Isolation of a human major histocompatibility complex class I gene encoding a nonubiquitous molecule expressed on activated lymphocytes.

The human major histocompatibility complex is a multigene family containing at least 20 class I genes. Included within this family are the loci encoding the highly polymorphic HLA-A, -B, and -C antigens present at the surface of most nucleated cells. The large number of genes detected with class I probes by Southern blot analysis and the existence of serological reagents defining nonubiquitous, non-HLA-A,B,C class I antigens suggest that products other than HLA-A,B,C antigens are encoded within the class I gene family. These products might be the human counterparts of the murine Qa and TL antigens. In order to identify non-HLA-A,B,C genes, we have developed a probe, JF11, located in noncoding regions flanking the HLA-A locus. This probe detects only a limited number of class I genes and does not detect HLA-A,B,C-associated restriction fragments on Southern blots. This probe was used to screen a human cosmid library. Some of the cosmids isolated with this probe were then transferred into mouse fibroblasts expressing human beta 2-microglobulin. One of the transfectants specifically reacts with one alloantiserum (HA2) that detects HLA class I molecules specific to HLA-A2-positive, phytohemagglutinin-activated T cells and not found on resting T or B cells. Data presented in this paper provide evidence for the isolation and expression of a class I gene encoding a nonubiquitous class I antigen that could be a human analogue of the murine Qa antigens.

New methods for detection of HLA genes polymorphism useful for associated diseases studies.

We have studied in 22 informative families typed for HLA the segregation of DNA restriction fragments obtained with five restriction enzymes and hybridized with one class I and three class II probes. Most of the fragments correlate with serologically defined specificities. Many fragments can be grouped in clusters, whose genetic significance is discussed. The RFLP distribution in patients with insulin-dependent diabetes mellitus, multiple sclerosis or narcolepsy, three diseases known to be associated with some HLA-DR specificities, has been also studied. Many fragments allow to distinguish between patients and HLA-DR matched controls. Hybridization of genomic DNA with synthetic oligomers will refine moreover the understanding of the HLA system polymorphism.

Specific lysis of murine cells expressing HLA molecules by allospecific human and murine H-2-restricted anti-HLA T killer lymphocytes.

The lysis by human and murine anti-HLA cytolytic T lymphocytes (CTL) of murine cells expressing class I HLA molecule after gene transfection has been studied using two different murine cells: LMTK- and P815-HTR-TK-. Weak but significant HLA-A11-specific lysis was found occasionally with human CTL on the HLA-A11+ L cells. On the contrary, P815-A11 or P815-A2 cells were lysed strongly and specifically by HLA-A11 or HLA-A2-specific human CTL. The T8+T4- phenotype of the effector cells was confirmed and the reaction was inhibited by anti-HLA class I monoclonal antibodies. Despite their higher sensitivity to human CTL, the P815-HLA+ cells did not express higher levels of HLA antigens than L cells, and the presence or the absence of human beta 2 microglobulin was irrelevant. Anti-human LFA-1 antibodies abrogated the lysis of P815-A11+ cells showing that the LFA-1 receptor which is apparently lacking on the L cell surface was on the contrary expressed on P815 cells. On the other hand, murine anti-HLA CTL have been prepared by immunizing mice against syngeneic HLA-A11+ L cells. They lysed very efficiently and specifically these cells, but appeared completely devoid of activity against human HLA-A11 target cells. This barrier was apparently due to the H-2 restriction of these H-2k anti-HLA murine CTL, as shown by their inability to lyse allogeneic H-2d cells expressing HLA-A11, and by the blocking of their activity by anti H-2k antibodies. By contrast, xenogeneic anti-HLA CTL obtained by immunizing murine lymphocytes against human cells lysed both human and murine HLA+ cells but they reacted with a monomorphic epitope of the HLA molecule in a nonrestricted way. These results show that human cells lyse very efficiently P815 murine cells expressing HLA class I antigens; the higher sensitivity of P815 cells compared to L cells is probably due to the presence of a LFA-1 receptor on these cells; a class I molecule of human origin can be seen as an H-2-restricted minor histocompatibility antigen in another species.

Serological expression after sequential double transfection with purified HLA-A11 gene of mouse fibroblasts carrying human beta-2 microglobulin.

A genomic cosmid library constructed from DNA from a genotyped individual (JF = HLA-A11, Cw-, B38/A26, Cw7, B51) was screened for clones containing class I histocompatibility genes. Among these clones, one was found to carry a 4.8 kb Hind III fragment which is highly correlated with HLA-A11. This clone was used to transfect LMTK+ cultured mouse fibroblast transformants expressing human beta-2 microglobulin. The human beta-2 microglobulin heavy chain-associated determinant was positively detected by the M18 monoclonal antibody. HLA-A11 expression on these doubly transformed cells was specifically demonstrated by complement-dependent cytotoxicity with HLA-A11 + A3-specific but not with HLA-A3-specific monoclonal antibodies. Absorption studies with human alloantisera confirmed the presence on these cells of HLA-A11 determinants and of cross-reacting determinants which absorbed anti-HLA-A1 and -A3 alloantisera. The JF5-J27 transfected cell expressed both heavy and light chains of human class I histocompatibility genes.

Analysis of HLA class I genes with restriction endonuclease fragments: implications for polymorphism of the human major histocompatibility complex.

Cellular DNA from HLA-typed individuals was digested with the restriction endonucleases HindIII, EcoRV, and EcoRI. The separated restriction endonuclease fragments were hybridized with a HLA class I cDNA probe by using the Southern transfer technique. Digestion of cellular DNA with HindIII generated 22 restriction endonuclease fragments, 11 of which showed polymorphism for presence or absence in a population sample. With EcoRV, 13 fragments were identified; 6 showed polymorphism. EcoRI generated 11 fragments, of which 1 was polymorphic. Of these 18 polymorphic fragments generated by the three restriction endonucleases, each of 5 was found to be positively associated with one allele of the HLA-A or -B allelic series (HLA-Aw24, -B8, -B15, -Bw35, and -B40). One fragment was positively associated with two HLA-A series alleles (HLA-A1 and -A11). Another fragment was positively associated with five HLA-B series alleles (HLA-B5, -B7, -B14, -Bw16, and -Bw35) and one fragment was positively associated with alleles at two loci (HLA-B14 and -Cw5). The serologically defined allele HLA-Aw24 was associated with two polymorphic fragments, one association showing a positive correlation and the other a negative correlation. Each informative family studied thus far has shown segregation of the restriction fragment with the associated serologically defined allele. The fragments associated with serologically defined alleles occurred in the population sample studied at low or moderate frequencies. The remaining polymorphic fragments occur at high frequency, suggesting that class I genes not serologically detected show less polymorphism than serologically defined class I genes.