a single amino acid interchange. Therefore, the amino acid sequence in the vicinity of the disulfide bond in the C1 domain plays a prominent role in determining the H-4.2-specific immune response potential. These observations are the first to clearly demonstrate association between particular MHC gene product, amino acid sequences and immune responsiveness.     

The functional significance of two amino acid polymorphisms in the antigen-presenting domain of class I MHC molecules. Molecular dissection of Kbm3

The functional properties of two amino acid substitutions, characteristic of the bm3 mutation, in the Kb class I glycoprotein were analyzed in light of the HLA-A2 crystal model. The model predicts that amino acid residues extending into the proposed ligand-binding site or projecting up from the alpha-helices are functional with respect to peptide Ag presentation; whereas those residues pointing away from the site are silent. L cell clones expressing Kb, Kbm3, and derivatives of Kbm3, Kbm3-77 (Asp----Ser "ligand-binding") and Kbm3-89 (Lys----Ala "silent"), were generated for the analysis. Serologic characterization of this panel of cells by using the mAb B8-24-3, EH-144, 20-8-4, K9-136, and Y-25 (Kb but not Kbm3 specific) revealed the loss of the epitopes recognized by these mAb in the Kbm3-89 clone and the retention of these epitopes in the Kbm3-77 clone. Analysis of the L cell clones by using B6 anti-bm3 CTL demonstrated that L cell clones expressing Kbm3 or Kbm3-77 were lysed by these CTL, whereas clones expressing Kb, Kbm3-89, and Ld were not lysed. In reciprocal experiments, bm3 anti-B6 CTL lysed L cell clones expressing Kb or Kbm3-89 but were unable to lyse clones expressing Kbm3, Kbm3-77, and Ld. The results indicate that the substitution at amino acid 89 determines the Kbm3 serologic phenotype, whereas the Kbm3 alloreactive phenotype is primarily determined by the substitution at amino acid 77. These findings are in good agreement with the predictions derived from the x-ray crystal model of the HLA-A2 molecule.     

Characterization of the spontaneous mutant H-2Kbm29 indicates that gene conversion in H-2 occurs at a higher frequency than detected by skin grafting.

A spontaneous mutation of H-2Kb, Kbm29, was discovered among the progeny of F1 hybrid parents. Unlike other characterized spontaneous class I variants, this mutant was detected with the use of antibody, rather than tissue grafting. Although Kbm29 is serologically indistinguishable from the previously described mutant molecule Kbm3, it is identical to the parental Kb by skin grafting and CTL assays. A full length cDNA of Kbm29 was amplified by polymerase chain reaction with locus-specific primers, cloned, and sequenced. Two nucleotides were found to be mutated, resulting in a single amino acid change (Lys----Ala) at amino acid 89 of the mature glycoprotein. This is consistent with the observed serologic changes, as the same amino acid substitution is responsible for the serologic profile of Kbm3. The occurrence of a mutation which is not detectable by the methods normally used to screen for H-2 mutants provides evidence that the high spontaneous rate of structural mutation described for the Kb molecule is underestimated.     

Cytotoxic T lymphocyte response to minor H-42a alloantigen in H-42b mice: clonal inactivation of the precursor cytotoxic T lymphocytes by veto-like spleen cells that express the H-42a antigen

When (B10.BR X CWB)F1 (BWF1; H-2k/b) mice carrying the H-42b allele at the minor H-42 locus were injected with H-42a C3H.SW (CSW; H-2b) or C3H (H-2k) spleen cells (SC), self-H-2Kb restricted anti-H-42a pCTL in the BWF1 recipients were primed and differentiated to anti-H-42a CTL after in vitro stimulation with (B10.BR X CSW)F1 (BSF1; H-2k/b, H-42b/a) SC. In contrast, anti-H-42a pCTL in H-42b mice were inactivated by injection with H-42-congenic H-42a SC, and stable anti-H-42a CTL tolerance was induced. Preference of H-2Kb restriction of anti-H-42a CTL was strict, and self-H-2Kb-restricted anti-H-42a CTL did not lyse target cells carrying H-42a antigen in the context of H-2Kbm1. Involvement of suppressor cells in the anti-H-42a CTL tolerance was ruled out by the present cell transfer study and the previous cell-mixing in vitro study. Notably, treatment with anti-Thy-1.2 antibody (Ab) plus complement (C) wiped out the ability of CSW SC in the priming of anti-H-42a pCTL of BWF1 mice but left that of C3H SC unaffected, and injection of the anti-Thy-1.2 Ab plus C-treated CSW SC induced anti-H-42a CTL tolerance in the BWF1 recipients. Furthermore, H-42a/b, I-Ab/bm12 [CSW X B6.CH-2bm12 (bm12)]F1 SC could not prime anti-H-42a pCTL in H-42b, I-Ab (CWB X B6)F1 recipients, whereas H-42a/b, I-Ab (CSW X B6)F1 SC primed anti-H-42a pCTL in H-42b, I-Ab/bm12 (CWB X bm12)F1 recipients. The unresponsiveness of anti-H-42a pCTL in H-42b mice to H-42-congenic H-42a SC was sometimes corrected by immunization of H-42b female mice with H-42-congenic H-42a male SC. Taking all of the results together, we propose the following. Unresponsiveness of anti-H-42a pCTL in H-42b mice to H-42-congenic H-42a SC is caused by "veto cells" contained in the antigenic H-42a SC. Anti-H-42a pCTL in the H-42b recipients directly interacting with H-42-congenic H-42a SC, which bear H-42a antigen and H-2Kb restriction element, are inactivated or vetoed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Peptide interactions with the Kb antigen recognition site

The ability of OVA-specific H-2Kb-restricted CTL to recognize the defined OVA258-276 peptide in the context of the Kbm mutants and variants of these mutants was examined to determine how specific variations in the Ag recognition site-influenced peptide presentation to these CTL. L cells expressing Kb or Kbm10 were equally capable of presenting the OVA peptide to Kb-restricted, OVA-specific bulk CTL, whereas L cell clones expressing Kbm8 or Kbm1 showed little to no capacity to present this peptide. L cell transfectants expressing Kbm3 and Kbm23 consistently demonstrated an intermediate to low level of presentation to bulk OVA-specific CTL. Dissection of the Kbm8 mutant revealed that cells expressing Kbm8-22 (Tyr----Phe) and/or Kbm8-24 (Glu----Ser) presented the OVA peptide significantly less well than the Kb-presenting molecule. Presentation of OVA by cells expressing Kbm8-23,30 (Met----Ile) (Asp----Asn), Kbm8-23 (Met----Ile), and Kbm8-30 (Asp----Asn) was equivalent to Kb presentation. Another mutation designated as Kbm5, that has a substitution at position 116 (Tyr----Phe), demonstrated an intermediate to high ability to present OVA258-276 to an OVA-specific CTL line. The Kbm3, Kbm11, and Kbm23 mutants were unable to present the OVA peptide to this same CTL line. Dissection of these mutants showed that the substitution at position 77 (Asp----Ser), which is shared by all three mutants, was responsible for their inability to present the peptide. A second Kb-restricted CTL line was able to recognize OVA in the context of the Asp----Ser substitution at position 77. The results of this analysis suggest that the OVA258-276 peptide interacts with multiple regions within the Ag recognition site of the Kb class I protein.