Monoclonal antibodies against the 4-1BB T-cell activation molecule eradicate established tumors.

The 4-1BB glycoprotein is a member of the tumor necrosis factor receptor superfamily and binds to a high-affinity ligand (4-1BBL) expressed on several antigen-presenting cells such as macrophages and activated B cells. Expression of 4-1BB is restricted to primed CD4+ and CD8+ T cells, and 4-1BB signaling either by binding to 4-1BBL or by antibody ligation delivers a dual mitogenic signal for T-cell activation and growth. These observations suggest an important role for 4-1BB in the amplification of T cell-mediated immune responses. We now show that administration of anti-4-1BB monoclonal antibodies can eradicate established large tumors in mice, including the poorly immunogenic Ag104A sarcoma and the highly tumorigenic P815 masto cytoma. The immune response induced by anti-4- 1BB monoclonal antibodies is mediated by both CD8+ and CD4+ T cells and is accompanied by a marked augmentation of tumor-selective cytolytic T-cell activity. Our data suggest that a similar approach may be efficacious for immunotherapy of human cancer.

B cell activation via CD40 is required for specific antibody production by antigen-stimulated human B cells.

Costimulatory signals provided by T cells are required for B cells to produce specific antibody (Ab) to T-dependent antigen (Ag) bacteriophage phi x 174. In this study, we demonstrate that if cultured in the presence of anti-CD40, interleukin 10 (IL-10), and Ag, purified B cells can produce antiphage Ab in quantities comparable to those synthesized by B cells cocultured with Ag and T cells. Isotypes produced by B cells in this culture system correspond to those observed in sera of B cell donors. Culture of immunoglobulin (Ig)D- and IgD+ B cells reveals that Ag-induced production of antiphage Ab is restricted to IgD- subset of B cells. In the absence of Ag, anti-CD40/IL-10-stimulated B cells produce only minute amounts of antiphage Ab, indicating that Ag stimulation is indispensable and provides a signal that is synergistic with anti-CD40 and IL-10. Addition of a soluble form of the CD40 ligand (sgp39) to the culture system has a similar effect on specific Ab synthesis as anti-CD40; addition of the soluble construct, CD40 Ig, known to inhibit gp39/CD40 interaction, suppresses in vitro antiphage Ab production by Ag exposed peripheral blood mononuclear cells. Finally, in vivo requirement of gp39/CD40 interaction for specific Ab production was demonstrated by the finding that activated T cells from patients with x-linked hyper IgM syndrome express functionally defective gp39 and respond with depressed Ab titers and fail to switch from IgM to IgG after multiple phage immunizations. These observations illustrate that in vitro and possibly in vivo Ag-specific Ab synthesis requires the presence of Ag and IL-10, and activation signals via CD40.

Differential expression of Fas (CD95) and Fas ligand on normal human phagocytes: implications for the regulation of apoptosis in neutrophils.

Human neutrophils, monocytes, and eosinophils are known to undergo apoptotic cell death. The Fas/Fas ligand pathway has been implicated as an important cellular pathway mediating apoptosis in diverse cell types. We conducted studies to examine the importance of the Fas/FasL system in normal human phagocytes. Although Fas expression was detected on neutrophils, monocytes, and eosinophils, constitutive expression of FasL was restricted to neutrophils. The three types of phagocytes demonstrated differential sensitivity to Fas-induced apoptosis. Only neutrophils were highly susceptible to rapid apoptosis in vitro after stimulation with activating anti-Fas IgM (mAb CH-11). Fas-mediated neutrophil apoptosis was suppressed by incubation with G-CSF, GM-CSF, IFN-gamma, TNF-alpha, or dexamethasone, as well as the selective tyrosine kinase inhibitors, herbimycin A and genistein. Spontaneous neutrophil death in vitro was partially suppressed by Fas-Ig fusion protein or antagonistic anti-Fas IgG1 (mAb ZB4). In coculture experiments, neutrophils released a soluble factor inducing death in Fas-susceptible Jurkat cells via a mechanism sensitive to the presence of Fas-Ig or anti-Fas IgG1. Immunoblot analysis using specific anti-human FasL IgG1 (mAb No. 33) identified a 37-kD protein in lysates of freshly isolated neutrophils and a 30-kD protein in the culture supernatant of neutrophils maintained in vitro. Our results suggest that mature neutrophils may be irrevocably committed to autocrine death by virtue of their constitutive coexpression of cell-surface Fas and FasL via a mechanism that is sensitive to proinflammatory cytokines, glucocorticoids, and inhibitors of tyrosine kinase activity. Furthermore, neutrophils can serve as a source of soluble FasL, which may function in a paracrine pathway to mediate cell death.

T cell subsets defined by expression of Lyt-1,2,3 and Thy-1 antigens. Two-parameter immunofluorescence and cytotoxicity analysis with monoclonal antibodies modifies current views.

Using monoclonal antibodies and multiparameter fluorescence analyses, we show that the expression of Lyt-1, Lyt-2, and Lyt-3 on T cell subpopulations is more complex than was originally recognized by the cytotoxic depletion studies with conventional reagents that defined the Lyt-1+2+3+, Lyt-1+2-3-, and Lyt-1-2+3+ populations. We detect at least some Lyt-1 on all T (Thy-1-bearing) lymphocytes; however, in agreement with previous studies, we find that Lyt-2+3+ cells are more difficult to depelete with anti-Lyt-1 than Lyt-1+2-3- cells. Surprisingly, we found a small subpopulation of cells carrying relatively large amounts of Lyt-1 and no Thy-1 detectable by fluorescence-activated cell sorter analysis. We also detect cells with this phenotype histologically in B cell zones (primary follicles) and germinal centers in spleen and lymph nodes. In general, the Lyt-1 only population represents approximately 2% of spleen cells. The relative quantitative expression of Thy-1, Lyt-1, Lyt-2, and Lyt-3 changes systematically during T cell maturation. Among Lyt-1+2+3+ cells in the thymus, Thy-1 and Lyt-2 are high, whereas Lyt-1 is low. Among splenic T cells, in contrast, Thy-1 is low, Lyt-1 is high, and Lyt-2 and Lyt-3 are a little higher than in thymus. In general, Thy-1 expression is negatively correlated with Lyt-1. Thus, even among splenic and lymph node T cells subpopulations exist that tend to be either high Thy-1 and low Lyt-1 or vice versa. Lyt-2+3+ cells represent approximately 85% of thymocytes but only approximately 35% of splenic or lymph node T cells. The Lyt-2+3+ cells are found predominantly in the low Lyt-1, high Thy-1 subpopulation.

Expression of Lyt-1 antigen on certain murine B cell lymphomas.

Although the Lyt-1 antigen has previously been considered a T cell-specific marker, recent evidence suggests that a population of Thy-1-, Lyt-1+ cells exists in normal lymphoid tissues. In this study, we have observed that the WEHI-55, WEHI-259, and CH5 B cell lymphomas express high levels of the Lyt-1 antigen, as detected by monoclonal antibodies using the fluorescence activated cell sorter. Three other B cell lymphomas of the 11 examined also gave weak but detectable reactions with the anti Lyt-1 monoclonal antibody. Except for the expression of the Lyt-1 antigen, these lymphomas are typical of cells in the B cell lineage with respect to surface phenotype. The Lyt-1 glycoprotein immunoprecipitated from metabolically labeled WEHI-55 cells is similar in structure to the Lyt-1 glycoprotein on thymocytes. These findings are similar to recent reports that B-type human lymphocytic leukemia cells express the putative human homologue of Lyt-1, the Leu-1 antigen.

Lyt-2 and lyt-3 antigens are on two different polypeptide subunits linked by disulfide bonds. Relationship of subunits to T cell cytolytic activity.

Lyt-2 and Lyt-3 antigens are carried on separate disulfide-bonded subunits of the same cell surface macromolecules. These are present on thymocytes in a variety of multimeric forms consisting of disulfide-bonded dimers, tetramers, and hexamers of pairwise combinations of three subunits (30,000, 34,000, and 38,000 Mr). From reduced and alkylated Nonidet-P40 thymus extracts, a monoclonal anti-Lyt-3 precipitates only the 30,000 Mr subunit, but not the 30,000 Mr subunit. Almost all of the Lyt-2 and Lyt-3 subunits on the cell are covalently linked by disulfide bonds. However, small amounts of free Lyt-3 subunit was seen in some experiments. Similarly, small amounts of Lyt-2-3- material, consisting of dimers of the 38,000 and 34,000 Mr subunits were identified. Each of the three subunits migrated with a basic charge (pI greater than 8) on two-dimensional gels. Cytotoxic effector cells that are blocked by anti-Lyt-2 and anti-3 can be treated with trypsin and other arginine-specific proteases to remove these antigens. At low concentrations of these proteases, Lyt-3 antigens are selectively removed. After selective removal of Lyt-3 antigens, cytotoxic effector cells are still active and blocking of activity by anti-Lyt-3 is significantly reduced, whereas blocking of activity by anti-Lyt-2 is significantly increased. Neither Lyt-2 nor Lyt-3 is allelically excluded on thymocytes or T cells. These results suggested that the Lyt-2, Lyt-3 macromolecules are associated with but do not serve as the T cell antigen receptor.

Binding of the B cell activation antigen B7 to CD28 costimulates T cell proliferation and interleukin 2 mRNA accumulation.

A successful immune response requires intercellular contact between T and B lymphocytes. We recently showed that CD28, a T cell surface protein that regulates an activation pathway, could mediate intercellular adhesion with activated B cells by interaction with the B7 antigen. Here we show that CD28 is the primary receptor for B7 on activated peripheral blood T cells, that CD28 binds to B7 in the absence of other accessory molecules, and that interaction between CD28 and B7 is costimulatory for T cell activation. To characterize the binding of CD28 to B7, we have produced genetic fusions of the extracellular portions of B7 and CD28, and immunoglobulin (Ig) C gamma 1 chains. 125I-labeled B7 Ig bound to CD28-transfected Chinese hamster ovary (CHO) cells, and to immobilized CD28 Ig with a Kd approximately 200 nM. B7 Ig also inhibited CD28-mediated cellular adhesion. The function of CD28-B7 interactions during T cell activation was investigated with soluble fusion proteins and with B7-transfected CHO cells. Immobilized B7 Ig and B7+ CHO cells costimulated T cell proliferation. Stimulation of T cells with B7+ CHO cells also specifically increased levels of interleukin 2 transcripts. These results demonstrate that the CD28 signaling pathway could be activated by B7, resulting in increased T cell cytokine production and T cell proliferation. Cellular interactions mediated by B7 and CD28 may represent an important component of the functional interactions between T and B lymphoid cells.

Identification of amino acid residues important for ligand binding to Fas.

The interaction of Fas (CD95), a member of the tumor necrosis factor receptor (TNFR) family, and its ligand (FasL) triggers programmed cell death (apoptosis) and is involved in the regulation of immune responses. Although the Fas-FasL interaction is conserved across species barriers, little is currently known about the molecular details of this interaction. Our aim was to identify residues in Fas that are important for ligand binding. With the aid of a Fas molecular model, candidate amino acid residues were selected in the Fas extracellular domain 2 (D2) and D3 and subjected to serine-scanning mutagenesis to produce mutant Fas molecules in the form of Ig fusion proteins. The effects of these mutations on FasL binding was examined by measuring the ability of these proteins to inhibit FasL-mediated apoptosis of Jurkat cells and bind FasL in ELISA and BIAcore assays. Mutation of two amino acids, R86 and R87 (D2), to serine totally abolished the ability of Fas to interact with its ligand, whereas mutants K84S, L90S, E93S (D2), or H126S (D3) showed reduced binding compared with wild-type Fas. Two mutants (K78S and H95S) bound FasL comparably to wild type. Therefore, the binding of FasL involves residues in two domains that correspond to positions critical for ligand binding in other family members (TNFR and CD40) but are conserved between murine and human Fas.

In vivo CD40-gp39 interactions are essential for thymus-dependent humoral immunity. II. Prolonged suppression of the humoral immune response by an antibody to the ligand for CD40, gp39.

The ligand for CD40 has been recently identified as a 39-kd protein, gp39, expressed on the surface of activated CD4+ T helper cells (Th). In vitro, soluble CD40 and anti-gp39 have been shown to block the ability of Th to activate B cells, suggesting that gp39-CD40 interactions are important to T cell-dependent B cell activation. Here it is shown that in vivo administration of anti-gp39 dramatically reduced both primary and secondary humoral immune responses to erythrocytes and soluble protein antigens without altering responses to the T-independent type II antigen, trinitrophenyl-Ficoll. Treatment of mice for 4 d with anti-gp39 inhibited the anti-sheep red blood cell (SRBC) response for at least 3 wk and inhibited the expression of all immunoglobulin isotypes in secondary responses to the protein antigen, keyhole limpet hemocyanin. To examine the direct effect of anti-gp39 on Th function, SRBC-immune Th cells from anti-gp39-treated mice were adoptively transferred and shown to be fully capable of providing help. These results suggest that anti-gp39 treatment does not cause Th deletion or anergy. Anti-gp39 may mediate its profound immunosuppressive effects on humoral immunity by blocking gp39-CD40 interactions. Moreover, these studies establish gp39-CD40 as an important receptor-ligand pair for the targeting of therapeutic antibodies to control thymus-dependent humoral responses.

CTLA-4 is a second receptor for the B cell activation antigen B7.

Functional interactions between T and B lymphocytes are necessary for optimal activation of an immune response. Recently, the T lymphocyte receptor CD28 was shown to bind the B7 counter-receptor on activated B lymphocytes, and subsequently to costimulate interleukin 2 production and T cell proliferation. CTLA-4 is a predicted membrane receptor from cytotoxic T cells that is homologous to CD28 and whose gene maps to the same chromosomal band as the gene for CD28. It is not known, however, if CD28 and CTLA-4 also share functional properties. To investigate functional properties of CTLA-4, we have produced a soluble genetic fusion between the extracellular domain of CTLA-4 and an immunoglobulin C gamma chain. Here, we show that the fusion protein encoded by this construct, CTLA4Ig, bound specifically to B7-transfected Chinese hamster ovary cells and to lymphoblastoid cells. CTLA4Ig also immunoprecipitated B7 from cell surface 125I-labeled extracts of these cells. The avidity of 125I-labeled B7Ig fusion protein for immobilized CTLA4Ig was estimated (Kd approximately 12 nM). Finally, we show that CTLA4Ig was a potent inhibitor of in vitro immune responses dependent upon cellular interactions between T and B lymphocytes. These findings provide direct evidence that, like its structural homologue CD28, CTLA-4 is able to bind the B7 counter-receptor on activated B cells. Lymphocyte interactions involving the B7 counter-receptor are functionally important for alloantigen responses in vitro.

Coexpression and functional cooperation of CTLA-4 and CD28 on activated T lymphocytes.

T cell costimulation by molecules on the antigen presenting cell (APC) is required for optimal T cell proliferation. The B7 molecule on APC binds the T lymphocyte receptor CD28, triggering increased interleukin 2 (IL-2) production and subsequent T cell proliferation. CTLA-4 is a predicted T cell membrane receptor homologous to CD28, which also binds the B7 counter receptor, but whose distribution and function are unknown. Here we have developed monoclonal antibodies (mAbs) specific for CTLA-4 and have investigated these questions. mAbs were produced that bound CTLA-4 but not CD28, and that blocked binding of CTLA-4 to B7. CTLA-4 expression as measured by these mAbs was virtually undetectable on resting T cells, but was increased several hundred-fold during T cell activation. On activated lymphocytes, CTLA-4 was expressed equally on CD4+ and CD8+ T cell subsets and was coexpressed with CD25, CD28, and CD45RO. CTLA-4 expression was lower than that of CD28, reaching a maximum of approximately 1/30-50 the level of CD28. Despite its lower expression, CTLA-4 was responsible for much of the B7 binding by large activated T cells. Anti-CTLA-4 mAb 11D4 and anti-CD28 mAb 9.3 acted cooperatively to inhibit T cell adhesion to B7, and to block T cell proliferation in primary mixed lymphocyte culture. When coimmobilized with anti T cell receptor (TCR) mAb, anti-CTLA-4 mAbs were less effective than anti-CD28 mAb 9.3 at costimulating proliferation of resting or activated T cells. However, coimmobilized combinations of anti-CD28 and anti-CTLA-4 were synergistic in their ability to augment anti-TCR-induced proliferation of preactivated CD4+ T cells. These results indicate that CTLA-4 is coexpressed with CD28 on activated T lymphocytes and cooperatively regulates T cell adhesion and activation by B7.

Induction of alloantigen-specific hyporesponsiveness in human T lymphocytes by blocking interaction of CD28 with its natural ligand B7/BB1.

The specificity of T lymphocyte activation is determined by engagement of the T cell receptor (TCR) by peptide/major histocompatibility complexes expressed on the antigen-presenting cell (APC). Lacking costimulation by accessory molecules on the APC, T cell proliferation does not occur and unresponsiveness to subsequent antigenic stimulus is induced. The B7/BB1 receptor on APCs binds CD28 and CTLA-4 on T cells, and provides a costimulus for T cell proliferation. Here, we show that prolonged, specific T cell hyporesponsiveness to antigenic restimulation is achieved by blocking the interaction between CD28 and B7/BB1 in human mixed leukocyte culture (MLC). Secondary T cell proliferative responses to specific alloantigen were inhibited by addition to the primary culture of monovalent Fab fragments of anti-CD28 monoclonal antibody (mAb) 9.3, which block interaction of CD28 with B7/BB1 without activating T cells. Hypo-responsiveness was also induced in MLC by CTLA4Ig, a chimeric immunoglobulin fusion protein incorporating the extracellular domain of CTLA-4 with high binding avidity for B7/BB1. Cells previously primed could also be made hyporesponsive, if exposed to alloantigen in the presence of CTLA4Ig. Maximal hyporesponsiveness was achieved in MLC after 2 d of incubation with CTLA4Ig, and was maintained for at least 27 d after removal of CTLA4Ig. Accumulation of interleukin 2 (IL-2) and interferon gamma but not IL-4 mRNA was blocked by CTLA4Ig in T cells stimulated by alloantigen. Antigen-specific responses could be restored by addition of exogenous IL-2 at the time of the secondary stimulation. Addition to primary cultures of the intact bivalent anti-CD28 mAb 9.3, or B7/BB1+ transfected CHO cells or exogenous IL-2, abrogated induction of hyporesponsiveness by CTLA4Ig. These data indicate that interaction of CD28 with B7/BB1 during TCR engagement with antigen is required to maintain T cell competence and that blocking such interaction can result in a state of T cell hyporesponsiveness.

Tyrosine phosphorylation of CD6 by stimulation of CD3: augmentation by the CD4 and CD2 coreceptors.

When T cells are activated via the T cell receptor (TCR) complex a number of cellular substrates, including some cell surface proteins, become phosphorylated on tyrosine (Tyr) residues. Phosphorylation of cytoplasmic Tyr renders these cell surface receptors competent to interact with proteins that link cell surface receptors to protein in the intracellular signaling pathways. Here we show that Tyr residues in the cytoplasmic domain of CD6 become phosphorylated upon T cell activation via the TCR complex. Tyr phosphorylation was observed when the T cells were activated by crosslinking CD3 or by cocrosslinking CD3 with CD2 or CD4, but not when the cells were stimulated by crosslinking CD2, CD4, or CD28 alone. Unlike other Tyr kinase substrates, such as the phospholipase C gamma 1-associated pp35/36 protein, whose level of Tyr phosphorylation is highest when T cells are activated by cocrosslinking CD3 with CD2, the levels of CD6 Tyr phosphorylation are highest when T cells were activated by cocrosslinking CD3 with CD4.

gp39-CD40 interactions are essential for germinal center formation and the development of B cell memory.

gp39, the ligand for CD40 expressed on activated CD4+ T helper cells, is required for the generation of antibody responses to T-dependent (TD) antigens. Treatment of mice with anti-gp39 in vivo inhibits both primary and secondary antibody formation to TD, but not T-independent antigens. However, the role of this receptor-ligand pair in the development of germinal centers and the generation of B cell memory is as yet undefined. Using an antibody to gp39, this study examines the in vivo requirement for gp39-CD40 interactions in the induction of germinal center formation, as well as in the generation of B cell memory. Animals were immunized, treated in vivo with anti-gp39, and evaluated using immunohistochemical staining for the presence of splenic germinal centers 9-11 d after immunization. The results demonstrate that the formation of germinal centers was completely inhibited as a result of treatment with anti-gp39. Moreover, adoptive transfer experiments demonstrate that the generation of antigen-specific memory B cells is also inhibited as a consequence of blocking gp39-CD40 interactions. Taken together, the data demonstrate that gp39-CD40 interactions are critical not only for the generation of antibody responses, but also in the development of B cell memory.

T cell ontogeny. Organ location of maturing populations as defined by surface antigen markers is similar in neonates and adults.

Earlier studies have suggested that splenic T cell populations in nursling mice (less than 18 d of age) have Lyt cell surface antigens that identify them as less mature than their adult counterparts. Studies presented here, however, demonstrate that the expression of the Thy-1, Lyt-1, Lyt-2, and Lyt-3 T cell antigens is virtually identical in 14-d-old and adult T cell populations even though at 14 d, T cells constitute less than 10% of the total spleen cell population. Because the expression of these antigens on the immature (cortical) thymocyte population differs substantially from their expression on peripheral T cells, the maturity of splenic T cells as judged by these criteria is similar in nurslings and adults. Very few cells in the neonatal thymus 4 h after birth correspond, in terms of antigen expression, to the more mature (medullary) thymocyte population of adults, but such cells develop rapidly during the first few days of life. They are present, therefore, sufficiently early to serve as the immediate source of peripheral T cells, as they apparently do in the adult. This then suggests that the locations for the major T cell maturational events are established within the first 2 wk of life of the mouse and maintained as such thereafter. The use of monoclonal antibodies and quantitative immunofluorescence analysis in our studies probably explains the differences between our findings and those reported previously, which relied on cytotoxic depletion by alloantisera and complement to estimate the frequencies of cells carrying the Lyt differentiation antigens in nurslings.

Evolutionary conservation of surface molecules that distinguish T lymphocyte helper/inducer and cytotoxic/suppressor subpopulations in mouse and man.

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.

The relative contribution of the CD28 and gp39 costimulatory pathways in the clonal expansion and pathogenic acquisition of self-reactive T cells.

The zona pellucida (ZP), an ovarian extracellular structure, contains three major glycoproteins: ZP1, ZP2, and ZP3. A ZP3 peptide contains both an autoimmune oophoritis-inducing T cell epitope and a B cell epitope that induces autoantibody to ZP. This study investigates two major T cell costimulation pathways in this disease model. Herein we show that blockage of glycoprotein (gp)39 and CD40 interaction with gp39 monoclonal antibody (mAb) results in the failure to induce both autoimmune oophoritis and autoantibody production. Inhibition of ligand binding to the CD28 receptor with the fusion protein, murine CTLA4-immunoglobulin (Ig), also results in failure to generate antibody to ZP and significantly reduces disease severity and prevalence. Surprisingly, the frequencies of antigen-specific T cells in anti-gp39 mAb-treated mice, CTLA4-Ig treated mice, and in mice given control hamster IgG or control fusion protein L6, were equivalent as determined by limiting dilution analysis (approximately equals 1:5,000). These T cells, which produced comparable amounts of interleukin 4 and interferon gamma in vitro, were able to transfer oophoritis to normal recipients. When anti-gp39 mAb and CTLA4-Ig were given together, the effect was additive, leading to inhibition of T cell activation as determined by in vitro proliferation and limiting dilution analysis (approximately equals 1:190,000); disease and antibody responses were absent in these mice. By studying these two costimulatory pathways in parallel, we have shown that autoimmune disease and autoantibody production are inhibitable by blocking either the gp39 or the CD28 pathway, whereas inhibition of clonal expansion of the effector T cell population occurs only when both pathways are blocked.

Differential induction of apoptosis by Fas-Fas ligand interactions in human monocytes and macrophages.

Human monocytes undergo spontaneous apoptosis upon culture in vitro; removal of serum from the media dramatically increases the rate of this process. Monocyte apoptosis can be significantly abrogated by the addition of growth factors or proinflammatory mediators. We have evaluated the role of the endogenous Fas-Fas ligand (FasL) interaction in the induction of this spontaneous apoptosis and found that a Fas-immunoglobulin (Ig) fusion protein, an antagonistic anti-Fas monoclonal antibody and a rabbit anti-FasL antibody all greatly reduced the onset of apoptosis. The results indicate that spontaneous death of monocytes is mediated via an autocrine or paracrine pathway. Treatment of the cells with growth factors or cytokines that prevented spontaneous apoptosis had no major effects on the expression of Fas or FasL. Additionally, monocyte-derived macrophages were found to express both Fas and FasL but did not undergo spontaneous apoptosis and were not sensitive to stimulation by an agonistic anti-Fas IgM. These results indicate that protective mechanisms in these cells exist at a site downstream of the receptor-ligand interaction.

In vivo CD40-gp39 interactions are essential for thymus-dependent humoral immunity. I. In vivo expression of CD40 ligand, cytokines, and antibody production delineates sites of cognate T-B cell interactions.

T-B cell interactions have a central role in the development of antibody responses. Upon activation, T helper (Th) cells express the ligand for CD40, gp39, which is essential for Th cell-dependent B cell activation. The cytokines produced by activated Th cells have a regulatory role in B cell differentiation. In this study, we investigated, using immunohistochemical techniques, the in vivo time course and localization of gp39 expression and cytokine production in relation to the specific antibody production. Both the immunization with keyhole limpet hemocyanin (KLH), a thymus-dependent (TD) antigen, and trinitrophenyl (TNP)-Ficoll, a thymus-independent type 2 (TI-2) antigen, induced Th cells to express gp39. The expression of gp39 was restricted to Th cells in the outer periarteriolar lymphocyte sheaths (outer-PALS) and around the terminal arterioles (TA). Incidentally, gp39+ Th cells were found in the corona of follicles, whereas gp39+ cells were never found in the germinal centers or marginal zones of the spleen. Maximum frequencies of gp39+ cells were observed 3 and 4 d after primary and secondary immunization with KLH. After injection of TNP-Ficoll, a marked increase in gp39+ cells was observed, confirming previous observations that activated T cells are involved in TI-2 antibody responses. Analysis of the in vivo cytokine production revealed that interleukin 2 (IL-2)-, IL-4- and interferon gamma (IFN-gamma)-producing cells (IFN-gamma-PC) developed according to similar kinetics as observed for gp39+ cells. IL-2-PC and IL-4-PC were present in higher frequencies as were IFN-gamma-PC in the immune response against TNP-KLH. Double staining experiments revealed gp39+ Th cells producing IL-2, IL-4, or IFN-gamma, suggesting that these cells were involved in both the initial activation as well as the differentiation process of B cells into antibody-forming cells. Dual immunohistochemical analysis revealed gp39+ T cells and cytokine-PC in close proximity to antigen-specific, antibody-forming B cells. In conclusion, this study shows that in vivo gp39 is expressed on activated Th cells after immunization with TD and TI-2 antigens. Furthermore, the time course and compartmentalization of gp39+ expression, cytokine production and antibody formation after immunization suggest that cognate T-B cell interactions and T cell-regulated B cell differentiation occur in the outer-PALS and around the TA of the spleen.

Antibody and B7/BB1-mediated ligation of the CD28 receptor induces tyrosine phosphorylation in human T cells.

CD28 is an adhesion receptor expressed as a 44-kD dimer on the surface of a major subset of human T cells. The CD28 receptor regulates the production of multiple lymphokines, including interleukin 2 (IL-2), by activation of a signal transduction pathway that is poorly understood. Here we show that ligation of CD28 by a monoclonal antibody (mAb) or by a natural ligand, B7/BB1, induces protein tyrosine phosphorylation that is distinct from T cell receptor (TCR)-induced tyrosine phosphorylation. CD28-induced protein tyrosine phosphorylation was greatly enhanced in cells that had been preactivated by ligation of the TCR, or by pretreatment with phorbol esters. Rapid and prolonged tyrosine phosphorylation of a single substrate, pp100, was induced in T cells after interaction with B7/BB1 presented on transfected Chinese hamster ovary (CHO) cells. Anti-B7 mAb inhibited B7/BB1 receptor-induced tyrosine phosphorylation, indicating that B7-CD28 interaction was required. CD28-induced tyrosine phosphorylation was independent of the TCR because it occurred in a variant of the Jurkat T cell line that does not express the TCR. Herbimycin A, a protein tyrosine kinase inhibitor, could prevent CD28-induced tyrosine phosphorylation and CD28-induced IL-2 production in normal T cells. The simultaneous crosslinking of CD28 and CD45, a tyrosine phosphatase, could prevent tyrosine phosphorylation of pp100. These results suggest that specific tyrosine phosphorylation, particularly of pp100, occurs directly as a result of CD28 ligand binding and is involved in transducing the signal delivered through CD28 by accessory cells that express the B7/BB1 receptor. Thus, this particular form of signal transduction may be relevant to lymphokine production and, potentially may provide a means to study the induction of self-tolerance, given the putative role of the costimulatory signal in the induction of T cell activation or anergy.