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The B1 cell surface molecule (CD20) is a 35-kDa phosphoprotein expressed by B lineage cells during most stages of differentiation. Some monoclonal antibodies reactive with B1 induce activation while others, anti-B1a, inhibit B lymphocyte function. To further determine the requirement of B1 molecule function in proliferation and differentiation the effects of anti-B1a antibody binding on early cellular activation events were examined. Immunoglobulin secretion of lymphocyte cultures stimulated with pokeweed mitogen was maximally inhibited if the antibody (1-10 micrograms/ml) was added during the first 24 h of culture. However, even high antibody concentrations were unable to inhibit increases in free intracellular Ca2+ concentrations immediately following cross-linkage of cell surface immunoglobulin, or inhibit cell enlargement and the expression of transferrin and interleukin 2 receptors. Antibody binding to B1 inhibited RNA synthesis (37-80%) and progression through cell cycle following activation. In contrast, proliferation induced by phorbol myristate acetate was not inhibited by antibody binding to the B1 molecule. The findings that the earliest activation events and phorbol myristate acetate-induced proliferation were not inhibited by antibody binding to B1 suggest that inhibition is due to the blocking of a step of the activation process required for cell cycle progression and differentiation, rather than blocking initial signal transduction across the membrane or providing a negative or suppressive signal.

The intercellular adhesion molecule (ICAM-1) is a cell-surface molecule which binds to leukocyte function antigen-1 (LFA-1) and regulates both leukocyte adhesion to endothelial cells and immune functions requiring cell-cell contact. Membrane expression of ICAM-1 is highly regulated on all hematopoietic lineages. Cell membrane antigen is significantly expressed on a small subset of bone marrow (BM) progenitors but is weak or absent on all cell lineages once they enter the circulation. However, strong expression on tissue macrophages and germinal center B cells suggested that activated cells may show upregulated expression. When B cells, T cells, macrophages, or granulocytes were activated in vitro by suitable mitogens, ICAM-1 expression was induced in all cases. Parallel studies of hematopoietic tumors demonstrated a heterogeneity of expression which correlated with expression on their normal cellular counterparts. In particular, a striking correlation between expression on B-cell tumors and corresponding stages of B-cell differentiation was noted. The widely varying expression of ICAM-1 contrasts with LFA-1 which, while variable, is nevertheless significantly positive at all stages of differentiation. This suggests that the major regulation of homotypic adhesion mediated by the LFA-1/ICAM-1 linkage occurs through control of ICAM-1 expression. In keeping with this notion, ICAM-1 expression was also correlated with the "adhesiveness" of B-lymphoid tumors. Large solitary lymphoma masses showed intense expression of ICAM-1. Conversely, chronic lymphocytic leukemia (CLL) cells and lymphoma cells from tumors exhibiting diffuse, widespread lymph node disease showed weak expression. These observations are discussed in relation to the role of ICAM-1 in regulation of lymphoid recirculation and the biology of lymphoid tumors.

The role of intercellular adhesion molecule 1 (ICAM-1) in immune function was probed by using the Wehi-CAM-1 (W-CAM-1) monoclonal antibody. This antibody blocks aggregation of cell lines mediated by the ICAM-1 molecule and is here shown to block homotypic binding of purified populations of activated T and B lymphocytes (blasts) and also aggregation of mixed T- and B-cell blasts. We also demonstrate that W-CAM-1 inhibited T-cell adhesion to normal human endothelial cells, the first step in lymphocyte egress into the tissues. In tests of immune function, W-CAM-1 had a modest inhibitory effect on T- and B-cell activation by potent mitogens and no effect on the response of activated lymphocytes to lymphokines. By contrast, activation induced by cell-cell contact (mixed lymphocyte reaction, T-cell-mediated B-cell activation) was significantly inhibited. Moreover, the antibody was shown to block elements of both effector arms of the immune system (cytotoxic cell function and immunoglobulin production). These findings show that the ICAM-1 molecule is a central component of the mechanism of lymphocyte-endothelial cell adhesion. The studies of lymphoid function demonstrate a pivotal role for this molecule in both the T-cell/T-cell and T-cell/B-cell interactions, which underpin the regulation of the immune response, and in the mechanism of cell-mediated cytotoxicity.

Human thymocytes differed from peripheral blood T cells, in that they did not proliferate in response to mouse monoclonal antibodies specific for the T3 antigen complex (UCHT1 and OKT3), even in the presence of T-cell growth factor (IL-2) or with monocytes plus IL-2. The failure to respond was not the result of inhibition by cortical thymocytes, since OKT6-negative cells also did not respond when tested under conditions of limiting dilution. Of more importance, these antibodies rendered human thymocytes unresponsive to the lectin phytohaemagglutinin when added before, during, or immediately after addition of the lectin, an effect which was much more profound than the decrease in mitogenesis caused with blood mononuclear cells. These results illustrate a clear difference between medullary thymocytes and peripheral T cells in their ability to respond to signals transduced through the T3 antigen complex.

The characteristics of a novel T lineage-specific activation antigen, termed TLiSA1, are described. The antigen was detected with a mouse monoclonal antibody, LeoA1, that was raised against activated human T cells generated in mixed lymphocyte culture (MLC). The antigen became strongly expressed on T cells 48-72 h after stimulation with phytohemagglutinin, and retained expression on MLC-activated T cells after 10 d of culture. The antigen was absent from a range of human T, B, myeloid, fibroblast, and tumour cell lines, but was present on the surface of the interleukin 2 (IL-2)-dependent gibbon cell line MLA-144. Analysis of the antigen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates obtained from activated human T cells demonstrated a broad band in the region of 70 kD, whereas precipitates obtained from MLA-144 revealed a single narrow band of 95 kD. The molecule was expressed with a maximum density of 66,000 copies per cell on the surface of MLC-activated T cell blasts, as assessed by Scatchard analysis. TLiSA1 was distinguished from the IL-2 receptor bound by the anti-Tac monoclonal antibody by demonstrating that the antigens did not comodulate or coprecipitate, and by constructing an IL-2-independent human T X T hybrid that expressed the TLiSA1 but not the Tac antigen. MLC with B lymphoblasts was used to generate cytotoxic T lymphocytes (CTL) specific for the stimulating cell, and anomalous killer (AK) cells able to kill melanoma target cells. The presence of LeoA1 or F(ab')2 fragments of the antibody from the beginning of coculture did not affect proliferation in these cultures, but did inhibit the induction of both CTL and AK cells from their precursors. This inhibition of differentiation by LeoA1 was confirmed under conditions of limiting dilution, where it was shown that the antibody reduced the frequency of CTL produced, and greatly (fourfold) reduced the frequency of AK cells generated from their precursors. We discuss the possibility that human CTL may express a differentiation factor receptor that is distinct from the receptor for IL-2.

The selective destruction of the pancreatic islet beta cells in type 1 diabetes mellitus is thought to be mediated by a cellular autoimmune process, possibly triggered by virus infection in genetically susceptible individuals. Because of the potentially important role of cell-cell adhesion in the immune response, we investigated whether cytokine products of mononuclear cells, or virus infection, induced the expression of intercellular adhesion molecule 1 (ICAM-1) on human endocrine islet cells. By flow cytofluorimetry, control islet cells did not express detectable ICAM-1. However, after a 72-hr exposure of islets to interferon gamma (IFN-gamma) and/or tumor necrosis factor alpha (TNF-alpha) (each at 250 units/ml), ICAM-1 was induced on greater than 85% of islet cells. IFN-gamma was 50% more potent than TNF-alpha; together, their effects were additive. Class I major histocompatibility complex (MHC) protein expression, detected on control islet cells, was also stimulated by IFN-gamma and/or TNF-alpha. In contrast, infection with reovirus type 3 did not induce ICAM-1 on islet cells, although it stimulated the expression of class I MHC proteins. By double-label indirect immunofluorescence microscopy, ICAM-1 expression was identified on both beta (insulin-secreting) and delta (somatostatin-secreting) islet cells. Monoclonal antibody to ICAM-1 precipitated protein of Mr 97,000 from [35S]methionine-labeled islets exposed to IFN-gamma and TNF-alpha, but not from control islets. RNA blot analysis revealed a major species of 3.3 kilobases and a minor species of 2.2 kilobases induced in islets exposed to the cytokines. These findings have implications for the molecular mechanisms of beta-cell destruction in type 1 diabetes, in that expression of ICAM-1 by beta cells may facilitate adhesion of antigen-targeted immune cells.

The study of human leukocyte antigens, predominantly by monoclonal antibody techniques, is a rapidly changing area of basic research and clinical investigation. This review outlines some of the results and trends of research in this field. Of particular importance is the updating of the current nomenclature. The CD classification of these antigens has become the standard form in published literature and provides a basis for standardization of clinical reporting. The current CD classification is presented in the form of a list, with a brief summary of each antigen beside each entry. The results reviewed range from the section on CD1 antigen in which the data presented are primarily concerned with the underlying biology of the antigens to the section on clinical application which has little biological content.

Two independently produced monoclonal antibodies, UCHT1, an IgG1 antibody, and OKT3, an IgG2 antibody, which specifically identify all human peripheral T lymphocytes, were shown to possess similar functional properties. Both were highly mitogenic to peripheral blood T lymphocytes, and UCHT1 blocked specific T lymphocyte killing of autologous B lymphoblasts when present during either the induction or the cytotoxic phase of the reaction; this, too, is a property reported for OKT3 in an analogous situation. Both antibodies also inhibited the 6-day proliferative response to soluble antigens and mixed lymphocyte culture, although this may be attributable to their strong mitogenicity earlier in culture. These properties were not found with other monoclonal antibodies directed against human T lymphocytes, and suggested that UCHT1 and OKT3 recognize important functional structure(s) on the T cell surface. The identity of the antigen recognized by these two antibodies was demonstrated by 2-directional co-capping, by cold antibody inhibition of binding of radiolabeled antibody, and by immunoprecipitation and two-dimensional gel analysis of labeled T cell surface proteins. Both UCHT1 and OKT3 bound with similar affinities (Ka approximately 2 X 10(9) M-1) to the same number of binding sites (2.8 X 10(4) sites/activated T cell), and both precipitated the same glycoprotein complex with a m.w. of approximately 19,000 in the presence of reducing agent.

Distinct populations of human B lymphocytes can be identified by their expression and/or co-expression of the B cell-restricted antigens B1 and B2. Dual fluorochrome staining and flow cytometric cell sorting permitted the isolation of the B1+B2+ and B1+B2- cells to homogeneity. In contrast, very few B1-B2+ cells were obtainable from normal lymphoid organs. Virtually all B1+B2+ cells expressed IgM and IgD, but lacked IgG and the plasma cell antigens PCA-1 and PC-1, whereas the B1+B2- cells more frequently expressed IgG, PCA-1 and PC-1. Both populations were noncycling and were composed of similar percentages of small and large cells. The B1+B2+ cells proliferate to anti-mu or to anti-mu + PHA-LCM, but not to PHA-LCM alone. They require both T cells and PWM to produce Ig. In contrast, B1+B2-cells do not significantly proliferate to anti-mu, PHA-LCM, or anti-mu and PHA-LCM. They produce Ig in response to T cells alone without PWM. These phenotypic and functional observations provide preliminary evidence that these populations are distinct and that the B1+B2+ cell may be a "resting" B cell, whereas the B1+B2- cell appears to be more "differentiated." The present studies further suggest that they will also be helpful in characterizing B cells in some human disease states. We believe that the identification and isolation of these and similar subsets of B cells defined by differing cell surface phenotype should aid our understanding both of normal B cell differentiation and of B cell disease states.

The characterization of a new human B cell-restricted activation antigen (B5) is described in this report. With the use of a monoclonal antibody to B5, we show that B5 can be detected on peripheral blood or splenic B cells after 1 day of stimulation with either anti-immunoglobulin, protein A, Epstein Barr virus, or pokeweed mitogen. In contrast, B5 was not expressed on resting B, T, or myeloid cells. More important, B5 could not be detected on activated T cells or monocytes. The B5 antigen was expressed on some lymphoblastoid B cell lines and B cell neoplasms but was not expressed on leukemias or lymphomas of T or myeloid origin. The B5 antigen is distinct from previously reported B cell activation antigens by its m.w. and pattern of cellular expression. These studies suggest that B5 is a novel B cell-restricted activation antigen, which may be useful to study the events of early human B cell activation.

The B1 molecule is a 32,000 m.w. phosphorylated cell surface protein expressed exclusively by B cells from the mid pre-B until the plasma cell stage of differentiation. Two monoclonal antibodies (gamma 2a and mu) reactive with this molecule were used to assess the role of B1 in B cell activation, proliferation, and differentiation. The anti-B1 antibodies at concentrations ranging from 0.1 to 100 micrograms/ml significantly inhibited B cell proliferation induced by anti-mu antibodies, Staphylococcus aureus Cowan strain 1, activated T cells, and Epstein Barr virus. Although capable of inhibiting proliferation, anti-B1 antibody in soluble form or coupled to beads did not activate B cells or induce proliferation. Antibodies of comparable isotypes or against other B cell-restricted antigens, including B2, B4, B5, and HB-5, did not inhibit activation. Pretreatment of B cells with anti-B1 antibody did not inhibit activation, indicating that B cells had to be cultured with anti-B1 antibody for anti-B1-mediated inhibition to occur. Maximum inhibition was obtained when anti-B1 antibody was added at the initiation of culture. In agreement with this, growth factor-dependent proliferation of preactivated B cells was not inhibited by anti-B1 antibodies. Comparable inhibition of B cell activation was noted with antibodies reactive with class II antigens of the major histocompatibility complex with the exception that anti-B1 antibody inhibited immunoglobulin secretion in pokeweed mitogen assays, whereas anti-DR antibody did not. These results suggest that the B1 molecule may serve a central role in the regulation of B cell activation and differentiation.