The melanoma cell line FO-1 does not express HLA class I antigens and does not acquire them on the cell surface after incubation with IFN--y. Immunochemical studies showed that FO-1 cells synthesize HLA class I heavy chain, but do not synthesize t#2-microglobulin (02-it). The latter abnormality is associated with lack of 82-mRNA which remains undetectable in FO-1 cells incubated with IFN-'y. The defect was identified as a genetic lesion in the B2m gene, since DNA hybridization analysis detected a deletion of the first exon ofthe 5'-flanking region, and of a segment of the first intron of the B2m gene. HLA class I antigen expression was reconstituted on melanoma cells FO-1 after transfection with the wild-type mouse B2m gene, thereby confirming the abnormality of the endogenous B2m gene.The defect identified in FO-1 cells is distinct from that underlying the lack of HLA class I antigen expression by lymphoblastoid cells Daudi, but is remarkably similar to that causing lack of H-2 class I antigen expression by mouse lymphoblastoid cells R1 (TL-). These results suggest that genetic recombination in the 5' region of the B2m gene is a recurrent mechanism in B2m gene defects. In addition to contributing to our understanding of molecular abnormalities in HLA class I antigen expression by melanoma cells, FO-1 cells represent a useful model for analyzing the role of HLA class I antigens in the biology of melanoma cells and in their interaction with cells of the immune system. (J. Clin. Invest. 1991. 87:284-292.)

No abstract

The class-I mutants have provided a model system for understanding the generation of diversity of the genes encoding the histocompatibility molecules K, D, and L, and the relationship of their structure to function. The complex nature of the alterations found in Kb molecules from mutant mice has been documented at the nucleic acid level for eight mutants. The clustered changes in the mutant genes are consistent with the hypothesis that genetic recombination between class-I genes generates the Kb mutants. Techniques using synthetic oligonucleotide probes to mutant DNA sequence demonstrated that other class-I genes were available as donors for interaction with the Kb gene to produce the mutations. Intriguingly, donor genes found in the K region (K1) and the D region (Db), as well as the Qa regions (Q4, Q10), were capable of the interactions. The amount of genetic transfer to Kb from other class-I donor genes may range from a potential minimum of 5 nucleotides to a potential maximum of 95 nucleotides. Genealogical analysis of several bm mutants has further indicated that at least some, if not all, of the gene interaction events generating Kb mutations occurred during mitotic amplification of the germ cells. Genetic recombination among class-I genes occurring in nature to the extent observed for the Kbm mutants could readily generate mosaic transplantation genes containing sequences derived from other class-I genes. Thus, it seems likely that genetic interaction plays a major role in the diversification and ongoing evolution of the MHC. The localization of altered amino acids in the in vivo mutant Kb molecules has directed our attention to recognition regions on the Kb product that play a major role in determining alloreactivity and H-2 associative recognition. The replacement of one or a few amino acids in either of the postulated recognition regions located in the alpha 1 domain (residues 70-90) or alpha 2 domain (residues 150-180) can have marked effects on biological function. While the majority of monoclonal antibodies recognize epitopes in one or the other recognition region, CTL recognize determinants dependent on the apparent interaction of amino acids located in both regions. These overall conclusions are supported to a large extent by studies on mutants derived from several sources, i.e. spontaneous mutants, mutagen-induced somatic variants, and products of hybrid H-2 genes. Studies of in vitro variants can provide a more refined approach for analysis of structure-function relationships through the introduction of minimal biochemical changes.(ABSTRACT TRUNCATED AT 400 WORDS)

Kbm9 sequence is identical to that of another independently arising MHC mutant gene, Kb". As both the Kbm9 and Kb" genes were generated by recombination between the Kb and Q4 genes, our data indicate that the identical genetic interactions have occurred at least twice. The relatively large extent of identity between Q4 and Kb may be responsible for frequent recombination between the two genes. The parents of the original bm9 mutant mice had five identical mutant offspring, which can be explained by mitotic recombination in the germ cells, producing gonadal mosaicism in the C57BL/6 mother.Thus, mitotic recombination, and not meiotic recombination, appears to be responsible for the formation of at least some of the Kb mutants. Such a mechanism probably plays a major role in the generation of diversity in the major histocompatibility complex.

Phenotypic analysis of bone marrow cells from IL-7 knockout (KO) mice revealed that B cell development is blocked precisely at the transition between pro-B cells and pre-B cells. In contrast, the generation of pre-pro-B cells and pro-B cells appeared to be normal, as judged by total cell numbers, proliferative indexes, D-JH and V-DJH gene rearrangements, and mRNA for recombinase-activating gene-1 (RAG-1), RAG-2, TdT, Igμ, λ5, and VpreB. However, upon closer inspection, several abnormalities in pro-B cell development were identified that could be corrected by injection of rIL-7 in vivo. These included the absence of the subset of late pro-B cells that initiates cμ expression for pre-B cell Ag receptor (BCR) formation, and the failure of pro-B cells to up-regulate TdT and the IL-7Rα (but not the common γ-chain) chain. Similar defects were present in common γ-chain and Jak3 KO mice, but not in λ5 or (excluding cytoplasmic Ig μ heavy chain (cμ)) RAG-1 KO mice, all of which also arrest at the late pro-B cell stage. Consequently, up-regulation of TdT and IL-7Rα expression requires signaling through the high affinity IL-7R, but does not require cμ expression or a functional pre-BCR. Taken together, these results suggest that IL-7 and its receptor complex are essential for 1) up-regulating the expression of TdT and IL-7Rα, 2) initiating the production of cμ, and 3) promoting the formation of a functional pre-BCR in/on pro-B cells. These key events, in turn, appear to be prerequisite both for differentiation of pro-B cells to pre-B cells and for proliferation of these cell subsets upon continued stimulation with IL-7.