Although human la or class II histocompatibility antigens were initially thought to be restricted to cells of the immune system, studies have shown the presence of la antigens on malignant melanomas (1-3) and other cell types having no known immune functions (3-6). While la antigens can be detected on most melanoma biopsy specimens and melanoma cell lines (7,8), normal melanocytes lack constitutive la expression (9), either in vivo or in vitro . To investigate the relationship of la expression in melanomas to malignant transformation, we infected melanocytes with transforming amphotropic pseudotypes of Harvey murine sarcoma virus (Ha-MSV)' or Kirsten murine sarcoma virus (Ki-MSV) . The Ha-MSV and Ki-MSV retroviruses contain oncogenes of the ras gene family, and were chosen because our previous studies (10) indicated that 10% of cultured melanomas have an activated ras gene allele (either Ha-ras or N-ras), and no other conclusive perturbation (i.e., rearrangement or amplification) in 15 other known oncogenes (A . P. Albino, unpublished results) . Materials and MethodsTissue Culture. Melanocytes and other cell lines were derived as described previously (3) . Cultures were maintained in Eagle's MEM supplemented with 2 mM glutamine, 1 nonessential amino acids, 100 U/ml penicillin and 10% FCS . For culturing melanocytes, the medium was supplemented (11) with 10 -s M cholera toxin (Schwartz/Mann Biologicals, Orangeburg, NY) and 10 ng/ml 12-o-tetradecanoyl phorbol-l3-acetate (TPA) (Consolidated Midland Corp., Brewster, NY) . Melanocyte cultures were used during early passages (less than four) .Virological Techniques. Viral stocks were isolated from NIH 3T3 cells infected with 4070A amphotropic murine leukemia virus (MuLV) (12), or from Ki-MSV, Ha-MSV, or Ki-MSV temperature sensitive (ts) mutant-infected NIH 3T3 nonproducer clones superinfected with 4070A amphotropic helper MuLV (13, 14, and A. I. Oliff, unpublished data) . The Ki-MSV and Ha-MSV pseudotypes were collected as fresh 24-h cell-free supernatant fluids and frozen at -70°C until use .^-10 5 melanocytes were pretreated for 60 min with DEAE/dextran (25 Ag/ml), washed, then incubated with virus at a multiplicity

Human interferon-gamma (IFN-gamma) is an important immunomodulatory protein produced predominantly by T cells and large granular lymphocytes (LGLs). Whereas large amounts of data have been accumulated regarding IFN gamma gene expression in these two cell types, little information about IFN gamma expression in other cell types exists. In this study, we have analyzed the production of IFN gamma by the Epstein- Barr virus (EBV)-positive B-cell line, JLP(c), derived from a patient with Burkitt's lymphoma, and another human B-cell line, PA682BM-1, which was derived from an acquired immunodeficiency syndrome patient. Southern blot analysis indicates the presence of an Ig heavy chain gene rearrangement, but no rearrangement of the T-cell receptor beta chain gene or IFN gamma gene in these B-cell lines. Both cell lines were found to express surface IgD and other B-cell surface markers, thus confirming their B-cell lineage. Analysis for surface Ig, cytoplasmic Ig, and secreted Ig indicates that the two cell lines are in relatively early stages of the B-cell differentiation pathway. We now report that PA682BM-1 can be triggered by the protein kinase C (PKC) activators, phorbol 12-myristate 13-acetate (PMA) and (-)Indolactam-v, to secrete IFN gamma, whereas JLP(c) cells spontaneously produce low levels of IFN gamma that can be enhanced by PKC activators and interleukin-2 (IL-2). After activation of the cell lines with IL-2, (-)Indolactam-v, and PMA, increases in cytoplasmic messenger RNAs (mRNAs) of IFN gamma and the IL- 2 receptor chains were also observed. The induction of IFN gamma mRNA and protein by IL-2 was completely blocked by a monoclonal antibody to IL-2 receptor p75 (beta chain), but not by the monoclonal antibody to p55 (alpha chain). Analysis of IFN gamma genomic DNA indicates that the gene is not amplified, but that hypomethylation in the 5′ noncoding region of the IFN gamma gene has occurred in the B-cell line from the Burkitt's lymphoma patient that spontaneously produces IFN gamma. This finding suggests that the methylation state of the promoter region may play an important role in the control of IFN gamma gene expression in B cells.

Mouse monoclonal antibodies (MAb) have been generated against the anionic isozyme of human glutathione S-transferase (GST pi). MAb AGST I can inhibit 50-70% of GST pi enzymatic activity and reacts with a 3-dimensional epitope which includes a putative glutathione binding site on GST pi. A sandwich enzyme-immunoassay established using MAb AGST I and a polyclonal antibody displayed a sensitivity of 0.5 ng/ml. Immunohistochemical analysis of human tissues demonstrated marked increases in GST pi levels in cancers of the brain, cervix, endometrium, colon, rectum and testis and in fibro- and chondrosarcomas.

Sllmmal-yIn an attempt to identify a molecule in target recognition by CD3-large granular lymphocytes (LGL), we have generated a rabbit antiidiotypic (anti-ID) serum against a monoclonal antibody (mAb 36) that reacted with the cell membrane of K562. Flow cytometry analysis demonstrated that the anti-ID serum bound selectively to CD3-LGL and that F(ab')2 fragments of the anti-ID serum blocked both target cell binding and lysis by NK cells. Stimulation of CD3-LGL with F(ab')z fragments resulted in the release of serine esterases and the secretion of interferon % Furthermore, anti-ID F(ab')2 antibodies crosslinked to anti-DNP F(ab')2 mediated directed cytotoxicity of a non-natural killer (NK)-susceptible mouse target (YAC-1) via this surface ligand. These functional reactivities were only removed by adsorption with the specific idiotype. Protein analysis showed that the anti-ID serum immunoprecipitated 80-, 110-, and 150-kD proteins. Using this anti-ID, a partial cDNA was cloned and an antipeptide antiserum was made against the portion of the predicted amino acid sequence that corresponded to a portion of the ID binding region. This antipeptide serum exhibited similar functional and biochemical reactivities to those observed with the anti-ID serum. These data suggest that the cell surface moiety recognized by the anti-ID and anti-p104 is novel and is selectively involved in both recognition and triggering of NK-mediated lytic function.

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