An approach to the study of structure-function relationships of MHC class I molecules: isolation and serologic characterization of H-2Kb somatic cell variants

Somatic cell variants expressing an altered antigenic form of the H-2Kb molecule were isolated for the purpose of performing structure-function analysis of a class I MHC molecule. Over 25 independently isolated variants were derived from an Abelson virus transformed pre- B cell line (R8) by mutagenesis with ethyl methane sulfonate or ethyl nitrosourea. Negative selection was performed by complement-dependent cytotoxicity with anti-H-2Kb monoclonal antibodies subsequently followed by positive selection to separate the H-2Kb surface negative variants from structural variants. Biochemical characterization of a random selection of three independent variants indicated that the variant H-2Kb molecule was present in normal amounts in lysates, and was unchanged in size. Cytofluorometric analysis with the use of a panel of seven monoclonal antibodies against H-2Kb indicated that all of the variants had lost one or more alloantigenic determinants (monoclonal antibody binding sites). For these variants, the pattern of monoclonal antibody loss of recognition suggested that antibody defined alloantigenic determinants appear to be discretely localized to a single domain, either the alpha 1 or the alpha 2 domain, of the H-2Kb molecule. In contrast, CTL recognition of the Kb molecule of these variants depends on involvement of both alpha 1 and alpha 2 domains as shown in the companion paper.     

Evidence that multiple residues on both the alpha-helices of the class I MHC molecule are simultaneously recognized by the T cell receptor

Single amino acid substitutions at nine different positions on the H-2Kb molecules from in vitro-mutagenized, immunologically altered, somatic cell variants were correlated with their patterns of recognition by monoclonal antibodies (MAbs) and allogeneic cytotoxic T lymphocyte (CTL) clones. While MAbs were found to detect spatially discrete, domain-specific sites, CTLs interacted simultaneously with multiple residues on the alpha 1 and alpha 2 domains of the Kb molecule. The computer graphic three-dimensional Kb model structure showed that, of the seven CTL-specific residues analyzed, six residues were located on the alpha-helical regions of the two domains. Every CTL clone was found to interact with a distinct pattern of residues composed of a specific subset of the CTL-specific residues.     

Mapping of epitopes recognized by alloreactive cytotoxic T lymphocytes using inhibition by MHC peptides

To identify epitopes recognized by alloreactive CTL we have examined H-2Kb-specific CTL for their recognition of synthetic peptides with sequences derived from the native Kb class I molecule. Consecutive nested peptides spanning the immunogenic alpha 1 and alpha 2 domains of Kb were tested for their capacity to inhibit CTL clones in their recognition of cells expressing the native Kb molecule. Inhibition by these peptides was found to be an extremely rare event. One peptide (Kb.111-122) did inhibit recognition by one particular CTL clone, clone 13. Upon further investigation it was observed that clone 13 also recognized peptide Kb.111-122 when presented in the context of the syngeneic MHC molecule, Kd. Considering that residues 111 to 122 are located at the base of the antigen groove, and clone 13 is able to recognize Kb.111-122 when presented by syngeneic target cells, we suggest that inhibition of this CTL clone may be due to MHC restricted, self-presentation of peptide rather than to direct binding of free peptide to the TCR. Taken together, these results suggest inhibition of allospecific CTL by MHC peptides is a rare event at least for Kb recognition. Furthermore, they demonstrate the need for caution when interpreting inhibition by peptide as evidence for recognition by the TCR of the corresponding region on the native 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.     

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

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

Cytotoxic T lymphocyte 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.     

Clonal T lymphocyte recognition of the fine structure of the HLA-A2 molecule

A human alloimmune cytotoxic T lymphocyte (CTL) clone (4E4) was generated against the HLA-A2 molecule. Lysis of 51Cr-labeled HLA-A2 target cells was blocked by monoclonal antibodies (mAb), including mAb PA2.1 (anti-HLA-A2), mAb BB7.2 (anti-HLA-A2), mAb 4B (anti-HLA-A2-plus-A28), mAb MA2.1 (anti-HLA-A2-plus-B17), and mAb W6/32 (anti-HLA-A,B,C), which are directed against different serologic epitopes on the HLA-A2 molecule. However, HLA-A2 mutant lines lacking the serologic epitope recognized by mAb BB7.2 (anti-HLA-A2) were efficiently lysed by CTL 4E4. Thus, although mAb may block cytolysis, the HLA-A2 epitope recognized the 4E4 CTL clone is distinct from the HLA-A2-specific epitope recognized by serologic reagents. Moreover, analysis of HLA-A2 population variants revealed that only the predominant HLA-A2.1 subtype molecule was recognized by CTL 4E4. No cross-reactivity on other, biochemically related HLA-A2 population subtypes was observed, including HLA-A2.2 cells (Hill, CVE, ZYL, M7), HLA-A2.3 cells (TENJ, DK1), or HLA-A2.4 cells (CLA, KNE). This CTL clone appears to recognize a single epitope and, like monoclonal antibody counterparts, can be used to discriminate among immunogenic cellular and serologic epitopes on closely related HLA-A2 molecules. On the basis of the known sequence changes in mutant and subtype HLA-A2 molecules, it appears that the sequence spanning residues 147 to 157 may be critical for cellular recognition of this Class I MHC molecule.     

The alpha-3 domain of class I MHC proteins influences cytotoxic T lymphocyte recognition of antigenic determinants located within the alpha-1 and alpha-2 domains

An interspecies class I MHC molecule, Kb1+2/A2 (in which the alpha-1 and alpha-2 domains of the H-2Kb molecule have been linked to the alpha-3, transmembrane and intracytoplasmic domains of the HLA-A2 molecule) has been expressed on both human and mouse target cells by gene transfer. Maintenance of serologic determinants has been demonstrated. However, decreased lysis by allospecific CTL populations of cell lines that expressed a hybrid interspecies class I molecule, Kb1+2/A2, as compared with lines that expressed the native Ag, H-2Kb, has been described. An analysis with a limited panel of H-2Kb allospecific clones demonstrated that not all H-2Kb-specific CTL can lyse cells that express Kb1+2/A2 Ag. This suggested that the reduction of lysis by CTL populations was due to the loss of specific alloreactive clones in the population. Each clone used in this study was then defined as having high or low affinity characteristics. No correlation between the affinity of the CTL and the ability to recognize the interspecies hybrid molecule could be shown. Rather, these data suggest that antigenic determinants that are located within the polymorphic domains, alpha-1 and alpha-2, may be conformationally influenced by the alpha-3 domain.     

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.     

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.     

Somatic cell variants of H-2k: b: A point mutation in the first extracellular domain results in altered immune recognition

A cell-surface-associated variant H-2K product was expressed by an Abelson virus-induced pre-B-cell line after chemical mutagenesis with ethyl methane sulfonate. The variant cell line (R8.313) was previously demonstrated to have altered allodeterminants in K^b as demonstrated by both K^b-specific monoclonal antibody binding and alloreactive cytotoxic T lymphocyte (CTL) cytolysis. The mutant H-2K ^b gene from R8.313 was cloned and characterized in detail. DNA sequence analysis of the region of the gene corresponding to the three extracellular domains identified a single point mutation resulting in a leucine-to-phenylalanine substitution at amino acid residue 82. The site of mutation within the 1 domain was confirmed by oligonucleotide hybridization analysis. Mouse L-cell fibroblasts transfected with the mutant gene were recognized with the same monoclonal antibody binding and CTL lytic pattern as the R8.313 cell line, confirming that the altered phenotype of the mutant cell line was due to a point mutation in the H-2K ^b gene. These data further extend the hypothesis that the region of amino acid residues 70–90 in the 1 domain is important in the formation of both antibody and CTL-defined recognition structures on major histocompatibility complex class I molecules.     

An H-2Ld hybrid molecule with a Qa-2 alpha-3 domain and phosphatidyl-inositol anchor is not recognized by H-2Ld-specific cytotoxic T lymphocytes

Ld/Q7d, a hybrid molecule consisting of alpha-1 and alpha-2 domains from H-2Ld and alpha-3 and carboxy-end components from Q7d, was expressed on the surface of CRL-3A rat liver cells. This molecule retained serologic H-2Ld epitopes. The Ag is attached to the cell membrane through a phosphatidyl-inositol linkage, characteristic of Qa-2 molecules. Both bulk cultured and cloned H-2Ld alloreactive CTL as well as H-2Ld restricted vesicular stomatitis virus-specific CTL lyse CRL-3A cells which express H-2Ld but show little or no lytic activity on cells which express the Ld/Q7d hybrid. These cells also fail to act as cold target competitors for alloreactive anti-H-2Ld CTL. However, cells expressing Ld/Q7d are not resistant to CTL mediated lysis because they can be killed in the presence of lectin. These data indicate that recognition of polymorphic class I CTL epitopes in the alpha-1 and alpha-2 domains are influenced by the structure of the carboxy-end of the molecule.     

Immunoselection of structural H-2Kb variants: Use of cloned cytolytic T cells to select for loss of a CTL-defined allodeterminant

Functional studies concerning the unique interaction between class I H-2 allodeterminants and cytolytic T lymphocyte (CTL) antigen receptors have benefitted from the development of H-2K^b mutant mouse strains and somatic H-2 variants selected with monoclonal antibody. Here, we describe the development of a novel approach to immunoselection of somatic H-2K^b variants employing a K^b-specific CTL clone as the negative selective agent. The rationale for this method is that the use of an alloreactive CTL clone as the selective agent should enable us to detect the emergence of structural K^b variants based on their loss of the relevant CTL-defined allodeterminant. Thus, these structural variants are well suited to an in-depth analysis of the functional relationship between H-2 antigens and receptor recognition by CTL. Using this approach, we successfully isolated two types of structural K^b variants, as well as numerous Kb-loss variants. The functional studies described in this paper indicate that these structural variants exhibit alterations in expression of both CTL-defined and serologically defined H-2K^b allodeterminants. The structural characterization of such variants should enable us to identify the precise amino acid residues responsible for the creation of the relevant CTL-defined K^b allodeterminants.     

Recognition of an MHC class I-restricted antigenic peptide can be modulated by para-substitution of its buried tyrosine residues in a TCR-specific manner

Conformational dependence of TCR contact residues of the H-2Kb molecule on the two buried tyrosine side chains of the vesicular stomatitis virus (VSV)-8 peptide was investigated by systematic substitutions of the tyrosines with phenylalanine, p-fluorophenylalanine (pFF), or p-bromophenylalanine (pBrF). The results of peptide competition CTL assays revealed that all of the peptide variants, except for the pBrF analogues, had near-native binding to the H-2Kb molecule. Epitope-mapped anti-H-2Kb mAbs detected conformational differences among H-2Kb molecules stabilized with these VSV-8 variants on RMA-S cells. Selective recognition of the VSV-8 analogues was displayed by a panel of three H-2Kb-restricted, anti-VSV-8 TCRs. Thus, these substitutions result in an antigenically significant conformational change of the MHC molecular surface structure at both C and D pockets, and the effect of this change on cognate T cell recognition is dependent on the TCR structure. Our results confirm that the structure of buried peptide side chains can determine the surface conformation of the MHC molecule and demonstrate that even a very subtle structural nuance of the buried side chain can be incorporated into the surface conformation of the MHC molecule. The ability of buried residues to modulate this molecular surface augments the number of residues on the MHC-peptide complex that can be recognized as "foreign" by the CD8+ T cell repertoire and allows for a higher level of antigenic discrimination. This may be an important mechanism to expand the total number of TCR specificities that can respond to a single peptide determinant.     

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.     

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.     

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.     

Analysis of somatic cell H-2 variants to define the structural requirements for class I antigen expression

We have taken the approach of producing somatic cell variants with altered H-2 products to study the structural requirements for cell surface expression of class I histocompatibility molecules. H-2 antigen variants generated by chemical mutagenesis of a cell line expressing the H-2b haplotype were first selected with alloantisera for their loss of H-2Kb expression, and then were analyzed by radioimmunoassay for the appearance of intracellular Kb antigen. For one such variant (69.9.15), whereas the H-2Kb antigen was absent from the cell surface as assayed by antibody-mediated complement-dependent cytotoxicity, an H-2Kb molecule was detected within the cell lysate as confirmed by direct immune precipitation with Kb-specific monoclonal antibodies. The product had an altered antigenic phenotype, since it reacted with only two anti-Kb monoclonal antibodies (Y-3 and EH-144) and not with a third (5F1.2). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis identified the beta 2 microglobulin-associated, intracellular H-2Kb heavy chain to be slightly smaller in Mr than the H-2Kb of the parental cell line. Hybridization analysis revealed the Kb gene from the variant to be without gross alterations, and furthermore, identified a Kb mRNA species that was identical in size to wild-type Kb mRNA. Because complementation was not observed after somatic cell fusion of variant cells with BALB/c splenocytes, it appeared that the alteration in Kb expression was due to a cis-acting defect. In addition, DNA-mediated gene transfer of the wild-type Kb gene into the variant cell line resulted in expression of the Kb antigen on the cell surface, thus confirming that the defect in expression of the mutant Kb product was not due to other factors in the 69.9.15 cell line. Such findings are consistent with the conclusion that stable H-2Kb surface-negative somatic variants can arise due to limited alterations in the Kb gene, resulting in the synthesis of a class I molecule that is expressed only as an intracellular product.     

Inhibition of cytolytic T lymphocyte clones reactive with Moloney leukemia virus-associated antigens by monoclonal antibodies: a direct approach to the study of H-2 restriction

H-2 restriction in cytolytic T lymphocyte (CTL)-mediated lysis of syngeneic murine Moloney leukemia virus (MoLV)-induced tumor cells was studied at the clonal level by testing the inhibitory effect of monoclonal anti-H-2 antibodies on the lytic interaction between CTL clones and target cells. Large numbers of MoLV-specific CTL clones were generated by placing limiting numbers of C57BL/6 regressor (responder) spleen cells into micro-mixed leukocyte-tumor cell cultures. The clonal CTL populations thus obtained were split into 5 aliquots and tested for lytic activity in the presence (or absence) of 1 of 3 monoclonal antibodies or of an anti-whole H-2b haplotype antiserum. Two of the monoclonal antibodies were directed against H-2Db and one against H-2Kb determinants. Specificity of these reagents had been verified by demonstrating inhibition of lysis by CTL populations directed against H-2Db and H-2Kb alloantigens. In 44 of a total of 51 clones tested, results showed selective inhibition by the anti-H-2Db (and the anti-whole haplotype) reagents, and lack of inhibition by the anti-H-2Kb antibody., Of the remaining 7 clones, none was inhibited by the anti-H-2Db antibody, and 3 were inhibited by the anti-whole haplotype antiserum. These studies show that the recognition of MoLV-associated antigens by the majority of CTL clones was restricted to the H-2Db region, and that there exists limited heterogeneity in the H-2 restriction of such clones.     

Dissociation of H-2 recognition by antibody and cytotoxic T cells of a cloned murine leukemia cell line

The differential expression of H-2 specificities recognized by antibody and by cytotoxic T lymphocytes (CTL) has been studied using a clone (FY7) of the C57BL/6 leukemia cell line FBL-3 (H-2b/H-2b). Unlike C57BL/10 spleen cells, EL-4 lymphoma cells and Y57-2C leukemia cells (all H-2b/H-2b), FY7 failed to induce the primary in vitro generation of anti-H-2b CTL by (B10.A x A)F1 (H-2a/H-2a) or B10.D2 x BALB/c)F1 (H-2d/H-2d) responder spleen cells. In addition, FY7 was not lysed by, and did not competitively inhibit anti-H-2b CTL. Quantitative absorption tests with H-2Kb and H-2Db antisera revealed that FY7 expressed these antigens in quantitatively similar amounts to EL-4. The H-2Kb product of FY7 appeared to be identical with that of C57BL/10 spleen cells both in apparent molecular weight and isoelectric point. Yet FY7 failed to inhibit anti-H-2Kb CTL competitively in a cold target inhibition assay. Possible mechanisms are discussed for the lack of T-lymphocyte recognition of the H-2Kb-gene product expressed by FY7.