"CD3low" human thymocyte populations can readily be triggered via the CD2 and/or CD28 activation pathways whereas the CD3 pathway remains nonfunctional

We have investigated the role of the CD2 and the CD28 Ag-independent pathways of activation on CD3low thymocytes. We previously showed that anti-CD28 mAb synergized with anti-CD2 mAb directed against epitopes T11.1 and T11.2, in the activation of purified resting T cells or unseparated thymocytes. Proliferation induced via CD2 plus CD28 was mediated via an IL-2-dependent pathway and was not affected by prior modulation of the CD3-TCR complex. Here, we show that a subset of CD3low thymocytes, although unresponsive to CD3 activation, can be activated to proliferate through the CD2 or the CD28 pathways, in the presence of exogenous IL-2. The mitogenic combination of mAb to CD2 and CD28 induces a proliferation of thymocytes which, in absence of exogenous lymphokines, is restricted to the more mature intrathymic subpopulation, CD1a-. However, CD3low thymocytes can also be triggered through the CD2 plus CD28 activation pathways but require at least addition of exogenous IL-2 to proliferate. This study demonstrates that a fraction of immature CD3low thymocytes possesses functional CD2 and CD28 surface molecules at a time when CD3 is not yet functional.     

The role of CD11a/CD18-CD54 interactions in human T cell-dependent B cell activation.

The role of leukocyte function-associated Ag-1 (LFA-1, CD11a/CD18) and intercellular adhesion molecule 1 (ICAM-1, CD54) interactions in human T cell and B cell collaboration was examined by studying the effect of mAb to these determinants on B cell proliferation and differentiation stimulated by culturing resting B cells with CD4+ T cells activated with immobilized mAb to the CD3 molecular complex. In this model system, mAb to either the alpha (CD11a) or beta (CD18) chain of LFA-1 or ICAM-1 (CD54) inhibited B cell responses significantly. The mAb did not directly inhibit B cell function, inasmuch as T cell-independent activation induced by formalinized Staphylococcus aureus and IL-2 was not suppressed. Moreover, DNA synthesis and IL-2 production by immobilized anti-CD3-stimulated CD4+ T cells were not suppressed by the mAb to LFA-1 or ICAM-1. Although the mAb to LFA-1 inhibited enhancement of IL-2 production by co-culture of immobilized anti-CD3-stimulated CD4+ T cells with B cells, addition of exogenous IL-2 or supernatants of mitogen-activated T cells could not abrogate the inhibitory effects of the mAb to LFA-1 or ICAM-1 on B cell responses. Inhibition was most marked when the mAb were present during the initial 24 h in culture. Immobilized anti-CD3-stimulated LFA-1-negative CD4+ T cell clones from a child with leukocyte adhesion deficiency could induce B cell responses, which were inhibited by mAb to LFA-1 or ICAM-1. These results indicate that the interactions between LFA-1 and ICAM-1 play an important role in mediating the collaboration between activated CD4+ T cells and B cells necessary for the induction of B cell proliferation and differentiation, and for enhancement of IL-2 production by CD4+ T cells. Moreover, the data are consistent with a model of T cell-B cell collaboration in which interactions between LFA-1 on resting B cells and ICAM-1 on activated CD4+ T cells play a critical role in initial T cell-dependent B cell activation.     

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.     

Stimulation via the CD3 and CD28 molecules induces responsiveness to IL-4 in CD4+CD29+CD45R- memory T lymphocytes

Although both IL-2 and IL-4 can promote the growth of activated T cells, IL-4 appears to selectively promote the growth of those helper/inducer and cytolytic T cells which have been activated via their CD3/TCR complex. The present study examines the participation of CD28 and certain other T cell-surface molecules in inducing T cell responsiveness to IL-4. Purified small high density T cells were cultured in the absence of accessory cells with various soluble anti-human T cell mAb with or without soluble anti-CD3 mAb and their responsiveness to IL-4 was studied. None of the soluble anti-T cell mAb alone was able to induce T cell proliferation in response to IL-4. A combination of soluble anti-CD3 with anti-CD28 mAb but not with mAb directed at the CD2, CD5, CD7, CD11a/CD18, or class I MHC molecules induced T cell proliferation in response to IL-4. Anti-CD2 and anti-CD5 mAb enhanced and anti-CD18 mAb inhibited this anti-CD3 + anti-CD28 mAb-induced T cell response to IL-4. In addition, anti-CD2 in combination with anti-CD3 and anti-CD28 mAb induced modest levels of T cell proliferation even in the absence of exogenous cytokines. IL-1, IL-6, and TNF were each unable to replace either anti-CD3 or anti-CD28 mAb in the induction of T cell responsiveness to IL-4, but both IL-1 and TNF enhanced this response. The anti-CD3 + anti-CD28 mAb-induced response to IL-4 was exhibited only by cells within the CD4+CD29+CD45R- memory T subpopulation, and not by CD8+ or CD4+CD45R+ naive T cells. When individually cross-linked with goat anti-mouse IgG antibody immobilized on plastic surface, only anti-CD3 and anti-CD28 mAb were able to induce T cell proliferation. These results indicate that the CD3 and CD28 molecules play a crucial role in inducing T cell responsiveness to IL-4 and that the CD2, CD5, and CD11a/CD18 molecules influence this process.     

Role of CD47 in the induction of human naive T cell anergy

We recently reported that CD47 ligation inhibited IL-2 release by umbilical cord blood mononuclear cells activated in the presence of IL-12, but not IL-4, preventing the induction of IL-12Rbeta(2) expression and the acquisition of Th1, but not the Th2 phenotype. Here we show that in the absence of exogenous cytokine at priming, CD47 ligation of umbilical cord blood mononuclear cells promotes the development of hyporesponsive T cells. Naive cells were treated with CD47 mAb for 3 days, expanded in IL-2 for 9-12 days, and restimulated by CD3 and CD28 coengagement. Effector T cells generated under these conditions were considered to be anergic because they produced a reduced amount of IL-2 at the single-cell level and displayed an impaired capacity 1) to proliferate, 2) to secrete Th1/Th2 cytokines, and 3) to respond to IL-2, IL-4, or IL-12. Moreover, CD47 mAb strongly suppressed IL-2 production and IL-2Ralpha expression in primary cultures and IL-2 response of activated naive T cells. Induction of anergy by CD47 mAb was IL-10 independent, whereas inclusion of IL-2 and IL-4, but not IL-7, at priming fully restored T cell activation. Furthermore, CD28 costimulation prevented induction of anergy. Thus, CD47 may represent a potential target to induce anergy and prevent undesired Th0/Th1 responses such as graft vs host diseases, allograft rejection, or autoimmune diseases.     

CD30 expression identifies the predominant proliferating T lymphocyte population in human alloimmune responses

CD30 is an inducible member of the TNFR superfamily that is expressed on activated T and B cells and some lymphoid malignancies. We have previously shown that human CD30(+) T cells elicited with allogeneic APC are a major source of IFN-gamma and IL-5 production. In the present study we have used alloantigen, as well as anti-CD3 plus anti-CD28 mAb stimulation, to further characterize human CD30(+) T cells with respect to function and the expression of other activation-dependent cell surface molecules, including the related TNFR family members OX-40 and 4-1BB (CD137). Our results indicate that human CD30(+) T cells are a subset of activated T cells that also express CD25 and CD45RO. Moreover, we observed that allogeneic APC consistently induced a greater proportion of CD30(+) cells within the activated T cell population than did stimulation with plate-bound anti-CD3 plus anti-CD28 mAb or stimulation with soluble anti-CD3 plus anti-CD28 and autologous APC. The enhanced induction of CD30 expression by alloantigen was not common to other inducible TNFR family members because anti-CD3 plus anti-CD28 mAbs were far more effective in inducing expression of 4-1BB and OX-40. Furthermore, CD30 expression marked the predominant proliferating T cell population induced by alloantigen as determined by CFSE staining and flow cytometry. These results indicate that CD30, but not 4-1BB or OX-40, is preferentially induced by alloantigen, suggesting that CD30 may be important in human alloimmune responses.     

T cell activation via Leu-23 (CD69)

The CD69 (Leu-23) activation Ag is a phosphorylated 28 to 32-kDa disulfide-linked homodimer that is rapidly induced after lymphocyte activation. CD69 is not present on the surface of peripheral blood resting T cells, but is constitutively expressed by CD3bright thymocytes. Activation of protein kinase C (PKC) by stimulation of the TCR/CD3 or by phorbol esters directly induces CD69 expression on T cells. In the attempt to elucidate the function of CD69 we investigated the ability of the CD69 glycoprotein to transmit an activation signal. Cross-linking of CD69 by mAb induced a prolonged elevation of intracellular [Ca2+], mostly due to an influx of extracellular Ca2+. This signal alone was unable to effectively activate PKC. When PKC was simultaneously activated by PMA, stimulation of CD69 induced IL-2 and IFN-gamma gene expression, enhancement of CD25 expression, and ultimately IL-2-dependent T cell proliferation. Both CD4+ and CD8+ peripheral T cells responded to CD69-mediated activation. Stimulation of CD69 induced proliferation of thymocytes as well as peripheral T cells, but both required independent PKC activation by PMA. Cyclosporin A, which does not prevent PKC-induced CD69 expression, completely suppressed CD69-induced IL-2 and IFN-gamma gene expression. Although the signal delivered by the CD69 initiates T cell proliferation, it is unable to trigger cytotoxicity programs in CD69+-activated T cells or T cell clones.     

Central role for TCR/CD3 ligation in the differentiation of CD4+ T cells toward A Th1 or Th2 functional phenotype.

Activated CD4+ T cells can be classified into distinct subsets; the most divergent among them may be considered to be the IL-2 and IFN-gamma-producing Th1 clones and the IL-4 and IL-5-producing Th2 clones. Because Th1 and Th2 clones can usually be detected only after several months of culture, we used conditions that modulate the IL-2 and IL-4 production in short term culture. Here we show that freshly isolated and subsequently in vitro-activated CD4+ T cells that were cultured for 11 days with rIL-2 and restimulated showed a IFN-gamma+ IL-2+ IL-3+ IL-4- IL-5- pattern. Because these cells were not capable of providing B cell help for IgG1, IgG2a, or IgE in an APC- and TCR-dependent T-B cell assay, they expressed a phenotype typical for most Th1 clones. In contrast, activated T cells that were cultured for 11 days with IL-2 plus a mAb to CD3 and then restimulated produced a IFN-gamma- IL-2- IL-3+ IL-4+ IL-5+ pattern. These cells were capable of providing B cell help for IgG1, IgG2a, and IgE synthesis and thus presented a phenotype typical for Th2 clones. Similar results were observed when mitogenic mAb to Thy-1.2 or to framework determinants of the alpha beta TCR were used. The induction of Th1- and Th2-like cells did not depend on the relative expression of CD44 or CD45 by the T cells before activation in vitro. Because the incubation of activated T cells with anti-CD3/TCR mAb induced high unrestricted lymphokine production, the latter might be responsible for the Th2-like lymphokine pattern observed after restimulation. To address this point, TCR V beta 8+ and V beta 8- T cell blasts were co-cultured in the presence of mAb to V beta 8. After restimulation, V beta 8+ cells had a IL-4high IL-2low phenotype and V beta 8- cells had a IL-4low IL-2high phenotype. This demonstrates that TCR ligation but not lymphokines alone are capable of inducing Th2-like cells, and this points out a central role for the TCR in the generation of T cell subsets.     

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.     

Cutting edge: stromal cell-derived factor-1 is a costimulator for CD4+ T cell activation

Stromal cell-derived factor (SDF)-1 is a chemoattractant for T cells, precursor B cells, monocytes, and neutrophils. SDF-1alpha was also found to up-regulate expression of early activation markers (CD69, CD25, and CD154) by anti-CD3-activated CD4+ T cells. In addition, SDF-1alpha costimulated proliferation of CD4+ T cells and production of IL-2, IFN-gamma, IL-4, and IL-10. Stimulation with SDF-1alpha alone did not induce activation marker expression, proliferation, or cytokine production by the CD4+ T cells. SDF-1alpha-mediated costimulation was blocked by anti-CXC chemokine receptor-4 mAb. RANTES also increased activation marker expression by anti-CD3-stimulated peripheral CD4+ T cells, but less effectively than SDF-1alpha did, and did not up-regulate IL-2 production and proliferation. These results indicate that SDF-1 and CXC chemokine receptor-4 interactions not only play a role in T cell migration but also provide potent costimulatory signals to Ag-stimulated T cells.     

Evidence for the involvement of three distinct signals in the induction of IL-2 gene expression in human T lymphocytes

The regulation of IL-2 gene expression during T cell activation and proliferation has been investigated in primary cultures of purified human peripheral blood T cells. Prior results indicated that stimulation of T cells by anti-CD28 mAb plus PMA could induce IL-2 expression and T cell proliferation that was entirely resistant to cyclosporine. The present studies examined whether CD28 augments IL-2 expression by a unique pathway or merely acts at a point common to CD3-induced proliferation but distal to the effects of cyclosporine. The induction of maximal IL-2 gene expression required three signals provided by phorbol ester, calcium ionophore, and anti-CD28 mAb. Stimulation of cells by optimal amounts of calcium ionophore and PMA induced IL-2 mRNA that was completely suppressed by cyclosporine. The addition of anti-CD28 to T cells stimulated with PMA plus calcium ionophore induced a 5- to 100-fold increase in IL-2 gene expression and secretion that was resistant to cyclosporine. The CD28 signal was able to increase steady state IL-2 mRNA levels even in cells treated with maximally tolerated amounts of calcium ionophore and PMA. The three-signal requirement did not reflect differential regulation of lymphokine gene expression between the CD4 and CD8 T cell subsets or differences in the kinetics of IL-2 mRNA expression. The signal provided by CD28 is distinct from that of CD3 because although anti-CD28 plus PMA-induced proliferation is resistant to cyclosporine, anti-CD3 or anti-CD3 plus PMA-induced IL-2 expression is sensitive. Thus, these studies show that three biochemically distinct signals are required for maximal IL-2 gene expression. Furthermore, these studies suggest that lymphokine production in T cells is not controlled by an "on/off" switch, but rather, that CD28 regulates a distinct intracellular pathway which modulates the level of IL-2 production on a per cell basis. The observation that CD28 stimulation results in IL-2 concentrations that exceed 1000 U/m1 in tissue culture supernatants suggests that a role in vivo for CD28 might be to amplify immune responses initiated by the CD3/T cell receptor complex. Finally, the observation that CD28 interacts with the signals provided by PMA and calcium ionophore shows that the function of CD28 is not merely to act as a scaffold to stabilize or enhance signalling through the CD3/TCR complex.     

CD3/Ti gamma A: a functional gamma-receptor complex expressed on human peripheral lymphocytes

We have recently developed a mAb designated anti-Ti gamma A, which was found to immunoprecipitate from the well characterized CD3+ TCR alpha/beta- F6C7 fetal clone a CD3-associated disulfide-linked gamma-glycoprotein. This antibody recognizes approximately 3% of adult peripheral lymphocytes and delineates a CD2+ CD3+ TCR alpha/beta- CD4- NKH1- subset where expression of CD8 appears to vary widely from one individual to another. In the present study, we have used anti-Ti gamma A mAb to assess whether gamma-chains expressed on these adult lymphocytes are used as functional R. The two activities which have been associated thus far with TCR gamma+ cells, that is, IL-2-dependent proliferation and non-MHC-restricted cytotoxicity, were investigated here by using either resting or activated Ti gamma A+ lymphocytes. On the resting state, these cells (which appear as a very homogeneous population of granular lymphocytes) mediate little if any NK activity that could not be augmented by anti-Ti gamma A mAb. In contrast, after initial stimulation by PHA plus rIL-2 and subsequent culture in the presence of IL-2, activated Ti gamma A+ lymphocytes were strongly lytic against a series of conventional NK target cell lines. This cytotoxic function was either blocked or enhanced by anti-Ti gamma A mAb, depending upon experimental conditions. With respect to proliferation, it was possible to induce responses of resting Ti gamma A+ lymphocytes with antibody-coated CNBr beads only in the presence of exogenous IL-2, whereas, in culture, the same cells proliferated directly and secreted IL-2 after treatment by anti-Ti gamma A beads. Taken together, these data demonstrate that a major subset of circulating CD3+ TCR alpha/beta- lymphocytes use protein products of T cell gamma rearranging genes as functional R structures.     

Characterization of T cell proliferative responses induced by anti-Qa-2 monoclonal antibodies.

The MHC class I Qa-2 Ag are attached to the cell surface by a glycanphosphatidylinositol (GPI) anchor. Crosslinking of Qa-2 and several other cell surface Ag attached by the GPI linkage has been shown to lead to cell activation. We have developed 10 new anti-Qa-2 mAb and characterized their capacity to induce proliferation of spleen cells. In the absence of anti-Ig-mediated crosslinking, none of the mAbs alone could induce activation. However, mAb 23.1 which reacts with the alpha 3 domain of Qa-2, when combined with most of the other mAbs (alpha 1, alpha 2 domain reactive), activated cells in the absence of anti-Ig crosslinking. The mAb pair 23.1 plus 24.16 was the most proficient and induced proliferation in the absence of any exogenous second signals. Responses were greatly enhanced and equivalent to those seen with anti-CD3 by the addition of phorbol myristate acetate (PMA). Ionomycin, rIL-2, or rIL-4 also potentiated anti-Qa-2 responses but less efficiently than PMA. Significant strain variation in the magnitude Qa-2-mediated proliferative responses was observed correlating with the levels of Qa-2 expressed on the cell surface. Crosslinking of Qa-2 molecules by the mAb combinations was required because monovalent Fab fragments failed to activate cells. F(ab')2 fragments of mAb 23.1 plus 24.16 induced vigorous proliferation indicating that accessory cell presentation of the mAb via Fc receptors was not required. Immobilized (plate bound) anti-Qa-2 mAb induced proliferation suggesting that the Qa-2 pathway may be distinct from that of other GPI molecules such as Thy-1 and Ly-6. Populations enriched for T cells (approximately 95%) responded as well as whole spleen cells, whereas B lymphocytes failed to proliferate to anti-Qa-2. Both CD4+ and CD8+ cells were activated following crosslinking of Qa-2. Finally, T cell activation mediated by Qa-2 induced elevation of [Ca2+]i, IL-2R expression, and the release of IL-2. These data demonstrate that crosslinking of Qa-2 on T lymphocytes represents a potent pathway for inducing cell activation.     

CD28 delivers a costimulatory signal involved in antigen-specific IL-2 production by human T cells.

CD4+ T cells require two signals to produce maximal amounts of IL-2, i.e., TCR occupancy and an unidentified APC-derived costimulus. Here we show that this costimulatory signal can be delivered by the T cell molecule CD28. An agonistic anti-CD28 mAb, but not IL-1 and/or IL-6, stimulated T cell proliferation by tetanus toxoid-specific T cells cultured with Ag-pulsed, costimulation-deficient APC. Furthermore, the ability of B cell tumor lines to provide costimulatory signals to purified T cells correlated well with expression of the CD28 ligand B7/BB-1. Finally, like anti-CD28 mAb, autologous human APC appeared to stimulate a cyclosporine A-resistant pathway of T cell activation. Together, these results suggest that the two signals required for IL-2 production by CD4+ T cells can be transduced by the TCR and CD28.     

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.