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.     

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.     

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.     

Cross-reactive recognition of mouse cells expressing the bm3 and bm11 mutations within H-2Kb by H-2Kb-restricted herpes simplex virus-specific cytotoxic T lymphocytes

Cytotoxic T lymphocytes, generated in C57BL/6 mice in response to herpes simplex virus type 1 (HSV) and known to be restricted in their recognition of HSV-encoded antigen(s) in association with the class I H-2Kb gene product, were consistently found to contain a subpopulation that recognized and lysed uninfected, SV40-transformed cells that expressed the H-2Kbm3 and H-2Kbm11 mutant class I gene products on their cell surface. The mutant cell lines, designated Lgbm3SV and Kbm11SV, share a common amino acid substitution at position 77, with the bm3 mutation having an additional amino acid substitution at position 89. Cross-reactive lysis was observed only after in vivo priming with HSV, suggesting an important role for an antigen-dependent driving step in the expansion of these cross-reactive CTL. The phenotype of the cross-reactive effector population was further confirmed as a T lymphocyte by negative-selection techniques. Limiting dilution analysis of the frequency of cross-reactive CTL precursors suggested that cross-reactivity was mediated by a subpopulation of HSV-specific CTL, and this was confirmed by clonal analysis of the reactivity patterns of short-term, HSV-specific CTL clones. However, analysis of the specificity of the cross-reactive CTL population by cold-target inhibition of bulk culture-derived CTL, or by Spearman ranking analysis of limiting dilution-derived CTL, indicated that the specificity of the cross-reactive population for HSV-infected H-2b target cells and for uninfected bm3 or bm11 target cells was quite distinct. These findings suggested that the cross-reactive CTL population played little, if any, role in the HSV-specific CTL response as measured in vitro. The findings also suggested that the HSV-specific CTL clones able to mediate cross-reactive recognition of the bm3 and bm11 targets had a higher intrinsic avidity for the foreign target than for the inducing antigen.     

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.     

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.     

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.     

Expression of H-2Dd and H-2Ld mouse major histocompatibility antigen genes in L cells after DNA-mediated gene transfer

Among the more than 20 H-2-like genes in the BALB/c mouse genome, there are two classical transplantation antigens (H-2Dd and H-2Ld) encoded at the D-end of the major histocompatibility complex. Here we report the identification of a bacteriophage clone that encodes H-2Dd. The H-2Dd gene was identified by nucleotide sequence analysis and by characterization of the new H-2 antigen expressed when the cloned gene was introduced into mouse L cells by DNA-mediated gene transfer. The previously identified H-2Ld gene was then compared with the H-2Dd gene. The two genes appear to have the same general structure, and for the 854 nucleotides that have been compared, the two genes are 89% homologous. The H-2Ld and H-2Dd antigens expressed on mouse L cells after DNA-mediated gene transfer were examined by immunologic criteria. The stably transformed cell lines express apparently normal levels of H-2Dd and H-2Ld on the cell surface as measured by quantitative immunofluorescence by using monoclonal anti-H-2 antibodies. They synthesize H-2Dd and H-2Ld at normal rates as determined by endogenous labeling and immunoprecipitation of cell extracts. They evoke a strong specific serologic response when used to immunize C3H mice. The newly expressed antigens are able to serve as targets for alloreactive T cells. These cloned genes provide good substrates for examining the evolution of two closely linked H-2 antigen genes. Comparison of the structures of these genes provides clues to the basis for the differential expression of these antigens and their different biologic functions.     

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.     

Interaction between Kb and Q4 gene sequences generates the Kbm6 mutation.

Genetic interaction as a mechanism for the generation of mutations is suggested by recurrent, multiple nucleotide substitutions that are identical to nucleotide sequences elsewhere in the genome. We have sequenced the mutant K gene from the bm6 mouse, which is one of a series of eight closely related, yet independently occurring mutants known collectively as the "bg series." Two changes from the Kb gene are found, positioned 15 nucleotides apart: an A-to-T change and a T-to-C change in the codons corresponding to amino acids 116 and 121, resulting in Tyr-to-Phe and Cys-to-Arg substitutions, respectively. Hybridization analysis with an oligonucleotide specific for the altered Kbm6 sequence identifies one donor gene, Q4, located in the Qa region of the H-2 complex. The two altered nucleotides that differentiate Kbm6 and Kb are present in Q4 in a region where Kb and Q4 are otherwise identical for 95 nucleotides, delineating the maximum genetic transfer between the two genes. Because the Kbm6 mutation arose in an homozygous mouse these data indicate that the Q4 gene contains the only donor sequence and demonstrates that Q-region gene sequences can interact with the Kb gene to generate variant K molecules.     

Polymorphism of murine major histocompatibility Class I antigen: assignment of putative allodeterminants to distinct positions of the amino acid sequence within the first external domain of the antigen

Forty-five new monoclonal antibodies reacting with the mouse H-2Dd antigen have been established. The specificities of 34 of these antibodies were mapped into the first external domain (N) of the Dd antigen by testing reactivities with the products of mosaic H-2 genes in which the coding sequences of the first and/or the second external domains of the H-2Dd genes were recombined in vitro with the remaining portion of the H-2Ld gene. These antibodies reacted with at least 13 distinct allodeterminants located in the N domain, composed of 91 amino acids, as judged from panel tests carried out on various H-2 haplotypes. To assign possible positions of antigenic determinants of these and other anti-H-2Dd antibodies, we compared primary sequences of seven H-2 antigens and searched for correspondence between the pattern of amino acid substitutions in the N domain, allowing 15 positions to be assigned for the antigenic sites. These putative antigenic determinants were assessed for possible relationships with several parameters of protein secondary structure postulated according to predictive methods. Many of these sites appear to be associated with greatest local hydrophilicity, known to correlate with sites of antibody binding in various proteins. We therefore propose that some of the correspondences found in this work represent structural correlates of allodeterminants.     

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.     

Biochemical studies of H-2K antigens from a group of related mutants. I. Identification of a shared mutation in B6-H-2 bm5 and B6-H-2 bm16

Structural studies of the H-2 gene products from a group of five closely related but independent C57BL/6 H-2 mutant mice were undertaken. Each of the mutants exhibits reciprocal graft rejection with the parent. The group is remarkable, however, because each member of this group can accept skin grafts from any other member. The results of biochemical analysis of the H-2 glycoproteins from two of these related mutants, bm5 and bm16, are presented in this report. Evidence is given that the H-2K molecules from these two mutants are identical to each other based on comparative tryptic peptide mapping profiles with the parent. From partial amino acid sequence analysis, K products of both mutants have at least one common difference from the parental type located at residue number 116. Definitive studies established that in both bm5 and bm16 a tryosine found in the parent molecule is substituted with a phenylalanine in the mutant. These results show that a biochemical difference between the K products of the two mutants and of the parent can be detected, that the mutants appear to be identical with one another even though they arose independently, and that they differ from the other H-2K ^b mutants analyzed.Abbreviations used in this paper B6 C57BL/6Kh - bm5 B6-H-2^bm5 - bm6 B6-H-2 ^bm6 - bm7 B6.C-H-2 ^bm7 - bm9 B6.C-H-2 ^bm9 - bm16 B6-H-2 ^bm16 - D H-2D - K H-2K - MHC major histocompatibility complex     

Structural analysis of an HLA-B7 antigen variant detected by cytotoxic T lymphocytes

It has been demonstrated previously that lymphocytes of donor CF (HLA-A29,w33; B7,14) are not recognized by the HLA-B7-specific CTL clone HG-31. This report presents a structural comparison of the HLA-B7 antigen of donor CF with a "normal" HLA-B7 antigen, derived from the cell line JY. Isoelectric focusing showed that CF HLA-B7 heavy chains were more acidic than JY HLA-B7 heavy chains by the equivalent of a single charge. High pressure liquid chromatography and ion exchange chromatography comparisons of double-labeled tryptic peptides revealed a single detectable difference, which corresponded to the tryptic peptide spanning residues 112 to 121 on the HLA-B7 heavy chain. Although the complete amino acid sequence of this peptide was not obtained, the partial sequence indicates a substitution of an unidentified amino acid for tyrosine at position 116 of the heavy chain. This residue is found to vary among HLA specificities and to be altered in many H-2Kb mutants.     

Interaction of alpha 1 with alpha 2 region in class I MHC proteins contributes determinants recognized by antibodies and cytotoxic T cells

The structure-function relationship of individual coding regions of class I mouse major histocompatibility complex proteins was studied by a combination of recombinant DNA, gene transfer techniques, and serologic and functional characterization. To examine the role of alpha 1 and alpha 2 regions in antibody and CTL recognition, the third exon of H-2Dd, Kd, and Ld transplantation antigen genes was replaced by the homologous coding region of the Qa-2-coded class I gene, Q6. We have chosen to carry out the exon shuffling experiments between these two different types of class I genes, because they are structurally similar and did not evolve to carry out identical functions. Therefore, it is less likely that the hybrid proteins will fortuitously recreate alpha 1-alpha 2 controlled functionally important determinants. The replacement of H-2 alpha 2 coding region with its Q6 counterpart had different effects on the expression of the three genes. The mutant H-2Dd gene transfected into L cells was expressed at high levels and retained several of the serologic determinants found on parental H-2Dd and Q6 domains. The serologic epitopes on the mutant H-2Kd-transfected cells were detectable at very low levels, whereas the product of the mutant H-2Ld gene could not be identified at all. Analysis of cells transfected with mutant H-2Dd gene with alloreactive and minor antigen(s)-restricted cytotoxic T cells indicated that the hybrid proteins lost the ability to be recognized by T cells. Our data suggest that cytotoxic T cells recognize conformational determinants composed of amino acids from alpha 1 and alpha 2 regions. Alternatively, it could be proposed that T cell recognition sites located in a single alpha 1 or alpha 2 protein region are susceptible to distortion upon alpha 1-alpha 2 interactions. Such susceptibility to conformational changes of the amino-terminal domain of transplantation antigens could be of functional importance for H-2-restricted antigen presentation.     

Biochemical and functional analyses of a secreted H-2Ld molecule.

A truncated H-2Ld gene was constructed by deleting the transmembrane and cytoplasmic exons. The truncated H-2Ld gene was introduced into mouse L cells using the thymidine kinase gene as a selectable marker. Transformants were isolated and screened for the presence of truncated H-2Ld antigen. The truncated H-2Ld gene product was present in both the cytoplasm and culture medium, but not on the cell surface. The truncated H-2Ld antigen was stable in culture medium for at least 9 h and was secreted into the medium at a rate similar to the kinetics with which complete H-2 antigens reach the cell surface. Transformants expressing the truncated H-2Ld molecule were not recognized by cytotoxic T lymphocytes specific for the H-2Ld antigen.     

Possible mechanisms by which the H-2Kbm3 mutation may decrease cytotoxic T-lymphocyte recognition of vesicular stomatitis virus nucleoprotein antigen.

Spleen cells from C57BL/6 (B6) mice generate a strong in vitro cytotoxic T-lymphocyte (CTL) response specific for vesicular stomatitis virus (VSV). Spleen cells from VSV-primed B6-H-2bm3 (bm3) mice, which have a mutation in H-2Kb, require approximately 10-fold more UV-inactivated VSV to generate in vitro secondary anti-VSV CTL, compared with spleen cells from primed B6 mice. Anti-VSV CTL elicited in both bm3 and B6 mice are primarily specific for the viral nucleocapsid protein (N protein), as demonstrated by using recombinant vaccinia viruses that express the VSV N protein. bm3 CTL were found to exhibit only a very low level of lytic activity when tested against autologous VSV-infected concanavalin A spleen cell blasts as well as several H-2b tumor cell lines. The weak anti-VSV response of bm3 CTL was found to be the result of a combination of inefficient recognition of VSV-infected target cells and decreased elicitation of secondary effector cells. VSV-infected bm3 target cells were not killed as well as B6 targets by either bm3 or B6 effectors. This is because of the inefficient recognition of targets, as demonstrated by the fact that VSV-infected bm3 cells were unable to competitively inhibit the lysis of VSV-infected B6 target cells by either bm3 or B6 effectors. By using cells from recombinant mice, it was shown that the CTL response restricted by H-2Kb was low in the bm3 mice, compared with that of the B6 mice. However, the H-2Db-restricted CTL activity was similarly low in both the B6 and bm3 mice. The possibility that the low response to VSV-infected bm3 cells is caused by differences between the bm3 and B6 cells in expression of either viral antigens or H-2K was investigated by radiolabeling and immunoprecipitation. VSV-infected B6 and bm3 cells were found to express equivalent levels of both viral antigens and H-2K. These results indicate that the bm3 mutation alters a functional site on the H-2Kb molecule that is involved in the recognition of VSV-infected cells. The observation that elicitation of bm3 CTL can occur at high antigen doses further suggests that the bm3 mutation results in a lower affinity of H-2K either for viral antigen or for receptor sites on the CTL.     

The DNA sequence of the H-2kb gene: evidence for gene conversion as a mechanism for the generation of polymorphism in histocompatibilty antigens

We have determined the DNA sequence of the H-2Kb gene of the C57B1/10 mouse. Comparison of this sequence with that of the allelic H-2Kd shows surprisingly that the exons have accumulated more mutations than their introns. Moreover, many of these changes in the exons are clustered in short regions or hot spots. Additional comparison of these sequences with the H-2Ld and H-2Db sequences shows that, in several cases, the altered sequence generated at the hot spot is identical to the corresponding region of a non-allelic H-2 gene. The clustered changes are responsible for 60% of the amino acid differences between the H-2Kb and H-2Kd genes and suggest that micro-gene conversion events occurring within the exons and involving only tens of nucleotides are an important mechanism for the generation of polymorphic differences between natural H-2 alleles.