Characteristics of the conditions for memory cell differentiation--the initial and enriched precursors of secondary cytotoxic T-lymphocytes (pCTL-2) specific for the histocompatibility class I molecule

The in vivo induced pCTL-2 with phenotype L3T4- Lyt2- specific to the H-2Kb molecule, turn into effector CTL during 4 days in the mixed lymphocyte culture (with heat-treated donor stimulators) much more efficiently when donor and recipient are different from one another not only in MHC class I (anti-BIO, MBR BIO.AKM) but in I + II (Kb + Ib) ahti-C57BL/6 BIAD2(RIOI). The initial pCTL-2 differentiation in enhanced as a result of synergistic effect between the Kb alloantigen and rIL2. The anti-Kb pCTL-2, being separated from helper T cells by means of absorption onto the macrophage donor monolayer and elution from it, give rise to pronounced differentiation in simplified conditions, irrespective of the stimulator presence and without external rIL2. It is supposed that these phenomena are raised to secretion of the CTL differentiation factor by the eluted pCTL-2 themselves, and besides, rIL2 may promote for secretion of this factor additionally.     

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.     

Generation of the alloreactive T cell repertoire: K region homology between H-2b T cell precursors and T cell maturation environment is required for the generation of the Kbm6-specific cytotoxic T cell repertoire

The influence of T cell genotype and T cell maturation environment on the generation of the T cell alloreactive repertoire was evaluated in the H-2b cytotoxic T lymphocyte response to Kb mutant determinants expressed by the strain B6-H-2bm6. Specifically, by constructing radiation bone marrow chimeras with B6 or B10 (H-2b) donor cells and B10.BR, B10.A(4R), B10.MBR, and B6.C-H-2bm1 irradiated mice as recipients, it was possible to investigate the major histocompatibility complex (MHC)-encoded gene products of the host environment required for the generation of a bm6-specific H-2b CTL response. The results of such experiments confirmed the previous finding that the alloreactive T cell repertoire is influenced both by T cell MHC genotype and by the MHC gene products of the T cell maturation environment. In addition, the results of the present study further demonstrated that in the chimeric donor and host genetic combinations used, it was both necessary and sufficient that there be a homology of K region-encoded determinants for the generation of a bm6-specific CTL response. Experiments utilizing a mixed responder population of unresponsive B6----B10.D2 spleen cells and responsive Lyt-2 congenic B6.Lyt-2.1 spleen cell suggested that the cellular defect(s) underlying the unresponsiveness of the chimeric cells to bm6-encoded determinants was at the level of the CTL precursor. Together, these findings indicate that an interaction of the K region-encoded gene products of the T cell and its maturation environment play a critical role in the generation of the CTL repertoire specific for bm6 mutant determinants. We discuss here the possibility that this interaction may reflect a requirement that T cells recognize such mutant allodeterminants in association with self restriction elements present on the same mutant K region-encoded molecule.     

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.     

Induction of linked suppression in addition to the donor H-2 class I-specific unresponsiveness in recipient T cells by transfusing class I plus class II-disparate, but not class I alone-disparate, bone marrow cells

This study was undertaken to determine whether bone marrow (BM) cells contain a cell population with the capacity to induce an unresponsiveness of T cells specific to the BM self-H-2 class I antigens in vivo, i.e., veto cell population. Recombinant or congenic mice were infused intravenously with H-2-incompatible BM cells. One to several weeks later, donor H-2-and irrelevant H-2-specific responses in mixed lymphocyte reaction cultures of recipient T cells were assessed. Transfusion of H-2-incompatible BM of C57BL/10 (B10) recombinant strains caused a long-lasting cytotoxic T lymphocyte (CTL) unresponsiveness to the donor class I antigens in recipient lymph node cells. When class I plus class II-disparate BM cells were transfused, an anti-donor class I CTL response and a response against a third-party class I antigen, which was presented on the stimulator cells coexpressing the donor class I and class II, were significantly suppressed. This linked suppression lasted for less than 2 weeks after transfusion. Transfusion of class I-alone-disparate BM induced the donor class I-specific CTL unresponsiveness, but not the linked suppression. The induction of linked suppression was prevented considerably by transfusing nylon wool-nonadherent BM or by treating recipients with cyclophosphamide 2 days before transfusion. An anti-third-party class I CTL response, stimulated in vitro with fully allogeneic spleen cells, was not hampered by the BM transfusion. Coculturing the lymph node (LN) cells obtained from the class I plus class II-disparate BM recipient with normal LN cells interfered with the generation of both anti-donor class I and anti-linked third-party class I CTL, whereas, coculturing LN cells from the class I alone-disparate BM recipient inhibited neither specificity of CTL generation. Transfusion of class I plus class II-disparate BM resulted in a significant suppression of the donor class II-specific proliferative response. In contrast, transfusion of class I alone-disparate BM did not suppress any proliferative responses, including even a "linked" third-party class II-specific response. Transfusion of bm 1, (B6 X bm 1)F1, or (bm 1 X bm 12)F1 BM to B6 did not induce unresponsiveness in bm 1-specific CTL responses. However, the transfusion resulted in a significant suppression of bm 1-reactive proliferative response of recipient LN cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Allogeneic lymphocyte induction in vivo of highly active T-killers specific for the molecule of the H-2 class-I complex and the detection of their receptors in vitro

The optimal conditions are found for in vivo irradiated lymphocyte induction of high cytotoxic T lymphocytes (CTL) specific to the H-2Kb molecule with a subsequent monoculture differentiation. B10.D2(R101) CTL have a pronounced excess as compared to B10.A (4R) CTL, with respect to lysis intensity of the same target cells (TC), and requires a lower term of the monoculture incubation in spite of their specificity to the same H-2Kb molecule. As the susceptibility of TC for CTL lysis is higher (M phi as compared to EL-4 thymoma cells), CTL are much more inactivated with the monoclonal antibodies to Lyt-2 and Lyt-3 antigens without complement. Anti-H-2Kb CTL differentiated in the monoculture cross react to TC bearing third-party H-2 molecules (Kk, Dq, Dk). Unlike a stable CTL adherence to the donor M phi monolayer, nonspecific CTL adherence to the syngeneic M phi monolayer declines in the presence of EGTA, and as a result of repeated detachment of lymphocytes. The findings give rise to study receptor affinity expressed on the in vivo induced CTL surface, the CTL receptor monoclonal antibody and the CTL differentiation factor.     

Comparative study of conditions for demonstrating memory T-cell receptors and other T-lymphocyte subpopulations immune to antigens of the H-2 complex

The immunological memory T cells assayed by the cytotoxic T lymphocyte (CTL) generation in the secondary mixed lymphocyte culture bear H-2 antigen-binding receptors as shown by the technique of the specific lymphocyte absorption on target cell (TC) monolayers of different H-2 origin. Memory T cells and specific suppressor T cells are demonstrated to be capable of adhering to native and fixed TC in a similar fashion, whereas CTL absorption appears to be two-fold reduced when the TC monolayer is fixed. The primary CTL precursors differ from memory T cells by a poor adherence to native TC which is not demonstrable at all when TC are fixed. The findings evidence the differences in receptor affinity (or structure) among the primary and secondary CTL precursors and the CTL themselves.     

Failure or success in the restoration of virus-specific cytotoxic T lymphocyte response defects by dendritic cells

C57BL/6 (B6, H-2b) mice are CTL responders to both Sendai virus and Moloney leukemia virus. In the former response the H-2Kb class I MHC molecule is used as CTL restriction element, in the latter response the H-2Db molecule. B6 dendritic cells (DC) are superior in the presentation of Sendai virus Ag to CTL in comparison with B6 normal spleen cells. Con A blasts have even less capacity to present viral Ag than NSC, and LPS blasts show an intermediate capacity to present viral Ag. H-2Kb mutant bm1 mice do not generate a CTL response to Sendai virus, but respond to Moloney leukemia virus, as demonstrated by undetectable CTL precursors to Sendai virus and a normal CTL precursor frequency to Moloney virus. Compared to B6 mice, other H-2Kb mutant mice show decreased Sendai virus-specific CTL precursor frequencies in a hierarchy reflecting the response in bulk culture. The Sendai virus-specific CTL response defect of bm1 mice was not restored by highly potent Sendai virus-infected DC as APC for in vivo priming and/or in vitro restimulation. In mirror image to H-2Kb mutant bm1 mice, H-2Db mutant bm14 mice do not generate a CTL response to Moloney virus, but respond normally to Sendai virus. This specific CTL response defect was restored by syngeneic Moloney virus-infected DC for in vitro restimulation. This response was Kb restricted indicating that the Dbm14 molecule remained largely defective and that a dormant Kb repertoire was aroused after optimal Ag presentation by DC. In conclusion, DC very effectively present viral Ag to CTL. However, their capacity to restore MHC class I determined specific CTL response defects probably requires at least some ability of a particular MHC class I/virus combination to associate and thus form an immunogenic complex.     

Cross-reactivity patterns of vaccinia-specific cytotoxic T lymphocytes from H-2K b mutants

Limit-dilution cultures were used to select vaccinia-immune T-cell populations from bml and bm3 mutant mice that were not lytic for virus-infected targets expressing the K^b and D^b MHC glycoprotein. Approximately 30% of virus-immune CTL were restricted in each case to K^bm1 and K^bm3, rather than to D^b. Evidence of extensive cross-reactivity was found for these virus-immune CTL. Bm3 and bmll mice sharing one amino acid mutation from wild-type but differing by a second mutation seen only in bm3 are the most cross-reactive pair in their presentation of vaccinia. The bm1 and bm10 pair with dissimilar mutations from wild-type affecting the same CNBr fragment are also largely cross-reactive. However, 30% cross-reactivity is also found for bm1 and bm3, which differ in separate CNBr fragments. That mutants expressing amino acid substitutions in the same region of the peptide tend to show more evidence of cross-reactivity does not necessarily mean the T cells see linear arrays of amino acids on the MHC glycoprotein. For instance, K^bm1 and K^bm10 differ for three amino acids, but bm1 T cells are highly lytic for bm10 virus-infected targets. However, there is no cross-reactivity for K^bm1 and K^b, which differ at only two amino acids. The key to further understanding may rest with defining the nature of the conformational differences among the K^bm1, K^bm10, and K^b glycoproteins.     

Positive selection is limited by available peptide-dependent MHC conformations

Recent data suggest that the diversity of self peptides presented in the thymus during development contributes to positive selection of a diverse T cell repertoire. We sought to determine whether a previously defined "hole in the immunological repertoire" could be explained by the absence of an appropriate selecting self peptide. The repertoire defect in question is the inability of bm8 mice to make an H-2K-restricted response to OVA. Like other OVA-specific, H-2K-restricted receptors, OT-I-transgenic T cells are not positively selected in bm8 mice. Using criteria we had previously established for identifying positive selection ligands, we found peptides that could restore positive selection of OT-I thymocytes in bm8 mice. Thus, the T cell repertoire can be limited by a requirement for specific self peptides during development. Data with MHC-specific Abs suggested that peptides might be able to force MHC residues to adopt different conformations in Kb vs Kbm8. This shows that peptides can potentially contribute to ligand diversity both directly (via variability in the solvent-exposed side chains) and indirectly (through their effect on the MHC conformation). Our data support a model where self peptide diversity allows selection of T cells specific for a broad range of MHC conformations.     

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.     

The diversity of T cell receptors specific for self MHC gene products

Cytolytic and helper T cells exhibit, in addition to their specificity for foreign antigen, a restriction specificity for self MHC gene products. The present study was designed to assess the degree of diversity within the repertoire of receptors that are involved in T cell recognition of self MHC gene products. For this purpose, we generated a series of murine cytolytic T lymphocyte (CTL) clones specific for a hapten antigen and restricted to the self MHC gene product H-2Kb. An analysis of the hapten fine specificity of these clones by using hapten analogues revealed the presence of substantial diversity within the repertoire of CTL receptors specific for the hapten. The degree of diversity within the repertoire of self H-2 recognition structures on these clones was assessed by testing clones on panels of syngeneic, congenic H-2K disparate, and H-2Kb mutant target cells bearing varying amounts of antigen. A striking degree of heterogeneity in H-2K recognition was found among these H-2Kb restricted CTL. We estimate that there are probably a minimum of 65 different patterns of H-2K recognition among these clones. Our results suggest a high degree of diversity exists within the repertoire of self MHC recognition structures on antigen-specific T cells restricted to a single self MHC gene product.     

Immunobiology of cytotoxic T-cell resistant virus variants: studies on lymphocytic choriomeningitis virus (LCMV)

Replication of the genetically variable lymphocytic choriomeningitis virus (LCMV) gives rise to a pool of variant viruses. Under the selection pressure exerted by a strong but narrow repertoire of antiviral cytotoxic T-cells (CTL) i.e. monoclonal or polyclonal monoepitope, variant viruses emerge that contain point mutations in the nucleotide sequence encoding antigenic CTL epitopes; these variants can be selected in both infected mice and cell cultures. These mutations permit infected cells to escape CTL recognition by altering the ability of the mutant peptides to bind MHC class-I-molecules or by interfering with the ability of T-cell receptors to interact with the mutant peptide/MHC complex. Because viral infections often trigger a polyclonal repertoire of antiviral CTL to multiple epitopes, the likelihood of selection of CTL resistant variants is probably low, but not impossible. Our empirical observations suggest that antigenic variations, even if they only occur in a part of the available CTL epitope, may exert significant effects on the subtle biological equilibrium established between virus and host immune system. This can reduce immunological control of the pathogen population, and so permit persistence of viral infection and promote disease progression.     

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).     

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.     

Cytotoxic T lymphocyte recognition of novel allodeterminants expressed on in vitro selected H-2Kb mutants

In the present study, in vitro derived H-2Kb mutants have been examined by alloreactive CTL. Two mutants, R8.24 and R8.246, have been shown to express novel determinants detected by CTL generated against some but not all in vivo derived Kb mutants. BDF1 anti-bm3, anti-bm11, anti-bm19, anti-bm23, and anti-bm6 CTL populations lyse the two R8 variants. The novel determinants expressed on the R8 mutants detected by the bm3 and bm23-specific CTL appear to differ from the determinant recognized by the bm6-specific CTL. No new serologically defined determinants were detected on any of 18 independent R8 variants. However, these results do not rule out the existence of new determinants which could be recognized by antibodies. Finally, the relationship between the T cell recognition of the in vivo and in vitro derived mutants and their use in understanding the structure/function relationships between the immune response and class I Ag based on recent crystallographic analyses is discussed.     

Lethal graft-vs-host disease induced by a class II MHC antigen only disparity is not mediated by cytotoxic T cells

Treatment of C57BL/6J (B6) murine splenocytes with L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) selectively removes NK cells, CTL precursors, and the capacity to cause lethal graft-vs-host disease (GVHD) in irradiated B6 X DBA/2 F1 mice. In contrast, alloantigen-induced L3T4(+) Th cell function has been shown to be relatively preserved after exposure to this agent. The present studies assessed the effects of Leu-Leu-OMe treatment of donor cells on induction of lethal GVHD in other murine strain combinations. When irradiated B6 X CBAF1 mice were infused with T and NK cell-depleted B6 bone marrow cells and 3 to 30 X 10(6) B6 spleen cells, uniformly lethal GVHD was observed. However, B6 X CBAF1 recipients of T and NK-depleted B6 bone marrow cells and similar numbers of Leu-Leu-OMe-treated B6 spleen cells demonstrated 90 to 100% long term survival. In contrast, Leu-Leu-OMe treatment of B6 donor cells had no beneficial effect on mortality rates in irradiated (B6 X B6-C-H-2bm12)F1 (B6 X bm12F1) recipients. When B6 spleen cells were stimulated in vivo or in vitro with either B6 X CBAF1 or B6 X bm12F1 stimulator cells, the capacity to generate alloantigen-specific CTL was abolished comparably by Leu-Leu-OMe treatment. Thus, the dramatic difference between the effects of Leu-Leu-OMe treatment of B6 spleen cells on the course of GVHD in B6 x CBAF1 and class II MHC only disparate B6 x bm12F1 recipients could not be explained by unique resistance of bm12-specific CTL precursors to Leu-Leu-OMe. These findings indicate that T cell effector mechanisms distinct from classic cell-mediated cytotoxicity are sufficient to generate lethal GVHD in class II MHC only disparate B6----B6 X bm12F1 mice.     

Induction of cytotoxic T cells to a cross-reactive epitope in the hepatitis C virus nonstructural RNA polymerase-like protein.

Cytotoxic T lymphocytes (CTL) have been found to mediate protection in vivo against certain virus infections. CTL also may play an important role in control of infection by hepatitis C virus (HCV), but no CTL epitopes have yet been defined in any HCV protein. The nonstructural protein with homology to RNA polymerase should be a relatively conserved target protein for CTL. To investigate the epitope specificity of CTL specific for this protein, we used 28 peptides from this sequence to study murine CTL. Mice were immunized with a recombinant vaccinia virus expressing the HCV nonstructural region corresponding to the flavivirus NS5 gene (RNA polymerase), and the primed spleen cells were restimulated in vitro with peptides. CTL from H-2d mice responded to a single 16-residue synthetic peptide (HCV 2422 to 2437). This relatively conserved epitope was presented by H-2d class I major histocompatibility complex (MHC) molecules to conventional CD4- CD8+ CTL but was not recognized by CTL restricted by H-2b. Moreover, exon shuffle experiments using several transfectants expressing recombinant Dd/Ld and Kd demonstrated that this peptide is seen in association with alpha 1 and alpha 2 domains of the Dd class I MHC molecule. This peptide differs from the homologous segments of this nonstructural region from three other HCV isolates by one residue each. Variant peptides with single amino acid substitutions were made to test the effect of each residue on the ability to sensitize targets. Neither substitution affected recognition. Therefore, these conservative mutations affected peptide interaction neither with the Dd class I MHC molecule nor with the T-cell receptor. Because these CTL cross-react with all four sequenced isolates of HCV in the United States and Japan, if human CTL display similar cross-reactivity, this peptide may be valuable for studies of HCV diagnosis and vaccine development. Our study provides the first evidence that CD8+ CTL can recognize an epitope from the HCV sequence in association with a class I MHC molecule.     

Determinant selection in murine experimental autoimmune myasthenia gravis. Effect of the bm12 mutation on T cell recognition of acetylcholine receptor epitopes.

C57BL/6 (B6) mice respond to immunization with acetylcholine receptor (AChR) from Torpedo californica as measured by T cell proliferation, antibody production, and the development of muscle weakness resembling human myasthenia gravis. The congenic strain B6.C-H-2bm12 (bm12), which differs from B6 by three amino acid substitutions in the beta-chain of the MHC class II molecule I-A, develops a T cell proliferative response but does not produce antibody or develop muscle weakness. By examining the fine specificity of the B6 and bm12 T cell responses to AChR by using T cell clones and synthetic AChR peptides, we found key differences between the two strains in T cell epitope recognition. B6 T cells responded predominantly to the peptide representing alpha-subunit residues 146-162; this response was cross-reactive at the clonal level to peptide 111-126. Based on the sequence homology between these peptides and the T cell response to a set of truncated peptides, the major B6 T cell epitope was determined to be residues 148-152. The cross-reactivity of peptides 146-162 and 111-126 could also be demonstrated in vivo. Immunization of B6 mice with either peptide primed for T cell responses to both peptides. In contrast, immunization of bm12 mice with peptide 111-126 primed for an anti-peptide response, which did not cross-react with 146-162. Peptide-reactive T cells were not elicited after immunization of bm12 mice with 146-162. These results define a major T cell fine specificity in experimental autoimmune myasthenia gravis-susceptible B6 mice to be directed at alpha-subunit residues 148-152. T cells from disease-resistant bm12 mice fail to recognize this epitope but do recognize other portions of AChR. We postulate that alpha-148-152 is a disease-related epitope in murine experimental autoimmune myasthenia gravis. In this informative strain combination, MHC class II-associated determinant selection, rather than Ag responsiveness per se, may play a major role in determining disease susceptibility.     

MHC polymorphism can enrich the T cell repertoire of the species by shifts in intrathymic selection

The murine class I molecule H-2Kb and its natural gene conversion variant, H-2Kbm8, which differs from H-2Kb solely at 4 aa at the bottom of the peptide-binding B pocket, are expressed in coisogenic mouse strains C57BL/6 (B6) and B6.C-H-2bm8 (bm8). These two strains provide an excellent opportunity to study the effects of Mhc class I polymorphism on the T cell repertoire. We recently discovered a gain in the antiviral CTL repertoire in bm8 mice as a consequence of the emergence of the Mhc class I allele H-2Kbm8. In this report we sought to determine the mechanism behind the generation of this increased CTL diversity. Our results demonstrate that repertoire diversification occurred by a gain in intrathymic positive selection. As previously shown, the emergence of the same Mhc allele also caused a loss in positive selection of T cell repertoire specific for another Ag, OVA-8. This indicates that a reciprocal loss-and-gain pattern of intrathymic selection exists between H-2Kb and H-2Kbm8. Therefore, in the thymus of an individual, a new Mhc allele can select new T cell specificities, while abandoning some T cell specificities selected by the wild-type allele. A byproduct of this repertoire shift is a net gain of T cell repertoire of the species, which is likely to improve its survival fitness.