SummaryThe Apo-1/Fas antigen (CD95) mediates programmed cell death of lymphocytes when bound by Fas ligand or anti-Apo-1/Fas antibody. In contrast, the CD40 antigen provides a potent activation and survival signal to B lymphocytes when it is engaged by its T cell ligand (CD40L, gp39) or cross-linked by anti-CD40 antibody. In this study, we use human tonsillar B cells and the Ramos Burkitt's lymphoma B cell line, which serves as a model for human germinal center B lymphocytes, to study the effectors of Apo-1/Fas expression and apoptosis of human B cells. We found that Apo-1/Fas expression was upregulated on both malignant and normal human B lymphocytes after CD40 ligation induced by (a) cognate T helper-B cell interaction mediated by microbial superantigen (SAg); (b) contact-dependent interaction with CD40L +, but not CD40L-Jurkat mutant T cell clones; and (c) monoclonal anti-CD40, but not any of a panel of control antibodies. Enhanced B cell Fas/Apo-1 expression is functionally significant. Coculture of Ramos Burkitt's lymphoma line cells with irradiated SAg-reactive CD4 + T cells with SAg or CD40L + Jurkat T cells results in B cell apoptosis, evidenced by reduced cell viability and DNA laddering. This process is augmented by the addition ofanti-Apo-1/Fas monoclonal antibody, consistent with an acquired susceptibility to Apo-1/Fas-mediated apoptosis. These data support an immunoregulatory pathway in which seemingly contradictory signals involving the B cell proliferation/survival antigen CD40, as well as the Apo-1/Fas molecule, which mediates programmed cell death oflymphocytes, are linked in the process of human B cell activation.Po-1/Fas (CD95) is a 48-kD cell surface antigen of the TNF/nerve growth factor (NGF) 1 receptor superfamily that mediates apoptosis of T and B lymphocytes (1). The interaction of Apo-1/Fas and its ligand have been shown to play a central role in the regulation of programmed cell death of mature peripheral murine and human T lymphocytes. Defects in both Fas and its ligand have been described in murine models of lymphoproliferative disease characterized by features of SLE (2-4), suggesting that this pathway may be responsible for the deletion ofautAbbreviations used in this paper: CD40L, CD40 ligand; MAM, Mycoplasma arthritidis-derived microbial superantigen; MHC II, MHC class II; NGF, nerve growth factor; SAg, microbial superantigen; TSST-1, toxic shock syndrome toxin 1; TMAMxr, irradiated MAM-reactive CD4 § cells; TTSSTxF, TSST-l-reactive CD4 + cells. toreactive clones. In peripheral T lymphocytes, TCR signaling leads to upregulation of Apo-1/Fas and, on Thl cells, Fas ligand expression, resulting in fratricide or even suicide of antigen-activated T cells (5-8). Although Apol/ Fas expression occurs early after antigen stimulation, susceptibility to Apo-1/Fas-mediated apoptosis develops slowly (9), allowing for the clonal expansion and effector function of antigen-activated T cells before their apoptotic elimination. B lymphocytes also express the Apo-1/Fas receptor (10, 11), and they a...
Three independent mutations involving the apoptosis-1 (APO-1)/Fas receptor or its putative ligand have led to lupuslike diseases associated with lymphadenopathy in different strains of mice. To determine whether humans with SLE also have a defect in this apoptosis pathway, we analyzed the expression of APO-1 on freshly isolated blood mononuclear cells and on lymphocytes activated in vitro using flow cytometry and the monoclonal antibody anti-APO-1. Significantly higher levels of APO-1 expression were detected on freshly isolated peripheral B cells and both CD4' and CD8' T lymphocyte populations obtained from lupus patients when compared with normal controls (P < 0.001). Almost 90% of the cells that stained positive for APO-1 also expressed the CD29 antigen, suggesting that APO-1 was upregulated after lymphocyte activation in vivo. No defect in APO-1 regulation was detected after activation of SLE T (with anti-CD3) or B (with Staphylococcus aureus Cowan 1) lymphocytes in the presence of IL-2 in vitro. Similarly, the anti-APO-1 antibody induced apoptosis in 74±5% of activated SLE T cells in vitro compared with 79±6% of the normal controls (P > 0.05). These results reveal that, while APO-1 / Fas may play an important role in the regulation of lymphocyte survival in SLE, no consistent defect in the expression or function of the receptor could be detected in these studies. (J. Clin.
Previous studies (1-9) have suggested that peripheral human T cells can be divided into two mutually exclusive functional subsets by the two monoclonal antibodies, OKT8 and OKT4. The OKT8+ subset does not provide helper activity but contains cells capable of suppressing B cell differentiation . Importantly, the suppression observed with OKT8+ cells requires the presence of radiosensitive OKT4 + cells . Potent helper activity is found in a radiosensitive OKT4' subset . Irradiated OKT4+ cells also induced B cell differentiation, but only at high ratios of T cells to B cells . In our previous studies (7), we also found that the addition of graded numbers of radiosensitive OKT4 + cells to B cells eventually decreased the net helper activity observed . These experiments raised the possibility that precursors of suppressor cells may be contained within the OKT4 + population .The current study was undertaken to further investigate the functional heterogeneity within the OKT4+ population. In particular, we asked whether OKT4 + cells could be induced to differentiate into immunoregulatory cells capable of suppressing B cell differentiation . In the experiments reported here, we observed that although in vitro pokeweed mitogen (PWM) -activated' OKT4+ cells can function as radioresistant helper cells, these activated OKT4+ cells could also exert potent feedback suppression . This suppression mediated by activated OKT4+ cells required the presence of radiosensitive cells contained within the resting OKT4 + population. These data emphasize the potential role of interactions of T cell subsets contained exclusively within the OKT4 + population in the immunoregulation of B cell differentiation . Volume 154 August 1981 459-467 Materials and MethodsLymphocyte Preparation and Isolation ofHuman T and B Cells . Fresh peripheral blood lymphocytes were isolated from consenting healthy human volunteers by Ficoll-Hypaque density gradient centrifugation . Highly enriched population of T and B cells were then isolated by methods previously described in detail (10) . In brief, human mononuclear cells were washed in minimum essential medium (Grand Island Biological Co ., Grand Island, N . Y .) containing 5% fetal calf serum (FCS ; Microbiological Associates, Bethesda, Md .) and then separated into surface Ig* Supported in part by grants AI-14969 and AI-11524 from the National Institutes of Health, and by The Robert Wood Johnson, Jr . Charitable Trust and The Arthritis Foundation .'Abbreviations used in this paper: C, complement ; E+ , E rosette positive ; FCS, fetal calf serum ; PFC, plaqueforming cells ; PWM, pokeweed mitogen. J . Exp . MED .
Three different murine monoclonal antibodies to the human clonotypic T-cell antigen receptor (l) immunoprecipitate the -sp chain heterodimer; (it) induce comodulation of the clonotypic molecule with the T3 molecular complex; (iiW) stain small populations of normal polyclonal T cells, suggesting that they react with variable or joining region determinants of the clonotypic receptor; and (iv) induce proliferation of resting T cells. While two of these antibodies detect the clonotypic receptor in all individuals studied, the third antibody (OT145), described herein, does not detect the T-cell antigen receptor on T cells of all individuals. By indirect immunofluorescence, three groups can be distinguished within a population of individuals (n = 138) by OT145. The antigen receptor of T lymphocytes may be classified as a member of a large family of related molecules, the immunoglobulin supergene family (1). Several members of this supergene family display genetically transmitted polymorphisms. For example, using antibodies as a probe, early investigators detected allelic forms of immunoglobulin chains, termed allotypes (2, 3). Allotypic systems have been described on both the constant and variable (V) portions of immunoglobulin heavy chains and on constant portions of light chains (4-6). Allotypes are inherited in an autosomal codominant manner. In view of the remarkable structural similarities between the T-cell antigen receptor and immunoglobulins, it is reasonable to expect that allotypic determinants will be identified on the a and/or 83 chains of the T-cell antigen receptor. Recent studies have used restrictionfragment-length polymorphism (RFLP) to identify polymorphisms in the human a and 8 chain genes (7,8). While these RFLPs map to introns of both genes, similar polymorphisms of the exons, which could result in amino acid sequence differences responsible for the expression of allotypic determinants, have not yet been described.Our group and others have raised monoclonal antibodies (mAbs) against the T-cell antigen receptor, some of which react with small subsets of normal human T cells (9-13). In one case the determinant recognized by this type of mAb was found to be a 13 chain V region determinant (12). We have characterized the small subsets of peripheral blood T cells identified by two such mAbs, designated S511 and C37 (10, 11). In both cases S511-and C37-reactive (S511' and C37+) T cells may belong to either the T4' or T8' subset of T cells.In both cases the mAbs can be used as mitogens to selectively expand these small populations of T cells and derive interleukin 2-dependent polyclonal and cloned S511' or C37' T-cell lines (refs. 10 and 11; unpublished data
Chronic lymphocytic leukemia (CLL) is characterized by a clonal expansion of CD5+ B cells in the peripheral blood. Associated immune aberrations include abnormal Th-cell function and pathogenic autoantibodies. Under most circumstances, CLL B cells do not proliferate in culture and express a limited repertoire of surface antigens, including CD19, CD20, CD23, CD27, CD40, and CD70. In this report, we demonstrate that freshly isolated B cells from a subset of CLL cases constitutively express CD40 ligand (CD40L, CD154), a member of the tumor necrosis factor family which is normally expressed by activated CD4+ T cells and mediates T-cell–dependent B-cell proliferation and antibody production. The degree of CD40L expression varied considerably among the CLL cases examined. CD40L was detected in purified CLL B cells by immunofluorescence flow cytometry, by RT-PCR, and by immunoprecipitation. To demonstrate that CD40L in the CLL B cells is functional, we used irradiated CLL cells to stimulate IgG production by target, nonmalignant B cells in coculture. The CLL B cells induced IgG production by normal B cells to a similar degree as did purified T cells in a process which was partially inhibited by monoclonal antibody to CD40L. This is one of the first reports of CD40L expression in a B-cell tumor. The data suggest that CD40L in the tumor cells may be a factor in the generation of pathologic antibodies by normal B cells in some patients with CLL.
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