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We have investigated mRNA expression for nonclassical MHC class I genes (HLA-E,-F,-G) in human gametogenic cells. Testicular tissue was treated by collagenase and the resulting cell suspension was further purified by fractionation on Percoll gradients in a two-step procedure. Three gametogenic cell fractions were analyzed: purified heterogenous suspension of gametogenic cells, fraction of round spermatids and fraction of elongated spermatids. Total RNA isolated from each cell population was subjected to both reverse transcriptase/polymerase chain reaction and Northern blot analysis using oligonucleotides specific for HLA-E, -F and -G. Both method gave similar results. We have found a considerable level of HLA-E mRNA, very low amounts of reamplified cDNA for HLA-F and both a complete lack of mRNA and reamplified cDNA for the HLA-G gene in the analyzed gametogenic cell fractions. Additionally, we have localized HLA-E molecules on the cells of the adluminal compartment within seminiferous tubules using immunostaining with monoclonal antibodies specific for HLA-E heavy chain followed by confocal microscopy analysis. The unique expression pattern of HLA class I antigens in the male gonad could play an important role in an efficient protection against an autoimmunological attack toward germ cells.

T cell lines and clones were analyzed for surface expression of Ia antigens using monoclonal antibodies (mAb) that detect monomorphic and polymorphic epitopes on Ia molecules encoded by the HLA-DR and HLA-DQ gene clusters. All mAb bound to B lymphocytes or lymphoblastoid cell lines of the same individuals from whom the T cells were derived. Three mAb detecting monomorphic epitopes, primarily associated with HLA-DR, bound to all T cells showing that each clone or line expressed some type of Ia. Three other mAb defining polymorphic epitopes associated with HLA-DR products showed differential binding patterns. Two reagents, R3 and E15/4 recognizing the supertypic specificity DRw52 (formerly MT2), bound to every alloreactive clone, whereas the 16.23 mAb, detecting a private DR3-associated epitope, failed to bind to any clone. In contrast, the 16.23 epitope was detected on high percentages of T cells specific for purified protein derivative of tuberculin (PPD) or tetanus toxoid (TT). Biochemical studies showed that the 16.23 and DRw52-like epitopes can be present on distinct DR molecules on B cell lines and this may also be the case for T cells. Three other mAb, detecting epitopes associated with HLA-DQ, also revealed differential binding patterns when tested on various T cells. Two failed to bind to any alloreactive clone and to only low numbers of PPD- or TT-specific T cell lines, whereas the third bound distinctly to a CD4+/CD8+ alloreactive clone. Biochemical analyses have shown that these DQ epitopes can be present on different molecules. Combined, these observations indicate that differential expression of Ia molecules encoded by both HLA-DR and DQ occurs between B and activated T cells as well as among T cell populations of the same individual. Whether these differences reflect quantitative variations in expression of given DR or DQ molecules or, alternatively, are due to differential class II gene expression in activated T cells remains to be determined.

Monoclonal antibodies were produced against melanoma cell lines derived from patients presently disease free. Based on tissue distribution of binding, the antibodies obtained could be used to classify melanoma surface antigens into groupings similar to those obtained from studies of autologous and xenogeneic antibodies. Three antibodies (21.43, 15.75 and 15.95) reacted with the immunizing tumor but not with peripheral blood lymphocytes or a lymphoblastoid cell line derived from the tumor donor. When tested on a broader cell panel, antibody 21.43 reacted only with melanoma lines, while antibodies 15.75 and 15.95 reacted with carcinoma cell lines as well as with the majority of melanoma cell lines. Antibody 15.95 precipitated a 49000 dalton glycoprotein from both melanoma and carcinoma cells. Antibody 15.75 precipitated a 74 000 dalton glycoprotein from the surface of melanoma and carcinoma cell lines which is also found on freshly isolated tumor cells from melanoma and carcinoma metastases in vivo.

Murine monoclonal antibodies directed against polymorphic epitopes on HLA-linked antigens were readily obtained following a single immunization with DRw6-homozygous cells. Five antibodies directed against class II antigens were isolated. Two precipitated DC-like molecules and 3 DR-like molecules from the immunizing lymphoblastoid cell line. Analysis of association with HLA-DR antigens in a panel of 60 individuals revealed 2 antibodies directed against the DC1 specificity (S1, R1) and one against the MT2 specificity (R3). Antibodies S2 and S3 showed significant association with DRw6 (chi 2 = 30.27) and DRw6 and w8 (chi 2 = 18.86), respectively. An analysis of 29 haplotypes indicated that epitopes defined by these monoclonal antibodies appear to identify polymorphic determinants on three independent gene products which can be encoded by a single HLA haplotype.

The melanoma-associated antigen P3.58 is rarely found on benign proliferating melanocytes but is consistently expressed on advanced malignant melanomas which have a high probability of metastasis. Previous studies have shown that its expression on normal tissues is limited to vascular endothelia and lymphoid follicle germinal centers and that it is also expressed by activated monocytes in vitro. In the studies reported here, anti-P3.58 monoclonal antibodies (mAb) were shown to partially inhibit antigen-specific and anti-CD3-induced T cell proliferation and to completely block a lymphocyte/monocyte clustering which occurs in the absence of added antigen. This inhibition is highly specific for P3.58 mAb and was not affected by mAb directed to major histocompatibility complex or T cell antigens. P3.58 therefore seems to be involved in an antigen-independent attraction or adhesion of lymphocytes. P3.58 is the second example (HLA-DR being the first) of an association between the expression of an immune function-associated molecule and the development of metastatic disease in melanoma.

A series of human T lymphocyte clones showing specific responses for major histocompatibility complex-encoded alloantigens were isolated from a single culture. They were classified into distinct functional groups based on measurement of alloantigen-specific proliferation and cytotoxicity. Surface antigen expression on these clones was analyzed using a panel of monoclonal antibodies specific for T cell differentiation antigens and for Ia-like antigens. Four distinct groups were identified based on the Leu series of differentiation antigens: (a) Leu1+,2+,3+,4+; (b) Leu1+,3+,4+; (c) Leu1+,2+,4+; and (d) Leu2+,4+. Functionally distinct clones showed not only differential expression of Leu2 and Leu3 , antigens which had previously been shown to distinguish helper/inducer and cytotoxic/suppressor cells, but also of Leu1, an antigen classified as a common T cell marker. All of the clones were found to express Ia antigens as detected by three framework monoclonal antibodies, but none were found to express a determinant associated with the DR3 allospecificity , although it was found on normal B lymphocytes and a B cell line established from the HLA-DR3 individual from whom these clones were derived.