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The 6th International Workshop on Leukocyte Antigens (white cell differentiation antigens) continued the international cooperative effort aimed at characterization of all leukocyte cell surface antigens with respect to their biochemical properties, cell- and cell line expression, molecular and cellular function(s) and eventual disease relevance. Significantly, among 36 newly defined CD clusters identified with the aid of numerous monoclonal antibodies submitted to the workshop [17], 8 new clusters belonged to the endothelial section, 6 new CD clusters were identified within cytokine receptor section and 5 such clusters were characterized in the adhesion structure section, i.e. as antigens with the pattern of expression also on cells, tissues and cell lines outside the hematopoietic system (Tables 1-3). Minority of newly defined clusters appurtened to lineage-specific or non-lineage hematopoietic differentiation antigens (Table 4), i.e. 5 new CD clusters within myeloid section, 3 new clusters within both non-lineage and NK antigens, 2 T-cell antigens and one new CD cluster defined within both B-cell or platelet sections.

Human multidrug resistant ovarian carcinoma cells (A2780/ADR) exhibited increased in vitro penetration into the collagen-normal human fibroblasts matrix, increased cell surface expression of alpha 6 integrin (CD49f antigen) and slightly increased expression of alpha 2 (CD49b) integrin compared with that of parental drug-sensitive A2780 cells. Both, multidrug-resistant and parental, drug-sensitive, cell lines did not express the 67 kDa non-integrin high affinity laminin receptor on their cell surfaces. As there were no marked differences between metalloproteinase activity of both A2780 cell sublines (with similar intensity of 72 kDa and 92 kDa lysis bands in zymograms), the increased penetration of the drug-resistant subline into the collagen-fibroblast gel matrix might be associated with the increased expression of adhesion proteins (including collagen-binding alpha 2 integrin), or cell surface-associated collagenase-stimulating protein(s). This multidrug resistant ovarian carcinoma cell line might serve as an in vitro model of neoplastic cells with increased biological aggressiveness, molecular mechanisms of which require further analysis.

Phorbol ester (TPA)-induced increase in cell surface expression of adhesion structures, i.e. intercellular adhesion molecule-1 (ICAM-1, CD54), beta 2 integrin LFA-1 (CD11a), complement-regulatory cell membrane protein-protein (CD59) and leukocyte common antigen (CD45) in human erythroid/myeloid leukemia cell line K-562 was inhibited by staurosporine, an inhibitor with broad, non-selective protein kinase inhibitory profile, but not by CGP 41,251, a benzoylated staurosporine derivative with the selective protein kinase C (PKC) inhibitory activity. Neither staurosporine nor CGP 41,251 modulated TPA-induced down-regulation of transferrin receptor (CD71). These data suggest that phorbol ester-induced cell surface antigen modulations in K-562 cells are predominantly mediated by PKC-independent signalling pathways.

Flow cytometric and immunochemical studies performed with the CD66/67 panel antibodies from the Vth International Workshop on Leukocyte Antigens allowed to subdivide these antibodies into five groups, according to the molecular weights of polypeptides recognized by these antibodies on pooled healthy donor granulocytes. Although some monoclonal antibodies recognized either 95-110 kDa, or 160-180 kDa polypeptides, majority of the examined antibodies reacted with epitopes shared by both 95-110 kDa and 160-180 kDa polypeptides. Differential TPA-induced modulation of recognized antigens was observed with HL-60 and U-937 leukemia/lymphoma cell lines.

Phorbol ester (PMA) induced decrease in cell surface expression of CD4 antigen was potentiated on human monoblastoid cell line U-937 cultured and induced in protein-free culture medium. Down-regulation of the CD4 antigen was partially inhibited by the isoquinoline protein kinase inhibitor H7 and unaffected by the calmodulin inhibitor R24571 (calmidazolium). PMA-induced increase of the monocyte-associated CD14 antigen was dependent the presence of fetal bovine serum (FBS) in culture medium and partially abolished by calmodulin inhibitor R24571. H7 inhibitor partially abrogated this up-regulation in 4 out of 8 induction experiments. PMA-induced down-regulation of MHC class I antigen was found in both FBS-supplemented and protein-free culture media. This effect was partially inhibited by both H7 and R24571 inhibitors.

The ability of cytokines (IFN alpha, IFN gamma, TNF alpha, IL-1 alpha, IL-6), all-trans retinoic acid, 1,25(OH)2-vitamin D3 and the tumor promoting phorbol ester TPA to regulate cell surface expression of protectin (CD59 antigen) on human hematopoietic and non-hematopoietic neoplastic cell lines was examined with the aid of immunocytofluorometric measurements. The tumor promoting phorbol ester TPA induced a marked up-regulation of protectin in all examined cell lines with the exception of promyelocytic leukemia HL-60, where TPA significantly decreased protectin cell surface expression. All-trans retinoic acid weakly down-regulated cell surface protectin on K-562, while 1,25(OH)2-vitamin D3 produced such effect on HL-60 cells. None of the examined cytokines induced a significant protectin down-regulation in the examined cell lines.

Cell surface expression of leukosialin (sialophorin, CD43 antigen) on human neoplastic hematopoietic cell lines K-562, U-937, HL-60 and REH was determined with the aid of a new CD43 monoclonal antibody (Bra7G) by the immunochemical (radioimmunoprecipitation, immunoblotting) and immunocytofluorometric techniques. Interferon-gamma and TNF-alpha were utilized as the "physiological" inducers of differentiation-associated markers. The "non-physiological" inducer phorbol ester PMA induced down-regulation of leukosialin cell surface expression on immature erythroid-myeloid leukemia cell line K-562, but up-regulation of CD43 antigen on the promyelocyte leukemia cell line HL-60 and, to a lesser extent on the monocyte-like U-937 and CALLA+ ALL cell line REH. Retinoic acid down-regulated leukosialin on both U-937 monocyte-like cells and the CALLA+ ALL cell line REH. In contrast to these data, interferon-gamma, TNF-alpha, retinoic acid and 1,25(OH)2-vitamin D3 induced the up-regulation of leukosialin in a promyelocytic leukemia cell line HL-60.

Monoclonal antibodies allowed recognition and identification of a series of hematopoietic cell membrane antigens expressed on plasma membranes of leukemia patients and on cells of healthy subjects. Monoclonal antibodies produced in different laboratories were clustered on the basis of their reactivity with normal and malignant hematopoietic cells into CD (cluster of differentiation) groups, with defined properties of recognized antigens (glycoprotein, phosphoprotein or glycolipid nature, molecular weight, isoelectric point, amino acid or carbohydrate sequence, etc.) and with functional characteristics of the corresponding antigen and its distribution on cells of the hematopoietic system. This "CD system" of human hematopoietic cell differentiation antigens was established in a series of international laboratory workshops (1982-1989). The present state of the CD classification system, as assessed after the IVth International Workshop on Hematopoietic Cell Differentiation Antigens (1988/89), in which more than 100 laboratories analyzed over 1,000 monoclonal antibodies, is described. An overview of the data concerning properties of monoclonal antibodies produced in the authors' laboratory (monoclonal antibodies series Bra-) is presented.

Two new murine monoclonal antibodies were prepared by hybridoma technique after immunization with the immature pluripotent leukemia cell line K562. The monoclonal antibody Bra10G (IgG2b) reacted in a non-lineage pattern with all examined hematopoietic neoplastic cell lines and peripheral blood cells (granulocytes, lymphocytes, erythrocytes) of healthy donors, with the exception of monoblastoid cell line U-937 and B lymphoma cell line Daudi. This monoclonal antibody immunoprecipitated an 18-20 kDa cell surface protein expressed also on the cell surface of examined non-hematopoietic (malignant glioma, melanoma and breast carcinoma) cell lines. These properties and the efficient inhibition of Bra10G binding to the cell surface of K562 cells by the reference CD59 monoclonal antibody (MEM-43) indicated that Bra10G belongs to the CD59 cluster of monoclonal antibodies which identify the human protectin molecule. The monoclonal antibody Bra7G (IgM) reacted with a 95 kDa cell surface protein expressed on hematopoietic cells (with the exception of erythrocytes) and was absent on the examined non-hematopoietic neoplastic cell lines. These data together with a partial inhibition of Bra7G binding by the reference CD-43 monoclonal antibody suggested the CD43 (leukosialin, sialophorin) specificity of this monoclonal antibody.

Pretreatment of MOLT-4 T-cell line with 12-0-tetradecanoylphorbol 13-acetate (TPA) potentiated the interferon alpha-induced cell surface expression of class I HLA. Simultaneous combined action of TPA and rIFN-alpha resulted in a synergistic effect upon the up-regulation of class I HLA. Neither TPA nor rIFN modulated the cell surface expression of HLA class II (DR and DP) antigens. Leucocyte common antigen (CD45) cell surface expression was quantitatively increased on MOLT-4 cells by TPA induction, unaltered by rIFN and up-regulated by combined action of TPA and rIFN. CD8 antigen was down-regulated by both TPA and rIFN.

Leukocyte common antigen (LCA) isoform expression patterns were examined on different human hematopoietic cells and cell lines with the aid of the immunoblotting technique utilizing two anti-LCA monoclonal antibodies elicited by an ALL cell line and able to detect four isoforms (180, 195, 205 and 220 kDa) of LCA on peripheral blood lymphocytes from healthy donors and some neoplastic hematopoietic cell lines. Fourteen leukemia/lymphoma cell lines of different origin and immunophenotype, as well as three nonhematopoietic human tumor cell lines were examined by this technique. Human hematopoietic cell lines expressed two to four different polypeptide chains (180, 195, 205 and 220 kDa). Non-T, non-B cell lines, some B-cell lines and monocyte lymphoma cell line U 937 predominantly expressed higher molecular weight isoforms. In myeloid leukemia cell lines examined the expression of lower molecular weight isoform(s) predominated. Individual variability in LCA isoform patterns on cell lines precluded the strict correlation between these patterns and immunophenotype of the examined cell lines.