We describe the biochemical properties and cell surface distributions of three human T cell antigens (Leu-1, Leu-2a, and Leu-2b) which we postulate to be the homologues of the Lyt-1, Lyt-2, and Lyt-3 antigens that distinguish functional T cell subsets in the mouse. Leu-l, like Lyt-1, is on all thymocytes and peripheral T cells and is present in greater amounts on the helper/inducer subset than on the cytotoxic/suppressor subset. Both antigens increase in parallel fashion during T cell maturation in the thymus and each antigen is carried on a single 67,000-molecular weight (relative) (M(r)) polypeptide chain. Surprisingly, Leu-1 and Lyt-1 each are also expressed in readily detectable amounts on some B celI Ieukemias but not detectably so on normal B cells. Leu-2a and Leu-2b are antigens found only on suppressor/cytotoxic cells in the human and are very similar to the murine Lyt-2 and Lyt-3 antigens. In both species, the two antigens are on the same disulfide- linked multimeric molecules. Disulfide-bond reduction in both species yields subunits of similar size and charge. Lyt-3 and Leu-2b are extremely sensitive to trypsin digestion on viable cells whereas Lyt-2 and Leu-2a are much less so. A different membrane antigen, Leu-3, is an exclusive marker of the helper/inducer subset in man. No mouse homologue for this 55,000-M(r) protein is known. The maintenance of the homologous molecules on functionally distinct T cell subpopulations in two evolutionarily distant species suggests that the Lyt and Leu antigens perform essential functions for the cells on which they are found.

Measurement of intracellular ionized calcium concentrations ([Ca2+]i) has been indispensable in elucidating the central role of [Ca2+]i as a trigger of cellular responses to activating stimuli. Such studies have employed the dye quin2, which has not been readily adapted to analysis of individual small cells. We show here that the calcium response of large numbers of single cells can be analyzed with the use of flow cytometry and the recently described dye, indo-1. Such analyses demonstrate for the first time the heterogeneous nature of the [Ca2+]i response to mitogenic stimuli within populations of peripheral blood lymphocytes (PBL). By simultaneous quantitation of one- or two-color surface immunofluorescence labels, some of this heterogeneity of [Ca2+]i response in PBL is shown to be related to cellular immunophenotype. Almost all T cells responded to anti-CD3 antibody; however, the response is greater among CD4+ than CD8+ cells, and within the CD4+ population the rate of response to stimulation by antibody to CD3 differed between subpopulations defined by expression of the common leukocyte marker p220. In contrast, not all T cells responded to phytohemagglutinin (PHA), even at very high doses. As with anti-CD3, after stimulation with PHA, CD4+ cells showed a larger proportion of responding cells than did CD8+ cells. In separate experiments, indo-1 was found not to impair reproductive viability of PBL, thereby providing the potential for analysis of functional activity after the separation of cells by sorting on the basis of the [Ca2+]i response to stimuli. Mixing experiments indicated that a response of a subpopulation representing as little as 0.3% of total cells could be readily detected. Thus, the flow cytometric assay with indo-1 is the first technique that allows the quantitative analysis of response differences of small subpopulations of cells and intercellular variation in [Ca2+]i.

Human polymorphonuclear leukocytes (PMN) preincubated overnight with 100 U/mL gamma-interferon (IFN-gamma) had an increased metabolic response, as measured by iodination and/or superoxide production, to stimulation by tumor necrosis factor (TNF), granulocyte-macrophage colony-stimulating factor (GM-CSF), formylmethionyl-leucyl- phenylalanine (FMLP), opsonized zymosan, and lipopolysaccharide (LPS), as compared with cells comparably preincubated in the absence of IFN- gamma. The decline in the staphylocidal activity of the stored PMN was also prevented in part by IFN-gamma, as was the depressed adherence of PMN stimulated with phorbol myristate acetate (PMA), FMLP, TNF, GM-CSF, and LPS. This protective effect of IFN-gamma on PMN function was associated with the prolonged surface expression of the complement receptor three (CR3) alpha-chain (CD11b), CR3 beta-chain (CD18), FcRII (CD32), and FcRIII (CD16), and the appearance of surface FcRI (CD64). The polymerase chain reaction (PCR) was used to amplify neutrophil RNA- derived cDNA recognized by synthetic oliogonucleotides designed from published nucleotide sequences for specific proteins. Using this procedure, mRNA for gp91-phox, p67-phox, p47-phox, CD64, two forms of CD32, CD16, CD11b, CD18, and actin were found to be depressed after overnight storage of neutrophils, and this decrease in steady-state mRNA levels was in part or totally prevented by IFN-gamma. CD64 and gp91-phox mRNA were generally increased by IFN-gamma to a level greater than that of freshly isolated neutrophils. Northern analysis of CD64 and p47 phox mRNAs confirmed the findings with the PCR method. These findings suggest that storage of PMN in a functionally active state is favored by the presence of IFN-gamma.

We have investigated the expression of the common acute lymphoblastic leukemia antigen (cALLA) by nonlymphoid cells, using a new murine monoclonal antibody (designated 24.1), specific for cALLA. This antibody completely blocks the binding of monoclonal anti-cALLA antibody J-5. Furthermore, antibody 24.1 binds to cALLA with greater affinity and induces a greater degree of antigenic modulation than antibody J-5. With antibody 24.1, we have demonstrated that normal cultured marrow and skin fibroblasts and mature granulocytes express cALLA. By immune precipitation and polyacrylamide gel electrophoresis, cALLA from fibroblasts has a slightly lower molecular weight and cALLA from granulocytes a slightly higher molecular weight than cALLA from cultured human leukemic cell lines. Quantitative studies indicate, however, that cALLA expression is approximately 3-fold and 20-fold lower, respectively, on normal granulocytes and skin fibroblasts than on NALM-6 cells. cALLA may be expressed by cells with widespread distribution in multiple organ systems. These findings emphasize that differentiation markers that appear to be tumor or tissue specific may be found on cells of diverse origin.

The past few years have seen a surge of interest in B cell depletion therapy for patients with rheumatoid arthritis. This paper outlines the possible mechanism(s) by which B cell depletion therapy works. It is likely there is more than one mechanism and the relative importance of each mechanism depends on the target cell. These include CD20-induced apoptosis, complement dependent cytotoxicity, antibody dependent cell-mediated cytotoxicity, and selective targeting and depletion of B cell subsets. The implications of these mechanisms in the further improvement of B cell depletion therapy in rheumatoid arthritis and other autoimmune diseases are discussed.