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.     

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.     

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.     

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.     

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.     

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.     

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).     

Cell-mediated cytotoxicity against allogeneic mutant H-2 antigens: restricted and nonrestricted responses

Two "gain and loss" type mutations of the H-2D region, the H- 2bm13 and H- 2bm14 , resulted in the expression of noncross-reactive CML determinants that are unique to each mutation, the Dbm13 gains and Dbm14 gains, respectively. According to the results of direct cytolytic and competitive inhibition assays of in vitro induced primary cytotoxic T lymphocytes, allogeneic responses specific for Dbm13 gains are generated by responders bearing the H-2b ( KbIbDb ) haplotype, but not by responders bearing the H- 2bm14 ( KbIbDbm14 ), KbIbDd , KbIbDk , or KbIb / qDq haplotype. Responses by the non-H-2b responders against Dbm13 are limited to those determinants shared by the Dbm13 and Db molecules. Because congenic mice differing only at the H-2D region are either responsive or nonresponsive to Dbm13 gains, the responsiveness is controlled by gene(s) in the H-2D region. F1 hybrid offspring of responsive (H-2b) and nonresponsive (non-H-2b) parents are invariably responsive, indicating genetic dominance of the responsiveness. In contrast to the response against Dbm13 gains, cytotoxicity specific for Dbm14 gains is generated by responders bearing the H-2b, H- 2bm13 , KbIbDd , KbIbDk , or KbIb / qDq haplotype. These data indicate the existence of two types of allogeneic MHC determinants; one, represented by Dbm14 gains, is the classic type capable of eliciting CML responses in mice of a wide range of H-2 haplotypes, whereas the other, exemplified by Dbm13 gains, elicits CTL responses only in mice of a few related haplotypes. It is proposed that recognition of Dbm13 gains is restricted by structures shared by Db and Dbm13 but missing from other D (or L, R, etc.) molecules, such as Dbm14 , Dd, Dk, and Dq. Availability of various restricting structures in self MHC molecules may thus influence the alloreactive CTL repertoire.     

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.     

Synthesis and immunochemical study of the antigenic determinant of the H-2Db molecule of the murine major histocompatibility complex

Primary structure of murine class I histocompatibility antigens has been analysed to select possible antigenic determinant. Hexapeptide Leu-Gln-Gln-Leu-Ser-Gly, homologous to the region 95-100 of the H-2Db antigen heavy chain, was synthesised by stepwise elongation of peptide chain beginning from the COOH-terminal Gly. Rabbit anti-hexapeptide antibodies were obtained and shown to interact specifically with purified H-2Db antigen as well as with the native antigen on cell surface. These antibodies bind to lymphocytes of H-2b haplotype (C57BL/6 mice) but not H-2d (BALB/c) or H-2k (CBA). These data suggest that the region 95-100 is responsible for serologic differences between the alleles of H-2 antigens, i.e. it may be a xenotypic as well as an allotypic antigenic determinant. The latter was confirmed by study of interaction of the hexapeptide with allogeneic monoclonal antibodies specific to H-2Db antigen.     

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.     

Genetic analysis of the H-2D region using a new intra-D-region recombinant mouse strain

A rare D-region recombination event which gave rise to the B10.RQDB major histocompatibility complex haplotype has been examined to ascertain the nature of the crossover and to determine which class I genes are present in the new alignment of D-region genes. Serologic analysis have shown that the B10 . RQDB major histocompatibility complex recombinant mouse inherited the H-2Dd gene from the B10.T(6R) parental line and the H-2Db gene from the B10.A(2R) parental line, representing the first example of an intra-D-region crossover resulting from an intercross. Previous molecular genetic analyses of the d and b haplotypes revealed structural diversity in the organization of their D-region gene clusters. Hence, the D region is comprised of five class I genes in the d haplotype and only one in the b haplotype. Because allelic relationships among the various D-region genes are not defined, either a homologous or nonhomologous alignment of genes has generated the RQDB crossover. Therefore, the possibility that all three D-region antigen-presenting molecules (Dd, Ld, and Db) might be encoded by the RQDB haplotype was examined. Fluorescence-activated cell sorter and cytotoxic T lymphocyte analyses revealed no detectable levels of H-2Ld cell-surface expression, confirming earlier studies with antibody-mediated cytotoxicity and immunoprecipitation. Southern blot analysis localized the recombination point to within a 1-kb region at the centromeric end of the H-2Ld gene on the B10 . T(6R) chromosome in a region of high homology to the H-2Db gene on the B10 . A(2R) chromosome. Together, these studies define the D region of the RQDB haplotype as containing the five class I genes: Dd, D2d, D3d, D4d, and Db. In addition to providing insight into rare recombination events in the D region, the B10.RQDB mouse should be a useful tool for exploring the function of D-region genes.     

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     

Resistance to H-2-restricted but not to allo-H2-specific graft and cytotoxic T lymphocyte responses in lymphoma mutant

The lymphoma mutant RMA-S escaped graft rejection after transplantation over a minor histocompatibility barrier, whereas it was rejected in H-2 allogeneic mice. The parental control line was rejected in both situations. The mutant, which had been selected against MHC class I molecules retained 5 to 10% of the wild-type H-2Db, Kb, and beta 2-microglobulin expression on the cell surface. It remained sensitive to allo-H-2b CTL in vitro, but was completely resistant to minor histocompatibility antigen-specific, H-2b-restricted CTL. It was equally resistant to other H-2b-restricted responses against internally derived Ag, such as tumor-specific CTL or a CTL clone specific for the influenza virus nucleoprotein. The results indicate a target cell defect that selectively abolishes the sensitivity to H-2-restricted CTL directed against internally processed Ag. This appears sufficient to shift the transplantation response over a minor histocompatibility Ag barrier from rejection to acceptance. There are two possible explanations for the results: 1) a block in the MHC class I-directed pathway for internal Ag processing, and 2) subthreshold H-2/Ag ligand density in relation to triggering requirements of restricted CTL. Regardless of the type of defect, the results demonstrate a difference between allo-H-2-specific and H-2-restricted CTL recognition at the level of the target cell.     

Role of dendritic cells in the regulation of class I restricted cytotoxic T lymphocyte responses

Two class I MHC mutant mouse strains, bm14 and bm13, differ from the strain of origin B6 in one and three amino acids in the alpha 1 and alpha 2 domains of the H-2Db molecule, respectively. These alterations result in specific failure to generate a CTL (Tc) response to the male-specific Ag H-Y. Immunization and/or restimulation in vitro with syngeneic male dendritic cells (DC), expressing very high levels of class I MHC molecules, restored the H-Y-specific Tc response of bm14 but not of bm13 mice. Serologically Db determinants were lost in normal spleen cells of both mutants, because FACS analysis showed a decreased binding of Db domain-specific mAb. Although bm13 DC show a higher fluorescence than bm13 normal spleen cells it is still strongly reduced (30 to 50%) in comparison with B6 DC. Surprisingly, bm14 DC show an equally very strong binding compared with B6 DC with these mAb. The quantitative expression of class I molecules on APC thus appears to be a major determinant in the regulation of Tc responses. In addition, immunization with DC markedly influenced the target cell specificity of the ensuing Tc response. The combined data clearly demonstrate that besides the highly efficient class II-restricted presentation of Ag to Th, shown previously, DC are also superior in the presentation of Ag in the context of class I molecules to Tc. bm14 DC are capable of directly activating H-Y-specific Lyt-2+ Tc memory cells without the need for L3T4+ Th. These biologic effects of DC can at least in part be explained by their very high class I MHC expression. Moreover, these results reiterate that class I MHC Db mutants and different APC can be used to study the contribution of specific class I domains to Tc recognition and restriction specificity.     

Interaction ofH-2D b with mutant histocompatibility geneH (KH-11) in the mouse

The detectable presence ofH(KH-11)^b, a mutant non-H-2 histocompatibility gene, was previously shown to depend upon the simultaneous presence, in the skin-graft donor, of both the mutant gene and theH-2 ^b haplotype. The experiments reported here demonstrate thatH-2D ^b is the essential element ofH-2 ^b for this interaction. Of twoH-2D ^b histocompatibility mutations,H-2 ^bm13 can replaceH-2D ^b in this interaction, butH-2ibm14 cannot.     

A new cytotoxic lymphocyte-defined antigen coded by a gene closely linked to the H-3 locus

F1 complementation results indicate that a new gene, putatively controlling a minor histocompatibility antigen, is closely linked to the minor histocompatibility gene, H-3, in the fifth linkage group of chromosome 2 of the mouse. This gene controls a product that was capable of inducing as well as acting as a target for cytotoxic lymphocytes (CTL). The lytic activity of CTL developed in B10.LP-H-3D mice specific for the product of the new gene of B10 was restricted to target cells possessing H-2Db antigens. This contrasts to the H-2Kb-restricted activity of H-3.1 specific CTL.     

H-2 class I loci determine sensitivity to MCMV in macrophages and fibroblasts

Peritoneal (PM) and bone marrow-derived (BMM) macrophages and lung fibroblasts (LF) from inbred, intra-H-2 recombinant, H-2 mutant, and hybrid mice were infected with murine cytomegalovirus (MCMV) under centrifugal enhancement. At the concentration of virus employed, peritoneal macrophages from strains carrying Kd, Kb, D^d, KS and/or D^s, K4 and/or D4 alleles could be infected to a level of 80%–100%, as assessed by viral antigen expression or loss of Fc receptors. Cells lacking these haplotypes and carrying K^k, K^f, D^k, Df, or Db were resistant, yielding levels of infection below 20% . The background (non-H-2) and class II genotype and the S allele did not influence the proportions of cells infected. Furthermore, sensitivity was dominant in the F, progeny of H-2 b x H-2 k and H-2d x H-2 k crosses, and was not compromised by thebm1, bm3, bm10, or bm14 mutations in the al or2 regions of Kb orD ^b. The proportions of cells able to release infectious virus were low, but paralleled the frequencies of viral antigen expression. The class I genotype also determined susceptibility to MCMV infection in BMM and LF, although up to 35% of H-2 k BMM and 46% of H-2 k LF could be infected. The findings are consistent with an association between K and D antigens and a cellular receptor for MCMV on all three cell types.     

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.