IL-1 alpha is produced by T lymphocytes activated via the CD2 plus CD28 pathways.

In addition to activation via the TCR complex, resting purified T cells can be activated to proliferate by mAb directed against the two surface molecules, CD2 and CD28. We demonstrate here that only the CD2 plus CD28 combined activation induces the expression and secretion of IL-1 alpha, a cytokine classically considered as a monokine. In contrast, neither IL-1 beta nor IL-6 were produced. A second monokine, TNF-alpha was transiently expressed by T cells activated with either CD2 or CD28 mAb, but was expressed to higher levels and with a prolonged kinetics in cells activated by the CD2 plus CD28 combination. The prolonged expression of the IL-1 alpha gene could account, at least in part, for the monocyte-independent and long lasting T cell proliferation induced by the CD2 plus CD28 co-stimulation. Secretion of monokines, such as IL-1 alpha, by activated T cells, could play a regulatory role in immune responses, as well as contribute to autoimmune processes.     

CD63 as an activation-linked T cell costimulatory element

Dendritic cells (DC) are unique in their capacity to either stimulate or regulate T cells, and receptor/ligand pairs on DC and T cells are critically involved in this process. In this study we present such a molecule, which was discovered by us when analyzing the functional effects of an anti-DC mAb. This mAb, 11C9, reacted strongly with DC, but only minimally with lymphocytes. In MLR it constantly reduced DC-induced T cell activation. Therefore, we assumed that mAb 11C9 primarily exerts its functions by binding to a DC-structure. This does not seem to be the case, however. Preincubation of DC with mAb 11C9 before adding T cells had no inhibitory effect on T cell responses. Retroviral expression cloning identified the 11C9 Ag as CD63. This lysosomal-associated membrane protein (LAMP-3), is only minimally expressed on resting T cells but can, as we show, quickly shift to the surface upon stimulation. Cross-linkage of that structure together with TCR-triggering induces strong T cell activation. CD63 on T cells thus represents an alternative target for mAb 11C9 with its binding to activated T cells rather than DC being responsible for the observed functional effects. This efficient CD63-mediated costimulation of T cells is characterized by pronounced induction of proliferation, strong IL-2 production and compared with CD28 enhanced T cell responsiveness to restimulation. Particularly in this latter quality CD63 clearly surpasses several other CD28-independent costimulatory pathways previously described. CD63 thus represents an activation-induced reinforcing element, whose triggering promotes sustained and efficient T cell activation and expansion.     

CTLA-4 and CD28 mRNA are coexpressed in most T cells after activation. Expression of CTLA-4 and CD28 mRNA does not correlate with the pattern of lymphokine production.

Ag-presenting cells provide at least two distinct signals for T cell activation. T cell receptor-dependent stimulation is provided by presentation of a specific peptide Ag in association with MHC molecules. In addition, APC also supply costimulatory signals required for T cell activation that are neither Ag- nor MHC restricted. One such costimulatory signal is mediated via the interaction of B7 on APC with the CD28 receptor on T cells. Recently, CTLA-4 has been shown to be a second B7 receptor on T cells. In the present report, we have examined the expression of CD28 and CTLA-4 on a panel of resting and activated normal T cell subsets and T cell clones by RNA blot analysis in an attempt to determine whether their expression defines reciprocal or overlapping subsets. CD28 was detected in resting T cells, whereas CTLA-4 was not. After stimulation with PHA and PMA for 24 h, CTLA-4 mRNA was expressed in both the CD4+ and CD8+ subsets as well as in CD28+ T cells. We examined 37 human and six murine T cell clones that had been previously characterized for their cytokine production. After activation, CTLA-4 and CD28 mRNA were coexpressed in 36 of 37 human T cell clones and all six murine T cell clones. These included T cells of CD4+8-, CD4-8+, and CD4-8- phenotypes as well as clones with Th1 and Th2 cytokine profiles. In contrast, CD28 but not CTLA-4 mRNA was detected in leukemic T cell lines and myelomas. CTLA-4 and B7 mRNA but not CD28 mRNA was detected in two long term HTLV-I-transformed T cell lines. These data demonstrate that CD28 and CTLA-4 mRNA are coexpressed in most activated T cells and T cell clones, providing evidence that they do not define reciprocal subsets. Moreover, they are consistent with the hypothesis that B7 transmits its signal through a single receptor, CD28, on resting T cells, and multiple receptors, CD28 and CTLA-4, on activated T cells.     

CD28-independent costimulation of T cells by OX40 ligand and CD70 on activated B cells.

OX40 and its ligand (OX40L) have been implicated in T cell-dependent humoral immune responses. To further characterize the role of OX40/OX40L in T-B cell interaction, we newly generated an anti-mouse OX40L mAb (RM134L) that can inhibit the costimulatory activity of OX40L transfectants for anti-CD3-stimulated T cell proliferation. Flow cytometric analyses using RM134L and an anti-mouse OX40 mAb indicated that OX40 was inducible on splenic T cells by stimulation with immobilized anti-CD3 mAb in a CD28-independent manner, while OX40L was not expressed on resting or activated T cells. OX40L was inducible on splenic B cells by stimulation with anti-IgM Ab plus anti-CD40 mAb, but not by either alone. These activated B cells exhibited a potent costimulatory activity for anti-CD3-stimulated T cell proliferation and IL-2 production. Anti-CD80 and anti-CD86 mAbs partially inhibited the costimulatory activity, and further inhibition was obtained by their combination with RM134L and/or anti-CD70 mAb. We also found the anti-IgM Ab- plus anti-CD40 mAb-stimulated B cells exhibited a potent costimulatory activity for proliferation of and IL-2 production by anti-CD3-stimulated CD28- T cells from CD28-deficient mice, which was substantially inhibited by RM134L and/or anti-CD70 mAb. These results indicated that OX40L and CD70 expressed on surface Ig- and CD40-stimulated B cells can provide CD28-independent costimulatory signals to T cells.     

Expression of a 33-kDa antigen Tm 1 on lymphocyte surface after modulation of cell surface antigens by IgM monoclonal antibody

The mAb Tm 1 was obtained from a fusion of SP2/O tumor cells with spleen cells from CF1 mouse immunized with T cells modulated by an IgM anti-CD3 mAb.mAb Tm 1 reacted with IgM anti-CD3 modulated T cells (66.6%) but not with unmodulated T cells (4.4%). Tm 1 was not expressed on T cells modulated with either IgG2a or IgG1 anti-CD3 mAb. Immunoprecipitation from 125I-labeled CD3-modulated T cells showed that Tm 1 Ag is a single polypeptide of 33 kDa under reducing and nonreducing conditions. Kinetic studies revealed that Tm 1 was detectable on T cells 10 min after incubation and maximally expressed after 4 h of incubation with IgM anti-CD3 mAb. CD3 expression was markedly modulated by this anti-CD3 mAb after the same period of incubation. Studies with cycloheximide revealed that Tm 1 expression on T cells does not require new protein synthesis. Tm 1 expression persisted long after CD3-reexpression 24 h later. Tm 1 was present on a small fraction of circulating T cells, B cells, and monocytes and absent from granulocytes, platelets, E, and thymocytes. Tm 1 was not expressed on T cells after various activation stimuli but was expressed on B cells upon activation. Additional studies indicate that IgM mAb against other T cell differentiation Ag and IgM mAb against B cell Ag also lead to the expression of Tm 1 on these cells. Thus, modulation of surface Ag by IgM mAb externalizes this cytoplasmic Ag. However, one exception has been noted. Purified mAb Tm 1 was not mitogenic and was unable to block either the T cell proliferation induced by 12-O-tetradecanoyl phorbol-13-acetate plus anti-CD3 mAb and other T cell stimuli, or the B cell proliferation induced by B cell mitogens. The role of Tm 1 on lymphocyte function remains to be determined.     

A specific inhibitor of the p38 mitogen activated protein kinase affects differentially the production of various cytokines by activated human T cells: dependence on CD28 signaling and preferential inhibition of IL-10 production

Cytokine production upon T cell activation results from the integration of multiple signaling pathways from TCR/CD3 and from costimulatory molecules such as CD28. Among these pathways, the possible role of p38 mitogen activated protein kinase (MAPK) is the least understood. Here, we used a highly specific p38 MAPK inhibitor, the SB203580 compound, to examine the role of this enzyme in the induction of various cytokines in human T cells stimulated with anti-CD3 and anti-CD28 mAb together or in combination with PMA. Cytokine induction was monitored by ELISA and at the mRNA level. While SB203580 had little effect on IL-2 production and proliferation, it significantly reduced the production of several other cytokines. The secretion of IL-4, IL-5, IL-13, and TNF-alpha was inhibited by 20-50% with modes of T cell activation involving the CD28 pathway, whereas their mRNA expression was little affected. In contrast, IFN-gamma induction via CD28/PMA or CD3/CD28, but not CD3/PMA, was markedly diminished both at the protein and at the mRNA levels. Most interestingly, SB203580 also suppressed IL-10 secretion and mRNA induction via CD28-dependent activation by 75-85% (IC50 approximately 0.2 microM). Subset analysis suggested that this inhibition did not reflect a differential effect on T cell subsets. Therefore, p38 MAPK activity appears to contribute to cytokine production, mostly via CD28-dependent signaling. Moreover, IL-10 seems to rely more on this activity than other cytokines for its induction in T cells.     

Characterization of rat OX40 ligand by monoclonal antibody

OX40 (CD134) is a member of the tumor necrosis factor (TNF) receptor superfamily first identified as a rat T cell activation marker. We previously identified the rat ligand for OX40 (OX40L) by molecular cloning. In the present study, we newly generated an anti-rat OX40L mAb (ATM-2) that can inhibit the binding of OX40 to rat OX40L and thus efficiently inhibits the T cell costimulatory activity of rat OX40L. Flow cytometric analyses using ATM-2 and an anti-rat OX40 mAb (MRC OX40) indicated that OX40 was inducible on splenic CD4(+) T cells by stimulation with immobilized anti-CD3 mAb, while OX40L was not expressed on resting or activated T cells. OX40L was expressed on splenic B cells after stimulation with lipopolysaccharide (LPS), but not on peritoneal macrophages. Interestingly, splenic dendritic cells (DC) expressed OX40L constitutively, which was further upregulated by LPS stimulation. The potent costimulatory activities of splenic DC for anti-CD3-stimulated rat CD4(+) T cell proliferation and cytokine (IL-2, IFN-gamma, IL-10, and IL-13) production were substantially inhibited by ATM-2. These results indicated that OX40L is expressed on professional antigen-presenting cells (APC), and may be involved in humoral immune responses via T-B interaction and in cellular immune responses via T-DC interaction in the rat system.     

Antibody blockade of Thy-1 (CD90) impairs mouse cytotoxic T lymphocyte induction by anti-CD3 monoclonal antibody

Thy-1 (CD90) expressed by mouse T cells is known to have signal transducing properties, but the ability of Thy-1 to enhance cytotoxic T lymphocyte (CTL) development is not well understood. Here we show that stimulation of mouse T cells with monoclonal antibodies (mAb) to CD3, CD28 and Thy-1 (clone G7), which were coimmobilized on polystyrene microbeads, resulted in a greater proliferative response than stimulation with only anti-CD3 and anti-CD28 mAb, indicating that Thy-1 cross-linking enhanced T cell receptor/CD28-driven T cell activation. Consistent with this finding, Thy-1 blockade with a soluble nonactivating anti-Thy-1 mAb (clone 30-H12) inhibited anti-CD3-induced proliferation of CD4+ and CD8+ T cells, and the induction of cytotoxic effector cells in a dose-dependent fashion. Interleukin-2 synthesis and CD25 expression were also impaired by Thy-1 blockade. The inhibitory effect involved a defect at or before the level of protein kinase C activation because the addition of phorbol ester ablated the anti-Thy-1-mediated inhibition of anti-CD3-induced T cell activation. The CTL that were induced in the presence of blocking anti-Thy-1 mAb adhered to target cells but showed reduced expression of granzyme B and perforin. In contrast, Fas ligand expression and function was not affected by Thy-1 blockade. We conclude that Thy-1 signalling promotes the in vitro generation of CTL that kill in a granule-dependent fashion.     

Analysis of the mechanisms of T cell-dependent polyclonal activation of human B cells. Induction of human B cell responses by fixed activated T cells.

Coculture of resting human B cells with T cells stimulated with immobilized mAb to the CD3 molecular complex induces polyclonal activation and the production of Ig of all isotypes. The current experiments were carried out to determine the nature of the signals provided to B cells by the anti-CD3-activated T cells. For these experiments, fresh T cells or T cell clones were activated with immobilized mAb to CD3 and then fixed with 1% paraformaldehyde. Upon coculture, the fixed activated T cells or T cell clones induced B cell RNA synthesis and IL-2R expression, but only minimal DNA synthesis and no Ig production. Induction of B cell RNA synthesis by fixed activated T cells was not inhibited by mAb to the alpha-chain of the IL-2R, and was not enhanced by supplementing cultures with IL-2, IL-4, IL-6, or supernatants of mitogen-activated T cells. Upon the addition of IL-2, but not IL-4 or IL-6, to cultures of B cells and fixed activated T cells, sustained proliferation was noted along with the production of Ig. Control fixed T cells or T cell clones did not induce any of these responses. The presence of cycloheximide or cyclosporin A during the activation with anti-CD3 prevented T cells from developing the capacity to provide help for B cells. The use of mAb to a variety of cell surface molecules indicated that several T cell surface molecules including CD11a/CD18, CD44, CD54, and class I MHC molecules are involved in the induction of B cell responses. Among the mAb that inhibited B cell DNA synthesis and/or Ig production, only mAb to CD11a, CD18, or CD54 inhibited initial B cell activation as assessed by RNA synthesis. Even though mAB to CD11a/CD18 inhibited the capacity of fixed activated T cells to induce B cell responses, the finding that fixed activated CD18 deficit clones provided help for B cells indicated that expression of the beta 2 family of integrins by T cells was not necessary. These results indicate that activated T cells acquire the capacity to stimulate B cells polyclonally and induce cytokine responsiveness, proliferation, and Ig production by utilization of a variety of surface molecules. Moreover, these results indicate that the initial activation of the B cell is independent of the metabolic activity of the T cell and the production of cytokines.     

SC5 mAb represents a unique tool for the detection of extracellular vimentin as a specific marker of Sezary cells

Circulating malignant Sézary lymphocytes result from a clonal proliferation of memory/activated CD4(+)CD45RO(+) T lymphocytes primarily involving the skin. Recently, the CD158k/KIR3DL2 cell surface receptor has been identified to phenotypically characterize these cells. We previously described a mAb termed SC5 that identifies an unknown early activation cell membrane molecule. It is expressed selectively by T lymphocytes isolated from healthy individuals upon activation, and by circulating Sézary syndrome lymphocytes. In addition, we found that SC5 mAb was reactive with all resting T lymphocytes once permeabilized, indicating that SC5 mAb-reactive molecule might present distinct cellular localization according to the T cell activation status. In this study, we show for the first time that SC5 mAb recognizes the intermediate filament protein vimentin when exported to the extracellular side of the plasma membrane of viable Sézary malignant cells. We demonstrate that SC5 mAb is unique as it reacts with both viable malignant lymphocytes and apoptotic T cells. As vimentin is also detected rapidly at the cell membrane surface after normal T lymphocyte activation, it suggests that its extracellular detection on Sézary cells could be a consequence of their constitutive activation status. Finally, as a probable outcome of vimentin cell surface expression, autoantibodies against vimentin were found in the sera of Sézary syndrome patients.     

U5A2-13, an antigen originally found on mouse NK-like T cells,is an early inducible cell surface antigen during lymphoid activation

We have previously reported a monoclonal antibody (mAb), U5A2-13 mAb, which originally recognizes a phenotypically and functionally similar population of natural killer (NK)-like T cells. In this study, we found that U5A2-13 antigen (U5A2-13) was expressed not only on NK-like T cells but also on T and B cells during activation. In contrast to the low levels of U5A2-13 on freshly harvested T and B cells, the activation of these cells by various stimuli resulted in high levels of expression of U5A2-13 in vitro and in vivo. Similar to CD69, U5A2-13 is also expressed in most mouse lymphoid cell lines but not in nonhematopoietic cells. U5A2-13 on T cells reached maximal expression by 24h after stimulation and returned to baseline levels after 3 days. However, U5A2-13 differed from CD69 since its expression profile was different on CD4(+)- and CD8(+)-activated T cells, phorbol ester-activated EL-4 cells, and activated splenocytes in CD69-deficient mice. In addition, immunoprecipitation study indicated that U5A2-13 is not identical to CD69. Importantly, the U5A2-13-positive population of CD4(+) T cells exhibited significant levels of cytokine producing activity upon stimulation. Overall, U5A2-13 is an early inducible cell surface antigen that could be involved in lymphocyte activation.     

Superantigen and HLA-DR ligation induce phospholipase-C gamma 1 activation in class II+ T cells.

Bacterial enterotoxin superantigens bind directly to HLA class II molecules (HLA-DR) expressed on both APC and activated human T cells, and simultaneously bind to certain V beta chains of the TCR. In this report, we compared early T cell signaling events in human alloantigen-stimulated T cells when activated by HLA-DR ligation through antibody cross-linking or by direct enterotoxin superantigen binding. Both types of stimuli induced tyrosine phosphorylation of phosphatidylinositol-specific phospholipase C gamma 1 (PLC gamma 1) and an increase in intracellular calcium concentration; however, superantigen-induced signaling was stronger than class II ligation alone. Antibody-mediated ligation of HLA-DR with CD3 resulted in augmented PLC gamma 1 activation and increased calcium mobilization, consistent with a mechanism of superantigen activity through a combination of class II and CD3/Ti signals. In addition, down-modulation of CD3 receptors with antibody demonstrated that superantigen-induced signaling events were CD3-dependent. Superantigen signaling was also class II-dependent, in that resting T cells were not responsive to direct enterotoxin stimulation. To address how early signal transducing activity correlated with T cell responsiveness, alloantigen-primed T cells were activated with immobilized class II-specific mAb or soluble superantigen. Both HLA-DR mAb-stimulated T cells and enterotoxin-treated T cells proliferated strongly in response to co-stimulation by a combination of CD28 receptor engagement and PMA addition. In addition, superantigen-induced growth was induced by CD28 receptor ligation with antibody or the B7 counter-receptor expressed on Chinese hamster ovary cells. Taken together, these results indicate that class II molecules expressed on activated T cells are directly coupled to the PLC gamma 1 signal transduction pathway, and that coligation of HLA-DR with CD3 augments T cell signaling comparable to that induced by enterotoxin superantigen. Thus, we suggest that superantigen-induced early signaling responses in activated T cells may be due in part to class II transmembrane signals induced when HLA-DR and V beta are ligated in cis.     

CD28 is an inducible T cell surface antigen that transduces a proliferative signal in CD3+ mature thymocytes

The rearrangement of TCR genes during thymic ontogeny creates a repertoire of T cell specificities that is refined to ensure the deletion of autoreactive clones and the MHC restriction of T cell responses. Signals delivered via the accessory molecules CD2, CD4, and CD8 have a crucial role in this phase of T cell differentiation. Recently, CD28 has been identified as a signal transducing molecule on the surface of most mature T cells. Perturbation of the CD28 molecule stimulates a novel pathway of T cell activation regulating the production of a variety of lymphokines including IL-2. We have studied the expression and function of CD28 during thymic ontogeny, and in resting and activated PBL. A variable percentage of resting thymocytes were CD28+ (3 to 25%, n = 8), but it was found in high density only on mature CD3+(bright) CD4/CD8 cells. Both unseparated thymocytes and isolated CD3-CD28-/dull cells proliferated when stimulated with PMA plus IL-2 or PMA plus ionomycin. PMA treatment also rapidly up-regulated CD28 expression in the CD3- subset as these cells became CD3-CD28+(bright). Despite the ability of PMA to induce high density CD28 expression in CD3- cells, CD3- thymocytes did not proliferate in response to PMA plus anti-CD28 mAb, in contrast to unseparated cells. CD3+ thymocytes stimulated with immobilized anti-CD3 mAb also failed to proliferate in culture. However, the addition of either IL-2 or anti-CD28 mAb supported proliferation, suggesting that only CD3+ cells could respond to CD28 signaling. The comitogenic effect of anti-CD3 and anti-CD28 mAb was IL-2 dependent as it was abrogated by an anti-IL-2R mAb. Interestingly, the expression of CD28 on the cell surface of CD3+ cells was also inducible, as flow cytometric analysis demonstrated a 10-fold increase in cell surface CD28 by 24 to 48 h after anti-CD3 stimulation of both CD3+ thymocytes and peripheral blood T cells. This increase was accounted for by a commensurate increase in CD28 mRNA levels. Together, these results suggest that CD28 is an inducible T cell antigen in both CD3- and CD3+ cells. In addition, stimulation of the CD28 pathway can provide a second signal to support the growth of CD3+ thymocytes stimulated through the TCR/CD3 complex, and may therefore represent a mechanism for positive selection during thymic ontogeny.     

A novel pathway of human T lymphocyte activation. Identification by a monoclonal antibody generated against a rheumatoid synovial T cell line

Substantial evidence indicates that compartmentalized infiltrates of T lymphocytes are central to the pathogenesis of autoimmune diseases such as rheumatoid arthritis, but the mechanisms by which such cells become activated remain unknown. To define surface components of activation pathways important in the function of these cells, we have generated mAb against a rheumatoid synovial T cell line. One such antibody, termed anti-UM4D4, reacts with an Ag, termed UM4D4, which is strongly expressed on most rheumatoid synovial T cell lines and clones, and on a subset of peripheral blood T cells, resting or activated. Anti-UM4D4 is mitogenic in soluble form for PBMC and certain T cell clones, and is comitogenic with the phorbol ester PMA for purified resting T lymphocytes. These functional effects are similar to those previously observed with antibodies to epitopes of CD2 and CD3, surface Ag involved in two well defined pathways of human T cell activation. Binding of anti-UM4D4 to T cells is not, however, blocked by antibodies directed at various epitopes of CD2 and CD3. Moreover, UM4D4 does not comodulate with CD3, and is expressed on a T cell line that lacks CD2, CD3, and CD28. The data, therefore, indicate that anti-UM4D4 identifies a T cell activation pathway, distinct from those previously described, that could play a role in the pathogenesis of T cell-mediated autoimmune diseases.