Richard A. Zeff

The role of beta-2 microglobulin in temperature-sensitive and interferon-γ-induced exocytosis of HLA class I molecules

The passage of MHC class I heavy chains through the exocytic pathway is promoted by association with β2 microglobulin (β2 m). In order to analyze the structural basis of this phenomenon, processing and cell surface expression of HLA class I molecules have been investigated in the β2m null human melanoma cell line FO-1 transfected with either the human or mouse β2 m genes. These natural structural variants of β2m display 30% amino acid sequence divergence. In comparison with a human β2m transfectant of the FO-1 cell line (designated FO-1H), FO-1 cells transfected with the mouse β2m gene (FO-1C) express HLA class I molecules that are processed with grossly altered kinetics and are present on the cell surface at reduced levels. The suboptimal expression of HLA class I heavy chains encoded by FO-1C cells reflects a defect in heavy chain stability since cell surface expression of HLA class I antigens was increased following incubation at 30°C. The increased cell surface expression paralleled accelerated processing of HLA class I heavy chains by FO-1C cells. In contrast, no induction in either cell surface expression or processing of HLA class I heavy chains was observed for the β2m-negative FO-1 parent cell line, which remained HLA class I antigen null when cultured at 30°C, or the FO-1H human β2m transfectant, which expressed equivalent levels of HLA class I antigens on the cell surface at 37°C and 30°C. Further up-regulation of the temperature-sensitive induction of HLA class I antigen expression was accomplished by treatment of the FO-1C transfectant with interferon-°; this latter effect appears to be active at a posttranscriptional step for FO-1 cells since IFN-γ was not as potent a transcriptional activator at 30°C as it was at 37°C. These results indicate that HLA class I heavy chains expressed by FO-1C cells are subject to temperature-sensitive and cytokine-inducible stabilization that increases their affinity for the structural variant of β2m and promotes exocytosis of the HLA class I heterodimer to the cell surface. Furthermore, β2m non-conformed MHC class I heavy chains undergo stabilization that is not associated with enhanced cell surface expression, indicating that the exocytosis of putative “empty” HLA class I antigens is a process dependent upon association with β2m.

Failure of cell surface expression of a class I major histocompatibility antigen caused by somatic point mutation.

The ability to down-regulate major histocompatibility complex class I antigen expression on allografts prior to transplantation would be expected to improve their survival in immunocompetent recipients. In order to identify genetic mechanisms that mediate attenuation of MHC class I antigen expression, we have begun characterizing H-2Kb surface null somatic cell variants derived from an H-2 heterozygous tumor cell line (H-2bxH-2d). These variants have sustained a modification in cell surface MHC phenotype, as evidenced by their failure to be recognized by both anti-H-2Kb antibodies and cytotoxic T lymphocytes. The mutant phenotype for one such variant (designated 69.9.15) was marked by the expression of abundant H-2Kb mRNA and immunoprecipitable H-2Kb protein in cell lysates. The failure in cell surface expression of the H-2Kb antigen was caused by a single base change (G to A transition) in exon 3, encoding the second external domain (α2) of the H-2Kb molecule. The mutation resulted in the substitution of Tyr for Cys at amino acid position 164, thereby disrupting an intrachain disulfide linkage formed between Cys 101 and 164. In contrast to the wild-type H-2Kb gene, DNA-mediated transfer of the mutant H-2Kb gene into mouse L cell fibroblasts failed to result in cell surface expression of the H-2Kb antigen, although both the wild-type and mutant genes were transcribed to equivalent levels. These data indicate that a genetic event as limited as somatic point mutation can abrogate expression of a MHC class I antigen and provide support for the hypothesis that protein folding plays an important role in the cell surface expression of MHC class I molecules.

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 Kb as demonstrated by both Kb-specific monoclonal antibody binding and alloreactive cytotoxic T lymphocyte (CTL) cytolysis. The mutant H-2Kb 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 α1 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-2Kb gene. These data further extend the hypothesis that the region of amino acid residues 70–90 in the α1 domain is important in the formation of both antibody and CTL-defined recognition structures on major histocompatibility complex class I molecules.

MHC class I heavy chain-dependent expression of discontinuous antigenic epitopes on beta 2-microglobulinb is inducible with peptide-ligand.

Previously, we reported that expression of the murine beta 2-microglobulinb (beta 2mb) antigenic epitopes defined by the mAb S19.8 and 23 (SJL [beta 2ma] anti-B10.S beta 2mb]) was dependent upon association of beta 2m with MHC class I heavy chains. We have further explored the antigenic properties of beta 2m under circumstances requiring the induction of MHC class I surface expression with heavy chain-specific peptide-ligand. For the RMA-S cell line, which is class I surface null due to a defect in the TAP-2 peptide transporter, treatment with the H-2Kb-specific vesicular stomatitis virus-derived N p52-59 peptide resulted in the cell surface expression of the epitopes defined by the anti-H-2Kb mAb Y-3, as well as equally strong expression of the epitopes defined by the anti-beta 2mb mAb S19.8 and 23. Similarly, the FLU-NP p366-374 peptide induced H-2Db on the surface of RMA-S cells as determined by cytofluorometry with the mAb MKQ8; however, expression of the epitope defined by S19.8 was only partially recovered and no reactivity was observed for mAb 23. That the H-2Db heavy chain was assembled with beta 2mb on the cell surface was established from immunoprecipitation experiments with 125I-surface-radiolabeled RMA-S cells treated with FLU-NP p366-374; MKQ8 immunoprecipitated prominent heavy chain and beta 2m bands, whereas S19.8 and 23 isolated a weak beta 2m band (12-15% of that co-immunoprecipitated with MKQ8). These results are consistent with the observation that human beta 2m-deficient cells (designated FO-1) transfected with the B2mb allele were induced, in combination with the endogenous HLA class I heavy chains, to express the epitope defined by S19.8, but not mAb 23, whereas both were expressed when contransfection was performed with the H-2Kb gene. That the determinants recognized by S19.8 and 23 were formed by a discontinuous cluster of amino acids within beta 2m was established from experiments demonstrating that H-2Kb heavy chain assembled with a chimeric beta 2m molecule (comprising human beta 2m from 1-69 and mouse beta 2m from amino acid 70-99, including the polymorphic residue Ala 85) did not lead to expression of the S19.8 and 23 epitopes. The results of this study provide evidence that heavy chain polymorphism can affect the antigenic properties of beta 2m and offer insight into the basis by which CTL may react against beta 2mb when assembled with the H-2Kb molecule.

Failure in expression of structurally altered (CYS164-->TYR) H-2Kb molecules is mitigated with high affinity peptide-ligand.

A C164Y somatic mutation in the H-2Kh class I molecule causes a disruption of the α2 domain disulfide bond and results in a loss of H-2Kb cell surface expression by the 69.9.15 cell line. In vitro culture of the somatic cell variant at 30°C induced weak, but reproducible, expression of the H-2Kb mutant molecule on the cell surface, which suggests that a temperaturesensitive mutation was contributing to the H-2Kb null phenotype. Based on the inherent structural instability of the mutant H-2Kb molecules synthesized by 69.9.15 cells, we sought to determine the ability of high affinity peptide-ligand to counteract the null expression of H-2Kb. Treatment of 69.9.15 cells was performed with acid-eluted cell-derived peptides, as well as synthetic H-2Kb-restricted peptides, ovalbumin (OVA) p257–264 (YSIINFEKL), and vesicular stomatitis virus-nuclear protein p52–59 (RGYVYQGL). Whereas the endogenous and vesicular stomatitis virus peptides were ineffective at inducing H-2Kb expression at either 37°C or 30°C, treatment with the OVA peptide at 30°C gave rise to dose-dependent enhancement in H-2Kb expression, an effect that was independent of exogenous sources of bovine β2-microglobulin at the time of peptide treatment. By comparison, expression of H-2Kb remained unaltered when cells were treated with the OVA peptide at 37°C, consistent with the temperature-sensitive expression of the mutant molecules. Decay of H-2Kb from the cell surface was similar for both 69.9.15 and RMA-S cells, an indication that binding of OVA p257–264 provided the same level of stability for class I molecules with either a cis(69.9.15) or trans-acting (RMA-S) defect in heavy chain transport. These data provide novel evidence that transport-defective MHC class I molecules, similar in nature to those encoded by class I genes isolated from human genomic libraries, i.e., the 12.4 pseudogene with a polymorphism at amino acid position 164 (C → F), are subject to high affinity peptide-induced stabilization which reverses the class I null phenotype.

Diversification of H-2 Genes through Recombination: A Study of Kb Mutants

H-2 genes, members of the MHC class I multigene family, exhibit extreme allelic sequence diversity. Spontaneous, histogenic mutants of the H-2Kb gene have been found to arise at a relatively high frequency. Sequence analysis of the Kb mutants indicate that they consist of clustered, multiple nucleotide substitutions that are identical to nucleotide sequences in other class I genes. Such characteristics suggest that the mutant Kb genes are generated by recombination of the Kb gene with other class I genes. Oligonucleotide probes were utilized to detect donor genes in the K, D, and Qa regions of the MHC with the exact sequence substituted into the mutant Kb genes. Recombination between Kb and donor genes involves short stretches of DNA and can be explained by gene conversion or double crossovers. Genealogical analyses indicate that some of the Kb mutants were generated by mitotic recombination in germ cells. Thus, the Kb mutants serve as a model system to understand the generation of diversity in the MHC.

The Analysis of H-2 Mutants: Molecular Genetics and Structure/Function Relationships

The class I genes of the murine major histocompatibility complex are located in four regions (K, D, Qa, Tla) along chromosome 17. The K and D regions contain the genes encoding the classical H-2 transplantation antigens (K, D, L). Alleles of each of the K, D and L loci exhibit substantial sequence diversity (1–3). Further, H-2 loci are highly polymorphic, with over 100 alleles identified per locus (3). The high sequence diversity and polymorphism of H-2 genes are thought to play a role in the function of their products as antigen presenting molecules, thus enabling a population to respond to many different pathogens. In contrast, Qa and Tla region loci are much less polymorphic and alleles of each locus appear to be much more conserved (4,5). Qa and Tla gene products have a limited tissue distribution and their function is unknown.