Functional characteristics and differential expression of class II DR,DS, and SB antigens on human melanoma cell lines

Although most cultured melanoma cell lines express DR Class II molecules, many of these do not also express the DS (MB) Class II molecules as detected by a monoclonal antibody specific for DS. Cells lacking either DR or DS molecules or both could only be induced to express DR antigens in rare cases by combined incubations with azacytidine and Interleukin-2 conditioned medium, although the expression of DR molecules on fibroblasts or U937 monocytes could more easily be induced under the same culture conditions. Melanoma cells expressing DR antigens could function in antigen presentation for the histocompatibility antigens themselves and for DR specific presentation of TNP determinants to allogeneic T-cells sensitized to TNP modified lymphocytes and showing restriction in their responses to the specificity of the DR molecules expressed on the original, autologous senzitizing cells. DR positive melanoma cells could not, however, be demonstrated to function in the presentation of any of the soluble antigens tested. All DR positive melanoma cells also expressed SB antigens, but these were not detected on DR negative melanoma cells. These studies collectively indicate that the expression of Class II histocompatibility antigens on diverse cell types is subject to differential regulatory control and is associated with differences in their functional activities.     

Loss of polymorphic restriction fragments of class I and class II MHC genes in a malignant melanoma

DNAs from human malignant melanoma cells and autologous peripheral blood lymphocytes were evaluated by Southern blot analysis with probes for class I and II HLA genes. DNA of melanoma cells digested with PvuII, EcoRI and BglI and hybridized with a DR beta probe showed a loss of several fragments when compared with DNA from lymphocytes. The same DNAs were not distinguishable when hybridized with a DQ beta probe. Analysis of melanoma and autologous lymphocyte DNAs from the same patient with a class I cDNA, after digestion with several endonucleases, revealed a further loss of fragments in melanoma cells. Comparison of restriction fragment patterns of melanoma and lymphocytes with those of homozygous, serologically-typed cell lines indicated that melanoma cells have lost fragments diagnostic of DR2 and A1 antigens. A densitometric analysis of signals of several oncogenes in comparison with that of DR indicated that a duplication of the remaining DR allele had occurred in melanoma cells.     

Expression of class I and class II markers on populations of leukemic cells

The study of class I and class II antigen expression on leukemic cells brought the following conclusions: most of the leukemic cells show a slower number of class I antigenic sites than normal peripheral blood lymphocytes (PBL) but, in most cases, this does not hinder HLA typing; contrarily to normal PBL, leukemic cells seem to carry "non HLA" antigens (and/or non classical HLA antigens) which are probably responsible of the false positive reactions frequently observed at the time of HLA typing; most of the leukemic cell types express DR antigens (except those belonging to the T lineage) but DQ antigen expression (and in some cases MT antigen expression) varies depending on the cell type studied: well defined on mature B hemopathies, DQ expression is often lower than DR expression on acute leukemic cell types.     

Monoclonal antibodies defining serologically distinct HLA-D/DR related Ia-like antigens in man

Four monoclonal antisera-identifying antigens with the identical tissue distribution and molecular weight of previously described Ia-like antigens were characterized. Two of these antisera, I-1 and I-2, identified antigens expressed on the HLA-D/DR positive cells from all HLA heterozygous individuals. Further characterization on homozygous typing cells (HTC's) demonstrated that I-2 was not reactive with most Dw7 and Dw11 HTC's.Monoclonal antisera, termed 1-LR1 and 1-LR2, defined polymorphic Ia-like antigens that demonstrated restricted expression of cells from HLA heterozygous individuals. Antigen I-LR1 was expressed on cells from 60% of HLA heterozygous and its reactivity with HTC's did not conform to any previously described monotypic or supertypic HLA-D/DR pattern. In contrast, I-LR2 was expressed on 40% of HLA heterozygotes and identified only HLA-DR3, 5, and 6 HTC's. Studies of families with HLA recombinants permitted the demonstration that the 1-LR1 and 1-LR2 antigens are tightly linked to the HLA-D/DR locus. These experiments permit the direct demonstration by immunoprecipitation, linkage studies, and MHC recombinant families that the p29,34 complex in man is closely linked to or is within the HLA-D/DR locus. These studies suggest that the human Ia-like antigens are more heterogenous than previously demonstrated and that monoclonal antisera will be useful in further defining the structural, genetic, and functional characteristics of these molecules.     

Expression of heterophile, Paul-Bunnell and Hanganutziu-Deicher antigens on human melanoma cell lines

Expression of heterophile antigens was studied on 6 human melanoma cell lines. Paul-Bunnell and Hanganutziu-Deicher (H-D) antigens, but not Forssman antigen, were demonstrated on these cell lines. H-D antigen was also demonstrated on melanoma cells isolated from primary and metastatic lesions. Evidence was also presented that HLA class I but not class II (DR and DQ) molecules are expressed on these cell lines. H-D antibodies of IgG class were detected in 10 of 23 (42%) melanoma patients suggesting the possibility that H-D antigen might have been expressed in an immunogenic form in the patients.     

Techniques used to define human MHC antigens: serology

The classical, routine test employed for definition of HLA antigens expressed in humans (tissue typing) is the complement-mediated cytotoxicity assay developed by Terasaki and McClelland in the early 1960s. In both healthy persons and patients, the assay target cells are usually lymphocytes obtained from peripheral blood, but when typing cadaver donors, splenic or lymph node lymphocytes can be used. HLA-A, B, Cw (class I) antigens are expressed on all nucleated cells while HLA-DR, DQ (class II) are restricted to B lymphocytes and immune activated cells. Tissue typing has been achieved using culture cells from amniocentesis and typing of cell lines is possible with small modifications to the standardised cytotoxicity assay. Usually, target cells are incubated under oil with typing antisera at 22 degrees C in a 60- or 72-well Terasaki tray. After 30 min rabbit serum is added as a source of complement. After a further 60 min incubation the test is stained. A positive reaction results in target cell death. There are local variations to this test. Automation of the assay is now commonplace, from reagent dispensing to automated reading of finished assay. The use of antibody-coated magnetisable microspheres has enabled separation of pure B lymphocyte samples for class II typing and has reduced incubation times through antigen modulation. It is possible to define antibodies to HLA antigens in the same assay using target cells with known HLA phenotypes.     

A supertypic HLA class II determinant shared by DR1 and DRw9, and crossreactive with DR2, defined by human monoclonal antibody

A human monoclonal antibody Pez.2F5, produced by a lymphoblastoid cell line, has been established in vitro by Epstein-Barr virus (EBV) transformation of B lymphocytes isolated from the blood of a volunteer immunized with allogeneic peripheral blood leukocytes (PBLs). The antibody reacted with a new supertypic determinant expressed on all lymphoblastoid cell lines homozygous for HLA-DR1, -2, and -w9. The genetic linkage of the Pez.2F5 determinant to the HLA region was demonstrated by family segregation studies. Quantitative absorption studies indicated that DR2-positive cells required more Pez.2F5 antibody for lysis, and since their absorption capacity was significantly lower than that of DR1- or DRw9-positive cells, it is likely that the Pez.2F5 determinant of the DR2 haplotype is crossreactive but not identical with the determinant found on the latter haplotypes. In addition, on a test panel of HLA-typed B lymphocytes, Pez.2F5 showed perfect correlation with DR1 and DRw9, but reacted with only a fraction of DR2-positive cells. The Pez.2F5 determinant was found to be absent from resting T lymphocytes, but its expression could be identified on IL-2-dependent T-cell lines by cytotoxicity and flow cytofluorometric analysis. By sequential immunoprecipitation and SDS gel analysis of antigens of DR1 cells it was determined that the Pez.2F5 determinant is carried by HLA class II DR molecules. Thus, the Pez.2F5 is the first described human monoclonal antibody able to immunoprecipitate HLA class II-related molecules.     

Proliferative T cell clones directed at a human minor antigen

Clones directed against a human minor antigen were isolated from an interleukin-2-dependent cytotoxic T cell line. As expected, some clones exhibiting cytotoxicity for HLA-identical minor antigen-positive cells were detected. In addition, some clones were detected which, though not cytotoxic, were able to proliferate in the primed lymphocyte typing (PLT) test in response to stimulation by cells from some HLA-identical sibs and certain DR 2+/ve unrelated cells. These clones expressed the Leu 3a cell surface antigen. DR 2 was one of the class II determinants expressed by the responding cell used to generate the cytotoxic T lymphocyte line. These results are consistent with the interpretation that helper T cells are able to recognize minor antigens in the context of self-class II HLA determinants and respond in PLT.     

Typing of human fetal organs for the histocompatibility antigens A, B and DR

In the transplantation of human fetal pancreatic explants into diabetic man, the importance of matching the histocompatibility antigens of donor and recipient to decrease the chances of rejection is unknown. Before this question can be answered human fetuses must be tissue typed. We have shown that lymphocytes harvested from fetal liver, thymus, bone marrow and spleen can be successfully HLA DR typed in 64% and A and B typed in 57% of 58 fetuses aged 15 wk or more. Typing should ideally be carried out on unseparated T and B cells. Best results were achieved if all four of the above organs were available and more than one million viable cells were able to be harvested for typing. Whilst the DR antigens could be typed from all tissues, the A and B antigens could be typed, with few exceptions only from thymus, spleen and bone marrow. The efficacy of matching the histocompatibility antigens of recipient and donor fetuses, especially the DR antigens can now be tested in the human diabetic being transplanted with pancreatic explants.     

Lack of shared lymphocyte-stimulating determinants between H-2I and HLA-D/DR using mouse primed lymphocyte typing cells

A number of anti-H-2 alloantisera containing antibody reactive with I region gene products (Ia) of the major histocompatibility complex cross-react with determinants expressed by human peripheral blood B lymphocytes. Such data have led to the conclusion that Ia and DR antigens share cross-reacting determinants. We have attempted to generate mouse primed T lymphocyte populations specific for defined I region gene product determinants which concomitantly recognize DR determinants on human peripheral blood leukocytes in primed lymphocyte typing (PLT) analysis. Mouse PLT cells were generated in primary MLC using strain combinations identical to those in which positive mouse/human cross-reacting antisera have been obtained. The resulting PLT cells exhibited strong, yet specific, secondary MLC responses against mouse cells expressing the Ia determinants used as the primary stimulus. In contrast, when examined on panels of human peripheral blood leukocytes, no reactivity was detected. This lack of cross-reactivity suggests that mouse T cells primed toward Ia determinants do not regularly recognize cross-reacting determinants of DR or D-associated antigens expressed on human PBLs. Consequently, mPLT cells are not a useful reagent in defining HLA-D region polymorphism.     

The detection of HLA-DR, MB and MT determinants on purified class II molecules by inhibition of microcytotoxicity

An assay has been developed which makes it possible to determine the HLA allospecificities carried by molecules in purified fractions of detergent lysates from EBV-transformed human lymphocytes. It is based on inhibition of the standard microlymphocytotoxic test used for identifying HLA class I and II antigens with alloantisera. Soluble cell membrane products from EBV-transformed cell lines homozygous for the HLA region gave specific inhibition of standard typing antisera. The test requires preincubation of microliter volumes of soluble antigen preparations maintained in 0.05% NP-40 with selected antisera prior to adding EBV-transformed cells as target cells. It was possible using this assay to follow isolation of the structurally related human class II molecules bearing the MB and DR specificities. Detergent lysates of cells were fractionated on affinity columns prepared from monoclonal antibodies directed against distinct class II antigens. Eluates from these columns contained the expected DR and MB specificities. The assay is easy to perform, highly reproducible and allows multiple determinations.     

Production in vitro of antibodies directed against alloantigen-specific recognition sites on T cells and on lymphocytotoxic HLA antibodies.

We have examined the mechanism of immunological unresponsiveness in a recipient (P.S.) with a long-term functioning renal allograft. P.S., whose HLA type is A1, A30; B14, B18; DR1, w8; DRw52; DQw1 and in whose serum we had earlier demonstrated the presence of antiidiotypic antibodies, received a kidney from a cadaver donor of HLA type A1, A10, B8 in March, 1970. Peripheral blood B lymphocytes from the patient were transformed with Epstein-Barr virus (EBV), and by the cluster-picking technique a B cell line was propagated with continuous production of antibodies. Antiidiotypic antibodies with two distinct biological functions were demonstrable; one specifically inhibiting the lymphocytotoxic activity of anti-HLA-B8, B5, and DR3 reference typing sera, and the other specifically inhibiting proliferative responses in MLC of the recipient's lymphocytes and of third party cells sharing B14, DR1, DQw1 with the patient against stimulator cells carrying B8, DR3 antigens. Immunodepletion experiments demonstrated that the inhibitory activity was associated with the IgM fraction. Absorption experiments suggested that different antibodies may be responsible for the inhibition of lymphocytotoxic activity of anti-HLA sera and of the proliferative responses in MLC. Antiidiotypic antibodies have been postulated to be important in maintaining allograft tolerance in vivo, thereby enhancing renal allograft survival. The availability of such antibodies in large quantities, produced in vitro, could provide antisera for the immunochemical characterization of specific idiotypic receptors on immunoglobulins and T lymphocytes.     

Specificity of HLA restricting elements for human nickel reactive T cell clones

In order to study the fine specificity of HLA class II restriction, we have established nickel specific T cell clones from a nickel allergic patient. Cells were cloned by limiting dilution after primary stimulation and selection of nickel specific blasts. Several clones were established which were all shown to carry the CD4 marker. All clones were shown to be completely blocked by monoclonal antibodies directed against DR antigens, but unaffected by antibodies against DQ or DP, thus demonstrating their DR specificity. For the study of HLA class II restriction, a panel of cell donors was carefully HLA typed by including the use of DRB and DQB cDNA probes. Specificity analysis, using allogeneic antigen presenting cells, revealed that the clones were either restricted to DR3- or DR4-like molecules, which is consistent with the fact that the donor was DR3, DR4 positive. However, the studies also revealed that the fine specificity of the DR3 and DR4 restriction could not be completely assessed by serological and genomic typing of panel cells. This indicates that cellularly defined HLA restriction elements recognized by T cells cannot be defined properly with available class II typing methods, and the results of these experiments documented the additional polymorphism of class II restriction elements. The clonal specificity analysis has shed further light on the biologically relevant level of DR polymorphism.     

“Bare lymphocytes” without immunodeficiency

“Bare lymphocytes”, which cannot be typed for the HLA-A,B, and C antigens were observed in two siblings, nine and six years of age. The elder child presented with aplastic anemia and was being considered for bone marrow transplantation. The younger child was healthy. The inability to phenotype both children for these three gene products persisted throughout the 21-month period of observation. The DR antigens were demonstrable which rendered it possible to deduce their A, B, and C genotype from the typing of the other four family members. Although alloantisera failed to detect the antigens on peripheral blood lymphocytes, monoclonal antibodies demonstrated reduced amounts of the HLA-A,B, and C antigens on the cells. The reduced level was confirmed following EBV transformation of the cells. After prolonged culture, HLA antigens immunoprecipitated from the cell extracts were normal in amount, molecular weight, and polypeptide composition. Southern blot analysis did not reveal gross genomic rearrangements. A regulatory defect leading to the expression of these Class I antigens is postulated.     

HLA typing used with cultured amniotic and chorionic villus cells for early prenatal diagnosis or parentage testing without one parent's availability

Like fetal fibroblasts and amniotic fluid cells, cultured chorionic villus cells can also be HLA typed with selected typing sera after preincubation with gamma interferon to promote better antigen expression. A modified procedure now in use would also allow any of these cell types to be tested for the presence or absence of all known HLA A,B,C, and DR antigens with standard preplated typing trays. This procedure was used to confirm that an on-going pregnancy had resulted from the successful in vitro fertilization and implantation of an anonymous donor's ovum and could also be of major use in rape or artificial insemination cases when the identity of the possible father(s) is not known.     

Typing of HLA class II and class I antigens using PHA-activated, IL-2-propagated T lymphocytes

We describe here a simple procedure, by which HLA class II antigens can be accurately and reliably identified in those patients where there is minimal or absent expression of HLA-DR,DQw antigens on B cells, or when the total number of leukocytes recovered from the patients do not permit reliable typing. Ficoll-Hypaque-separated peripheral blood mononuclear leukocytes, fresh or cryopreserved, were activated by PHA and then propagated in IL-2-containing medium until enough cells for typing were obtained (usually 7-14 days). At this stage, the cultured cells were shown to be primarily T cells (greater than 90% CD3+). Since the activated T cells propagate in the presence of IL-2, even a small number (10(4] of fresh or cryopreserved patients' cells suffice for this protocol. To date we have been able to successfully HLA-DR,DQw type 34/34 bone marrow transplantation candidates and 12/12 long-term dialysis patients, who were untypable using fresh cells. HLA-DR,DQw antigens on activated T cells from normal individuals were identical to those found on their uncultured B cells. In addition, class I antigens that were undetectable on the uncultured cells of one patient could be identified on activated T cells. The HLA antigens identified on the patients' activated T cells were confirmed by phenotypic analysis of cells from family members.     

Differential expression of HLA class II antigens on human fetal and adult lymphocytes and macrophages

A panel of monoclonal antibodies to monomorphic determinants of the MHC class II subregion locus products: DP, DR and DQ, was used to investigate the expression of these antigens on early lymphocytes and macrophages from human fetal liver (13-20 weeks), placenta (16 weeks and term) and cord blood, in relation to the class II phenotype of cells from adult tonsil and peripheral blood. Fetal liver sections and cell suspensions showed differential expression of class II antigens. DP was expressed at a higher frequency (11.0% of nucleated cells) than DR on lymphoid cells and macrophages from fetal liver, and DQ was either absent or expressed on less than 0.3% of nucleated cells. Consistent with this finding, DP but not DR or DQ antigens were observed on vascular elements and macrophages in the villi of 16-week placenta. At term, all three subregion locus products were expressed. Adult tonsil and peripheral blood B lymphocytes expressed DP, DR and DQ antigens with similar frequency; however, DQ was expressed at a lower frequency than DP and DR on cord blood B lymphocytes. In contrast, 30-50% macrophages from cord blood and adult peripheral blood expressed DP and DR, but fewer (5% and 18%, respectively) expressed DQ. These data suggest that class II antigens are expressed in the sequence DP, DR, DQ on developing lymphocytes. A similar sequence is suggested for macrophages.     

HLA disease association and protection in HIV infection among African Americans and Caucasians

In a previous investigation, we demonstrated an increased progression of overt AIDS in the African American population compared to the Caucasian population as reflected by the significantly lower absolute number of CD4+ lymphocytes detected in the African American population in an earlier study. The present study elucidates some of the possible genetic factors which may contribute to disease association or protection against HIV infection. The HLA phenotypes expressed as A, B, C, DR and DQw antigens were revealed by the Amos-modified typing procedure. NIH scoring was utilized to designate positive cells taking up trypan blue. A test of proportion equivalent to the chi 2 approximation was used to compare the disease population (n = 62; 38 African Americans, 24 Caucasians) to race-matched normal heterosexual local controls (323 African Americans, 412 Caucasians). Significant p values were corrected for the number of HLA antigens tested. HLA markers associated with possible protection from infection for African Americans were Cw4 and DRw6, whereas Caucasians expressed none. Disease association markers present in the African American population were A31, B35, Cw6, Cw7, DR5, DR6, DRw11, DRw12, DQw6 and DQw7, whereas in the Caucasian population A28, Aw66, Aw48, Bw65, Bw70, Cw7, DRw10, DRw12, DQw6 and DQw7 were demonstrated. The highest phenotypic frequency for a disease association marker in the study was for HLA-DR5 (62.9%) in the HIV-infected African American population without Kaposi's sarcoma compared to a frequency of 28.9% for the regional control group (p = 0.0012). We conclude that genetic factors do have a role in HIV infection since only 50-60% of those exposed to the AIDS virus will become infected.     

Lymphocyte Subsets and Activation Antigens in a Reference Population: A Flow Cytometric Study Using Single and Double Antibody Staining

The transferrin receptor (TR), the HLA DR antigen (DR), and the antigen binding OKT10 (T10) are present on activated lymphocyte populations. The authors have studied their expression and that of antigens defined by eight commercial monoclonal antibodies on peripheral blood lymphocytes of 50 healthy hospital workers aged 23–60 years. A whole blood lysis technique was employed and cells were enumerated on a flow cytometer. The percentage of cells bearing the three activation antigens were generally low: mean values for T10 being 7.2%; for TR 1.8%; and for DR 8.8%. There was, however, considerable variability, with occasional subjects having 20% or more cells positive for one of the three antigens. High values of one activation antigen did not correspond with high values of another in the same subject. Nor was there correlation of the presence of activation antigens with the occurrence of cells bearing T11, T4, T8, Leu 1, Leu 2a, Leu 3a, or Leu 7. Double labelling with the following pairs of fluorescein (FITC) and phycoerythrin (PE) labeled antibodies: Leu 2a, DR; Leu 3a, DR; Leu 3a, Leu 2a; TR, DR, indicated that simultaneous expression of the corresponding antigens do not normally occur on lymphocytes of healthy individuals. Double labelling with B1 and DR in five subjects indicated the presence of B1 (+) DR (-) cell population. No pattern of relationship could be detected among common clinical variables or HLA type and an increased expression of activation antigens.     

A new supertypic HLA class II determinant (PL2) differentially expressed with DR7, DRw11(5), DRw13(w6), DRw14(w6), DR3, and DR2 and biochemically localized to DR7 molecules

A monoclonal antibody, PL2, has been produced that reacts with a new supertypic determinant expressed on the peripheral blood B lymphocytes and B-leukemic cells (B-CLL) from all individuals who are HLA-DR7 and some individuals who are HLA-DR5 positive. The genetic linkage of the PL2 determinant to the HLA region was demonstrated by family segregation studies. When cultured Epstein-Barr virus (EBV) transformed B cell lines were examined, PL2 was again found to be expressed on all cell lines homozygous for HLA-DR7 and the DRw11(5) subtype of HLA-DR5 positive cells, while one DRw12(5) cell line was negative, suggesting PL2 may distinguish between these DR5 subtypes. In addition, using the panel of EBV-transformed B-cell lines, PL2 was also found to be weakly expressed on HLA-DRw14(w6), -DRw13(w6), -DR3, and -DR2 positive cells but was completely absent from HLA-DR1 and -DR4 positive cells, and is probably absent also from DRw8- and DRw10-positive cells. From titration analysis and quantitative absorption studies the PL2 determinant was found to be expressed at quantitatively different levels in the following order: DR7 greater than DRw11, DRw14 greater than DRw13 greater than DR3 greater than DR2. The molecules carrying the PL2 determinant on DR7 cells have been characterized biochemically to be a subpopulation of HLA class II molecules recognized by the DR specific monoclonal antibody, L243. Furthermore, by two-dimensional gel analysis, PL2 immunoprecipitated only two of three beta chains associated with the DR-apha chain, which are the same two chains that carry the DR7 allodeterminants.(ABSTRACT TRUNCATED AT 250 WORDS)