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Nonhuman primates represent phylogenetic intermediates for studying the divergence of human and murine beta 2Ms. We report the nucleotide sequences of B2m cDNA clones from a baboon cell line, 26CB-1 (Papio hamadryas; primates: Cercopithecoidea), and a cotton-top tamarin cell line, 1605L (Saguinus oedipus; primates: Ceboidea). The baboon and tamarin B2m sequences indicate a very slow rate of B2m evolution in primates relative to that in murid rodents. Phenotypic evolution of beta 2M has also been very conservative in primates, with only 9-14 substitutions separating baboon or tamarin beta 2Ms from those of humans or orangutans. Analyses of silent and amino-acid-altering nucleotide substitutions provide evidence that negative selection has acted to limit variability in beta strands of primate beta 2Ms, while positive selection has promoted diversity in non-beta-strand regions of murine beta 2Ms. No evidence for the action of selection upon beta 2M residues that contact the class I heavy chain was found in primates or mice. The finding that different selective forces have operated upon primate and murine beta 2Ms suggests that beta 2M may have evolved to serve distinct functions in primates and mice.

A simple, sensitive ELISA that is performed in 96-well microtiter plates and that requires less than 90 minutes to complete was developed for HLA-DRB oligotyping. The second exon of HLA-DRB1 was amplified using an unlabeled forward primer and a biotinylated reverse primer and the PCR product was immobilized in avidin-coated wells. Subsequent treatment included exposure to 0.4 N NaOH to remove the nonbiotinylated sense strand, addition of a fluorescein-labeled oligonucleotide probe, one or more 5-minute stringency washes, addition of an alkaline-phosphatase-labeled anti-FL FAB, and then alkaline-phosphatase substrate and amplifier. An intense red-violet color developed within 15 minutes in positive wells and could be quantitated by OD readings at 490-495 nm. To control for stringency and to establish threshold OD values for positive reactions, biotin-labeled antisense oligos that were complementary to the probe or that differed by one or more bases were immobilized in wells in place of PCR products. The assay was sensitive to < 0.05 pmol (approximately 4 ng)/well and required only standard incubators and waterbaths and an optional microplate reader. All reagents were commercially available. The method should facilitate oligotyping of both class I and class II alleles and is adaptable for analysis of other polymorphic gene products.

Differential DNA heteroduplex patterns were used to investigate inheritance of HLA class II region genes in a family where a living related kidney transplant was under consideration. Serologic typing of the family members for HLA class I (HLA-A, B, and C) and class II (HLA-DR and DQ) alleles indicated that the patient (109) and one sibling (126) had inherited the same maternal and paternal HLA alleles. However, a strong reciprocal mixed lymphocyte response implied that these two individuals were not completely HLA identical. Serologic typing for HLA-DQ was confirmed by allele-specific oligonucleotide typing family members for HLA-DQ alpha and beta genes. To assess a possible nonidentical gene(s), DNA was amplified from all family members at the second exon of the DR beta, DQ alpha, DP alpha, and DP beta genes and the products were analyzed by DNA heteroduplex formation. This method showed that individuals 109 and 126 were identical at DR and DQ but differed at DP. This difference was confirmed by allele-specific oligonucleotide hybridization and indicated that 126 had inherited a recombinant maternal chromosome with a cross-over occurring in the region between the DQ beta and DP alpha genes. These data demonstrate the applicability of DNA heteroduplex patterns in establishing identity-nonidentity of alleles in the major histocompatibility complex.

Two monoclonal antibodies (691-13-17 and 37-7) can precipitate DR antigens from radioiodinated, detergent solubilized melanoma cells. However, after complete depletion of lysates with one antibody (691-13-17) alpha-like chains can be still be precipitated from some melanoma cells by the other antibody (37-7). Antibody 37-7 also precipitates an additional distinct antigen from SK-MEL 37 but not from any five other melanoma cell lines.

Monoclonal antibodies reactive with polymorphic epitopes of HLA-B7 were analyzed by direct and indirect cytotoxicity assays on established panels of HLA typed lymphocytes. This permitted further refinement of their specificity and the identification of various novel reactions. The topographic relationship of polymorphic epitopes on the surface of the B7 molecule was assessed with various serological assays using cell surface B7 or papain solubilized B7 as the antigenic target. These studies focused on monoclonal antibodies recognizing B27 and B7. The results, in combination with those of previously published studies, are used to provide a current assessment of the epitope map of HLA-B7 as defined with mouse monoclonal antibodies. This is compared to the results obtained with alloantisera.

The Con-A--activated suppressor function of lymphocytes from polyglandular failure (PGF) patients in human mixed lymphocyte culture was compared to a normal population. As a group, PGF patients were found to have decreased suppressor activity: 67% of normal for autologous suppression, 45% of normal for heterologous. However, lymphocytes from most PGF patients have neither an absolute lack of suppressor activity nor an absolute inability to respond to suppression. The marked variability of assayed suppression, depending on the combination of stimulators and responder cells tested, limit the utility of this assay in defining individuals with abnormal suppressor function. One patient's lymphocytes were unique in that although suppression of heterologous cells was normal, suppression of her own cells was defective. Defective suppressor function may be related to the susceptibility of these patients to multiple autoimmune diseases.

The indigenous population of the Blue Nile Province, Sudan, is an Arab-Negroid admixture, although some isolates of west African origin (Fallata) have begun to intermix with the indigenous population. Consanguineous marriages are common in these Muslim families. Members of 22 nuclear families were typed for HLA class I and II antigens using complement-dependent microcytotoxicity with Ninth International Workshop (9th WS) and local reagents. Considerable polymorphism was observed at each locus with a majority of the World Health Organization (WHO)-recognized alleles represented in the parental sample, albeit at low frequency. Seven parental haplotypes carried A locus alleles which were not identical to WHO-recognized specificities. All appeared to be Aw19-related specificities. Five B locus serologic variants were observed; all appeared distinct from WHO-recognized specificities. In one family we observed a new DR-DQ association; DR4 segregated with DQw2, rather than with DQw3. As has been observed for several other genetic systems in these tribes, considerable polymorphism was found for all class I and class II gene products in spite of a high level of consanguinity. Tribal admixture and/or a selective advantage in protecting the population against disease may account for this unexpectedly high level of heterozygosity.

Melanoma cell lines treated with or without interferon-gamma were tested for the presence of DR alpha mRNA and protein. The six lines examined fell into three general categories: two that expressed high levels of DR alpha mRNA and protein before and after interferon-gamma treatment, one that expressed very low levels before treatment with interferon-gamma, but was induced to express high levels after interferon-gamma treatment, and three that expressed very low levels before treatment, and were only slightly inducible after treatment with interferon-gamma. The presence of DR-alpha protein on the melanoma cell surface was always positively correlated with the presence of DR alpha mRNA in the cells. Furthermore, in the cell line that was interferon-gamma-inducible, the time at which DR alpha mRNA and protein appeared and the doses of interferon-gamma needed to induce this appearance were directly correlated. Methylation patterns of the DR alpha gene in these cell lines were also studied in order to determine whether the degree of DR alpha gene methylation among the lines correlated with expression of the gene. Digestion of DNA with the restriction enzyme MspI, which recognizes the sequence 5'CCGG3' and 5'CmCGG3', led to the appearance of a 3.1 kb band from all lines tested. Hpa II digestion, which recognizes 5'CCGG3', but not 5'CmCGG3', led to the appearance of 3.1, 4.4, and 6.7 kb bands in all lines tested except for DUMEL 8, which showed only the 3.1 kb band. Interestingly, DUMEL 8 expressed very low levels of DR alpha mRNA and protein before and after interferon-gamma treatment. We conclude that interferon-gamma has a regulatory effect on DR alpha genes of various melanoma cell lines to varying degrees. This may reflect an effect of interferon-gamma on certain subpopulations of melanocytes in vivo. Our data also indicate that partial methylation of the DR alpha gene does not inhibit its expression. Furthermore, interferon-gamma does not appear to induce expression of the DR gene by altering methylation patterns within the region recognized by our probe.

The HLA-B5,B35 cross-reacting group is a large and serologically complex antigen family which includes the World Health Organization-recognized specificities HLA-B5,B51,Bw52,B35,Bw53,B18,Bw70,Bw71, and Bw72. In addition, several variants of antigens in this cross-reacting group have been described in the past but have not yet gained official recognition. A genetic basis for the complexity and the protein and molecular bases of this highly cross-reactive and polymorphic cross-reacting group have yet to be established. The potential contributions of shared amino acid sequences, the occurrence of multiple epitopes on a single HLA-B molecule, and the presence of new HLA-C antigens have been difficult to resolve. To address this issue, we have carefully examined the serologic reactions of more than 900 allo- and monoclonal antibodies (Tenth International Workshop, Third Asia-Oceanic Workshop, and local reagents) versus lymphocytes from 92 individuals of diverse ethnic origin (North American Caucasians, North American blacks, Amerindians, Middle Eastern Caucasians), 84 of whom were informative for the HLA-B5,B35 cross-reacting group and related antigens. Our results demonstrate that the HLA-B5,B35 gene products share different combinations of distinct epitopes. We have constructed a model for the evolution of this cross-reacting group by assigning polarity to distinct diversification steps utilizing principles of maximum parsimony.

An HLA class II-positive melanoma cell line, DU-Mel 17, expressing both DR and DQ was shown to be contaminated with Mycoplasma hyorhinis. After treatment of the cell line with anti-mycoplasma drugs (a tetracycline derivative and a pleuromutilin), both DR and DQ expression on the cell surface, as well as DR alpha mRNA, were greatly decreased and the cells were rendered mycoplasma-free. Interestingly, drug-treated cells became resistant to geneticin, an unrelated drug. To test whether drug treatment or elimination of mycoplasma caused the abolition of class II expression, contaminated DU-Mel 17 cells were inoculated into peritoneal cavities of nude mice and later removed upon growth of a solid tumor. The cells were shown to be mycoplasma-free, and they expressed DR alpha mRNA, as well as DR and DQ proteins on their surfaces. Although it appears that the drugs were responsible for abating class II molecule expression in DU-Mel 17 cells, the identical drug-treatment regimen on a class II-positive, mycoplasma-free B cell line did not alter DR alpha mRNA or class II protein levels. Based on restriction fragment lengths, the DR alpha gene appeared to be grossly intact after drug treatment, and alterations in methylation patterns were not observed. These studies indicate that anti-mycoplasma treatment of DU-Mel 17 cells resulted in selection of a class II-negative, drug-resistant clone(s). Such treatment may result in a phenotype that is divergent from the parental cell line in one or more characteristics.

Two DNA probes isolated from the region 5' to the HLA-B gene are described. One of these, pCH6, detects a TaqI polymorphism which is correlated with serological alleles of HLA-B. Both probes are used to show a high level of DNA sequence homology between the 5' flanking region of HLA-B and HLA-C.

A situation can arise in D typing in which the HLA (A,B,C,D, or DR) specificities of the responders are known and the specificities of the HTC's are unknown. The most powerful and direct method of detecting association between the unknown stimulator (HTC) and any given HLA specificity is by comparing the observed double normalized values (DNV's) of individuals known to be positive for the specificity with the DNV's of negative individuals. This can be done by comparing the two groups with a Kolmogorov-Smirnov test (K-S test), an established statistical procedure for evaluating correlation between continuous variables, such as the DNV, and discrete variables (such as presence of D type). The application of the K-S test will generate as a "cutoff" value of point that maximizes the average of the frequencies of correct assignments in D positives and D negatives. We also propose an alternative method of computing the "r" value. We have analyzed 49 HTC's from the 8th International Workshop and present the association observed with the D and DR specificities.

The self-specificity of human influenza virus-immune cytotoxic T cells has been analyzed in order to identify the relationship between the self-determinants which they recognize and the serologically defined HLA-A and -B antigenic determinants. Virus-immune T cells were generated in vitro by culture of normal adult peripheral blood lymphocytes with A/HK influenza virus. Virus-immune effectors from HLA-A2 positive donors were tested on panels of virus-infected target cells from donors who were either HLA-mismatched or matched only for the HLA-A2 specificity. Virus-immune T cells from 11/11 A2-positive donors lysed all A2-matched virus-infected target cells (and no HLA-mismatched targets), except that each of these effector cell populations consistently failed to lyse the virus-infected target cells from one A2-positive donor (designated M7). Although the A2 antigen of donor M7 could also be distinguished from the A2 antigen of other donors by alloimmune cytotoxic T cells, no differences in the A2 antigen of donor M7 could be defined by extensive serological analyses. Results of isoelectric focusing of A2 molecules from three individuals plus M7 demonstrated that the M7 A2 heavy-polypeptide chain is structurally distinct. These results indicate that: 1) there is a strong but incomplete association between a self antigen recognized by virus-immune T cells and the serologically defined HLA-A2 specificity; and 2) there may be at least two structurally and functionally distinct epitopes on the same A2 molecule: one is the serologically defined HLA-A2 antigenic determinant; the other is the self determinant recognized by T cells on HLA-A2 molecules.

Spontaneously arising class switch variants of the Genox 3.53 hybridoma cell line were isolated. They secrete IgG2a or IgG2b monoclonal antibodies of anti-DC1 specificity identical to that of the IgG1 secreting parental cell. In contrast to the parental monoclonal antibody, those secreted by the variants are cytotoxic to peripheral blood B lymphocytes of DC1 positive individuals and are thus compatible with existing HLA typing techniques. This provides a general method for converting noncytotoxic anti-HLA antibodies into cytotoxic typing reagents.

Three recently identified HLA specificities have been detected in a ten-member American Black family using 8th International Histocompatibility Testing Workshop and local antisera. Independent segregation of the two principal components of 8w59 (Bu and SV) was demonstrated. An Aw19-related specificity also segregated in the family.