Suml'l'lary A significant proportion of previously activated human T cells undergo apoptosis when triggered through the CD3/T cell receptor complex, a process termed activation-induced cell death (AICD). Ligation of Fas on activated T cells by either Fas antibodies or recombinant human Fas-ligand (Fas-L) also results in cytolysis. We demonstrate that these two pathways of apoptosis are causally related. Stimulation of previously activated T ceils resulted in the expression of Fas-L mILNA and lysis of Fas-positive target cells. Fas-L antagonists inhibited AICD of T cell clones and staphylococcus enterotoxin B (SEB)-specific T cell lines. The data indicate AICD in previously stimulated T ceils is mediated by Fas/Fas-L interactions. Mature peripheral T cells generally undergo activation . and proliferation when stimulated through the CD3/ TCR complex. Under certain circumstances, however, thymocytes, T cell hybridomas, and both CD4 + and CD8 + T cell clones (TCC) 1 undergo cell death when stimulated through the TCR with CD3 antibody in the absence of APC (1-6). This process is rapid and exhibits classic characteristics of apoptosis such as membrane blebbing, chromatin condensation, and the formation of DNA fragments of ",,200 bp. Deletion of T cells by apoptosis appears to be important not only in regulating autoreactive T cells in the thymus, but also in regulating the peripheral T cell pool (7,8). Little is known, however, about the mechanism that mediates the lytic process that has been termed activation-induced cell death (AICD).Fas/APO-1 (CD95) is a protein expressed on the surface of a variety of transformed cell lines and chronically stimulated T cells that can mediate apoptosis after ligation with a Fas-specific antibody (9-12). Under appropriate conditions Fas also transduces a stimulatory signal to certain B cell lines (13) and to .freshly isolated human peripheral blood T cells and thymocytes (14). To investigate a possible relationship between CD3-stimulated AICD and Fas-mediated T cell apoptosis, we have used a mAb directed against human Fas (Fas M3). Immobilized Fas M3 mAb is able to lyse Fas-expressing tumor cell lines in a manner analogous to Fas-ligand (Fas-L) or the prototypic Fas mAb, CH-11, whereas soluble Fas M3 blocks Fas-mediated killing (15).1 Abbreviations used in this paper: AICD, activation-induced cell death; Fas-L, Fas-ligand; SEB, staphylococcal enterotoxin B; TCC, T cell done, Materials and MethodsT Cell Lines and Clones. The alloreactive TCC used in this study were generated by establishing MLC in bulk culture for 7 d followed by limit dilution cloning in 96-well round-bottomed plates in the presence of 10 s irradiated allogeneic PBMC and 10 ng/mt of Ib2. TCC were maintained by stimulation with irradiated PBMC and soluble CD3 antibody (10 ng/ml) approximately every 2 wk and maintenance in IL-2 (10 ng/ml) between stimulations. Shortterm staphylococcal enterotoxin B (SEB)-specific T cell lines were established by stimulation of PBMC (106) with 5 /~g/ml SEB (Sigma Chemical Co., St. Loui...

SummaryCD40 is a member of the tumor necrosis factor (TNF) receptor family of cell surface proteins and was originally described as a B cell restricted antigen. Treatment of primary human monocytes with granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3), or interferon 3' (IFN-3') resulted in the induction of CD40 mRNA and enhancement of cell surface protein expression. CD40 was found to mediate monocyte adhesion to cells expressing recombinant CD40 ligand. CD40 ligand-transfected cells provided a potent costimulus for monocyte TNF-ot and IL-6 production in the presence of GM-CSF, IL-3, or IFN-3', and enhanced IL-8 production stimulated by GM-CSF or IL-3. In addition, CD40 ligand-transfected cells acting in the absence of a costimulus induced monocytes to become tumoricidal against a human mdanoma cell target. Collectively, these data indicate that CD40 ligand is pleiotropic with potent biological activity on monocytes.C D40 is a 50-kD molecule expressed on B cells, dendritic cells, some carcinoma cell lines, and human thymic epithelium (1-3). Abs specific for CD40 exhibit stimulatory effects on IL-4 activated B cells, including the induction of B cell growth, IgE secretion, and expression of CD23 (4-7). Human and murine forms of a ligand for CD40 (CD40L) 1 were recently cloned and demonstrated to be type II integral membrane proteins expressed primarily on activated CD4 + T cells (8)(9)(10)(11). CD40L provides a strong stimulatory signal to B cells from both species (8-10), however little is known about the regulation of expression of CD40 and the effects of CD40L on other cell types.Monocytes express a variety of cell surface proteins that are thought to play an important role in antigen presentation and the cell contact-dependent interaction of monocytes with other leukocytes. The expression of many of these proteins on monocytes can be regulated by a variety of cytokines, including GM-CSF, IL-3, IL-4, IFN-3", and . In this paper, we demonstrate that GM-CSF, IL-3, and IFN-'r enhance expression of CD40 by normal human monocytes. In addition, culture of monocytes with CD40L resulted in the stimulation or costimulation of cytokine production and in the induction of monocyte tumoricidal activity. Thus, CD40L is involved in the regulation of several critical aspects 1 Abbreviation used in this paper: L, ligand. of the immune response, and may be important in the cell contact-dependent activation of both T cells and monocytes during antigen presentation. Materials and MethodsMonocyte Purification and Culture. Monocytes were purified from normal donor PBMC by countercurrent elutriation (17) and were ~95 % pure by microscopic examination of Giemsa-stained cytocentrifuge preparations. Cells were cultured in RPMI 1640 medium containing 10% low endotoxin FBS, 50 U/ml penicillin, 50/zg/ml streptomycin, and 5 x 10 -s M 2-ME. Monocytes were cultured in polypropylene tubes (Falcon; Becton Dickinson Labware, Lincoln Park, NJ) for flow cytometry and mRNA analysis, in 24-well plates (Costar Corp., C...

SummaryThe Fas gene encodes a cell surface molecule that is a member of the the nerve growth factor/tumor necrosis factor receptor family of proteins and can mediate programmed cell death (apoptosis) in certain transformed cell lines . To characterize further the biological function ofFas, particularly with regard to its function in normal cells, a panel of monoclonal antibodies (mAbs) was generated against the extracellular portion of human Fas. Some of these mAbs induced apoptosis in transformed cell lines expressing Fas, but only when immobilized on the culture vessel . One of the new Fas mAbs (M38) was used for studies on normal lymphoid cells and found to stimulate the proliferation ofpurified human T cells and thymocytes when immobilized on culture wells along with CD3 antibody. T cell proliferation induced by Fas mAb was largely interleukin 2 independent and was demonstrated to be due to a direct effect on the precursor T cell. Thus, the data demonstrate that in addition to a role in the induction of apoptosis in certain transformed cell lines, the Fas protein may also play an important role in the activation and proliferation of normal T cells.

4-1BB was originally described as a cDNA expressed by activated murine T cells and subsequently demonstrated to encode a member of the tumor necrosis factor receptor family of integral membrane proteins. Recently, we identified and cloned a murine ligand for 4-1BB (mu4-1BB-L) and demonstrated it to be a member of an emerging family of ligands with structural homology to tumor necrosis factor. To characterize further the role of 4-1BB in the immune response we undertook to clone the human homologue of 4-1BB-L. However, attempts to isolate a cDNA encoding the human 4-1BB-L by cross-hybridization with the murine cDNA were unsuccessful. Therefore we first utilized cross-species hybridization to isolate a cDNA encoding human 4-1BB (hu4-1BB). A fusion protein consisting of the extracellular portion of hu4-1BB coupled to the Fc region of human immunoglobulin G1 (hu4-1BB.Fc) was then used to identify and clone a gene for human 4-1BB-L from an activated CD4+ T cell clone using a direct expression cloning strategy. Human 4-1BB-L shows 36% amino acid identity with its murine counterpart and maps to chromosome 19p13.3. Scatchard analysis demonstrated high-affinity binding of hu4-1BB.Fc to either native or recombinant human 4-1BB-L. Both monoclonal antibody to hu4-1BB and cells transfected with hu4-1BB-L induced a strong proliferative response in mitogen co-stimulated primary T cells. In contrast, ligation of 4-1BB on T cell clones enhanced activation-induced cell death when triggered by engagement of the TCR/CD3 complex.

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