CD40-CD154 interaction and IFN-gamma are required for IL-12 but not prostaglandin E2 secretion by microglia during antigen presentation to Th1 cells

IL-12 and PGE2 promote and inhibit, respectively, the development of Th1 responses. Production of these mediators by APC residing in the central nervous system (CNS) may be involved in the local regulation of the T cell phenotype during infectious and autoimmune CNS diseases. In the present study we have examined IL-12 and PGE2 secretion by cultured microglia and astrocytes from the mouse brain upon Ag-dependent interaction with I-Ad-restricted, OVA323-339 specific TCR transgenic Th1 and Th2 cell lines. We show that microglia, which restimulate efficiently both Th1 and Th2 cells, secrete IL-12 upon Ag-dependent interaction with Th1, but not with Th2 cells. Th1-driven IL-12 production depends on TCR ligation by MHC class II/peptide complexes, CD40 engagement on microglia, and IFN-gamma secretion by activated Th1 cells. Th1 and, to a lesser extent, Th2 cells also stimulate the production of PGE2 by microglia. T cell-mediated induction of PGE2 requires MHC class II/peptide/TCR interactions but does not depend on CD40 engagement or on the presence of IFN-gamma. Astrocytes, which preferentially activate Th2 cells, fail to produce IL-12 and secrete negligible amounts of PGE2 upon interaction with either Th1 or Th2 cells. These results suggest that during CNS infection or immunopathology, IL-12 produced by microglia upon Ag-specific interaction with Th1 cells may further skew the immune response to Th1, whereas the T cell-dependent production of PGE2 by microglia may represent a negative feedback mechanism, limiting the propagation of Th1 responses.     

Antigen-driven but not lipopolysaccharide-driven IL-12 production in macrophages requires triggering of CD40

We demonstrated that two distinct pathways exist for the induction of IL-12 in APC. The first pathway for IL-12 production occurred during responses to T cell-dependent Ags such as OVA and required triggering of CD40 molecules on the APC. IL-12 production in this T cell-dependent system increased in direct proportion to Ag concentration and required TCR ligation but not CD28 costimulation. The second pathway occurred when bacterial products such as LPS or heat-killed Listeria monocytogenes were used to activate macrophages to produce IL-12 in the complete absence of T cells. In this second pathway, IL-12 production was completely independent of CD40 triggering. In both pathways, the presence of IFN-gamma was not required for induction of IL-12 synthesis when splenic adherent cells (SAC) from normal mice were used. However, addition of IFN-gamma to cultures of Th2 T cells and SAC increased IL-12 production two- to fivefold, and addition of rTNF-alpha with IFN-gamma further enhanced IL-12 production. The addition of TNF-alpha in the absence of IFN-gamma, however, had no effect on IL-12 production in the T cell-dependent pathway. Similarly, addition of TNF-alpha in the presence or the absence of IFN-gamma to cultures of LPS or heat-killed Listeria and SAC did not increase IL-12 production, but addition of IFN-gamma alone greatly enhanced IL-12 production, consistent with the idea that bacterial stimuli induce significant quantities of endogenous TNF-alpha production. These results indicate that the requirements for the induction of IL-12 production in T cell-dependent and T cell-independent responses differs mainly with regard to CD40 triggering. Furthermore, these results suggest that IL-12 production can be induced by bacterial products in patients with hyper-IgM syndrome who lack CD40 ligand expression and in those treated with soluble gp39 to interrupt CD40-CD40 ligand interactions.     

RNA-binding protein conserved in both microtubule- and microfilament-based RNA localization

IL-12 is a key cytokine in the development of Th1 responses. IL-12 production by antigen-presenting cells (APC) can be induced by the interaction between CD40 on the APC and CD40 ligand (CD40L) expressed on T cells after activation. Our previous study indicated that in dendritic cells (DC), the only APC that can activate naive T(h) cells efficiently, the mere CD40 engagement is insufficient to induce IL-12 production. The aim of the present study was to dissect the conditions for efficient IL-12 production by DC further. Using populations of naive and memory Th cells, recombinant CD40L, neutralizing and blocking antibodies, and by determining IFN-gamma production and CD40L expression levels, we here show that T cell-induced IL-12 production by DC results from the action of two signals, mediated by CD40L and IFN-gamma, and that the inability of naive T(h) cells to induce IL-12 production resides in their inability to produce IFN-(gamma). Other factors than CD40L and IFN-gamma can provide the required signals for IL-12 production by DC, as either factor could be replaced by lipopolysaccharide (LPS). The two-signal requirement proved unique for the production of IL-12, since either CD40 engagement or LPS was sufficient for the efficient production of tumor necrosis factor-alpha, IL-8 and the p40 subunit of IL-12, and may be considered as a safety mechanism for optimal control of potentially harmful T(h)1 responses.     

Qualitative and quantitative effects of CD28/B7-mediated costimulation on naive T cells in vitro

The CD28/B7 system provides costimulatory signals necessary for optimal T cell activation. We have examined the effects of blocking B7.1 and/or B7.2 in an in vitro system using TCR transgenic T cells specific for myelin basic protein. Activation of naive T cells was found to be B7.2 dependent and not dependent on the presence of B7.1 molecules. However, increasing the strength of signal through the TCR using peptide analogues with higher affinity for MHC compensated for blockade of B7.2 molecules, suggesting that signal 1 alone can be sufficient for the activation of naive T cells. The role of B7 molecules in the differentiation of T cells was further investigated by restimulating T cells with fresh APC and peptide in B7-sufficient conditions. A down-regulation of IL-2 and IFN-gamma production by T cells primed in the presence of anti-B7.2 mAb was partially overcome when high affinity peptide analogues were used to restimulate T cells. In contrast, a significant down-regulation of the differentiation of cells producing Th-2 cytokines was observed in the presence of anti-B7 Abs. Differentiation of IL-4-secreting cells was influenced by both B7.1 and B7.2, while IL-5 secretion was totally dependent on B7.2. These results suggest that B7-mediated costimulation is essential for the development of Th-2-associated cytokines, the absence of which cannot be overcome by increasing the strength of the signal through the TCR.     

IL-15 promotes IL-12 production by human monocytes via T cell-dependent contact and may contribute to IL-12-mediated IFN-gamma secretion by CD4+ T cells in the absence of TCR ligation

At inflammatory sites, the number of activated bystander T cells exceeds that of Ag-activated T cells. We investigated whether IL-15, a monocyte-derived cytokine that shares several biologic activities with IL-2, may contribute to bystander T cell activation in the absence of IL-2 and triggering Ag. The addition of IL-15 to cocultures of monocytes and T cells stimulates CD4+ but not CD8+ T cells to produce IFN-gamma. IFN-gamma production requires endogenous IL-12, the production of which in turn is dependent upon CD40/CD154 interactions between CD4+ T cells and monocytes. Indeed, non-TCR-activated CD4+ but not CD8+ T cells express significant levels of CD154. IL-15 may enhance IFN-gamma in this system by up-regulating CD40 expression on monocytes and IL-12Rbeta1 expression on CD4+ T cells. Conversely, using neutralizing anti-IL-15 mAb, we show that the ability of IL-12 to augment IFN-gamma secretion is partly mediated by endogenous IL-15. Finally, in the absence of monocytes, a synergistic effect between exogenous IL-12 and IL-15 is necessary to induce IFN-gamma production by purified CD4+ T cells, while IL-15 alone induces T cell proliferation. It is proposed that this codependence between IL-12 and IL-15 for the activation of inflammatory T cells may be involved in chronic inflammatory disorders that are dominated by a Th1 response. In such a response, a self-perpetuating cycle of inflammation is set forth, because IL-15-stimulated CD4+ T cells may activate monocytes to release IL-12 that synergizes with IL-15 to induce IL-12 response and IFN-gamma production.     

IL-12 up-regulates CD40 ligand (CD154) expression on human T cells

IL-12 is a heterodimeric cytokine produced by APC that promotes the development of CD4+ Th1 cells and their IFN-gamma production after TCR/CD3 triggering. We here investigated the capacity of IL-12 to modify the expression on T cells of CD40 ligand (CD40L or CD154), a molecule transiently expressed on activated T cells and known to be of utmost importance for cognate interaction with B cells and for activation of dendritic cells and macrophages. Our data demonstrate that IL-12 up-regulates CD40L expression on anti-CD3-activated human peripheral blood T cells. For optimal induction of CD40L, IL-12 synergizes with IL-2 as well as with other costimulatory interactions, such as B7/CD28. The effect of IL-12 was observed at both the protein and the mRNA level. T cells costimulated by IL-12 provided more efficient help for IL-4-dependent B cell proliferation and for IgG production than when activated in the absence of IL-12. This helper activity was blocked by an mAb against CD40L, indicating that the effect of IL-12 on B cells is mediated indirectly through CD40L. The data thus suggest that the effects of IL-12 on cellular and humoral immune responses are partly mediated through CD40L induction.     

Modification of human T-cell responses by altered peptide ligands: a new approach to antigen-specific modification

Human CD4+ T-cells recognize antigenic peptides in the context of human leukocyte antigen (HLA) class II molecules and produce various lymphokines to proliferate and activate other cells. It was once considered that the T-cell response is an all or nothing type event, but recent studies have clearly indicated that T-cells show many different types of activation in recognition of altered ligands for T-cell receptors (TCR). In this review, we summarize our recent findings on the human CD4+ T-cell response to altered peptide ligands (APL); peptides carrying single residue substitutions in antigenic peptides. We observed the following: 1) TCR antagonism for T-cell clones reactive to non-self or autoantigenic peptides, 2) partial activation (agonism) without cell proliferation, including production of lymphokines and increases in cell size, and in expression levels of several cell surface proteins or survival time in the absence of antigenic stimulus, 3) augmentation in cell proliferation and production of interferon-gamma (IFN-gamma) and granulocyte monocyte colony stimulating factor (GM-CSF), 4) augmentation of interleukin (IL)-12 production by antigen presenting cell (APC) and the subsequent augmented production of IFN-gamma by T-cells. This information provides basic knowledge regarding the characteristics of T-cell recognition of antigens and the subsequent activation, and a novel method for modification of human T-cell responses by altered peptide ligands (APLs), as a possible candidate for antigen-specific immunopotentiating or immunosuppressive therapy against autoimmune diseases, allergies, infectious diseases and malignant tumors.     

Th1 cells induce and Th2 inhibit antigen-dependent IL-12 secretion by dendritic cells

Dendritic cells are the most relevant antigen-presenting cells (APC) for presentation of antigens administered in adjuvant to CD4+ T cells. Upon interaction with antigen-specific T cells, dendritic cells (DC) expressing appropriate peptide-MHC class II complexes secrete IL-12, a cytokine that drives Th1 cell development. To analyze the T cell-mediated regulation of IL-12 secretion by DC, we have examined their capacity to secrete IL-12 in response to stimulation by antigen-specific Th1 and Th2 DO11.10 TCR-transgenic cells. These cells do not differ either in TCR clonotype or CD40 ligand (CD40L) expression. Interaction with antigen-specific Th1, but not Th2 cells, induces IL-12 p40 and p75 secretion by DC. The induction of IL-12 production by Th1 cells does not depend on their IFN-gamma secretion, but requires direct cell-cell contact mediated by peptide/MHC class II-TCR and CD40-CD40L interactions. Th2 cells not only fail to induce IL-12 secretion, but they inhibit its induction by Th1 cells. Unlike stimulation by Th1, inhibition of IL-12 production by Th2 cells is mediated by soluble molecules, as demonstrated by transwell cultures. Among Th2-derived cytokines, IL-10, but not IL-4 inhibit Th1-driven IL-12 secretion. IL-10 produced by Th2 cells appears to be solely responsible for the inhibition of Th1 -induced IL-12 secretion, but it does not account for the failure of Th2 cells to induce IL-12 production by DC. Collectively, these results demonstrate that Th1 cells up-regulate IL-12 production by DC via IFN-gamma-independent cognate interaction, whereas this is inhibited by Th2-derived IL-10. The inhibition of Th1 -induced IL-12 production by Th2 cells with the same antigen specificity represents a novel mechanism driving the polarization of CD4+ T cell responses.     

Partial activation of naive CD4 T cells and tolerance induction in response to peptide presented by resting B cells

Tolerance is thought to occur when Ag is presented to T cells in the absence of costimulatory interactions from APC accessory molecules. Of the professional APC, the resting B cell may be the main tolerizing cell in vivo. We have analyzed several aspects of activation of naive transgenic CD4 cells stimulated with resting or activated B cells presenting peptide Ag. Similar results were obtained with stimulation from peptide presenting fibroblast APC lacking or expressing B7-1 with intracellular adhesion molecule-1. TCR ligation with little or no accessory molecule coreceptor engagement induced efficient blastogenesis; up-regulation of CD25, CD44, CD69, CD95 and CD71; and down-regulation of CD62L over a 48-h period. Accessory molecule help enhanced the expression of CD25, CD44, CD69, and CD71, but to very modest degrees. Only two molecules, CD40 ligand and IL-2, were found to be extremely dependent on accessory molecule help, with little or no expression evident with peptide presented on resting B cells or class II-positive fibroblasts. T cells induced on resting B cells expanded minimally over 3 days, and this was followed by extensive cell death and hyporesponsiveness of the resulting cells. These studies suggest that under tolerizing conditions, such as Ag presentation by resting B cells, much of the naive CD4 response is induced efficiently. Partial activation, however, may be the overall result due to the lack of CD40 ligand expression, which may regulate costimulatory activity in APC and, in turn, may contribute to limiting the production of IL-2 required for T cell expansion and survival.     

Microglia are more efficient than astrocytes in antigen processing and in Th1 but not Th2 cell activation

Microglia and astrocytes, two glial cell populations of the central nervous system, present Ag and stimulate T cell proliferation, but it is unclear whether they preferentially activate Th1 or Th2 responses. We have investigated the efficiency of microglia and astrocytes in the presentation of OVA peptide 323-339 or native OVA to Th1 and Th2 cell lines from DO11.10 TCR transgenic mice. Upon stimulation with IFN-gamma, microglia express MHC class II molecules, CD40, and ICAM-1 and efficiently present OVA 323-339, leading to T cell proliferation and production of IL-2 and IFN-gamma by Th1 and of IL-4 by Th2 cells. IFN-gamma-treated astrocytes, which express MHC class II and ICAM-1, present OVA 323-339 less efficiently to Th1 cells but are as efficient as microglia in inducing IL-4 secretion by Th2 cells. However, astrocytes are much less potent than microglia in presenting naturally processed OVA peptide to either T cell subset, indicating inefficient Ag processing. The capacity of astrocytes and microglia to stimulate Th1 and Th2 cells depends on their MHC class II expression and does not involve ICAM-1, B7-1, or B7-2 molecules. However, CD40-CD40L interactions contribute to Th1 activation by microglia. These data suggest that microglia may play a role in the activation of Th1 and Th2 cells, whereas astrocytes would restimulate mainly Th2 responses in the presence of appropriate peptides. This differential capacity of brain APC to restimulate Th1 and Th2 responses may contribute to the reactivation and regulation of local inflammatory processes during infectious and autoimmune diseases.     

Normal human CD4+ memory T cells display broad heterogeneity in their activation threshold for cytokine synthesis

CD4+ memory T cells coordinate immune responses against viruses and other pathogens via the Ag-induced secretion of potent effector cytokines. The efficacy of these responses depends on both the overall number of pathogen-specific memory T cells and the particular array of cytokines that these cells are programmed to secrete. Here, we provide evidence that heterogeneity in Ag triggering thresholds constitutes an additional critical determinant of memory T cell function. Using a novel assay that allows single-cell detection of Ag-specific T cell cytokine production, we demonstrate that CMV-specific CD4+ memory cells from human peripheral blood display pronounced differences in their costimulatory requirements for Ag-induced triggering of IFN-gamma and IL-2 secretion, ranging from cells that trigger with little costimulation (e.g., resting APC alone) to cells requiring potent costimulation through multiple pathways (resting APC plus multiple costimulatory mAbs, or activated APC). These differences in costimulatory requirements are independent of clonal differences in TCR signaling intensity, consistent with an intrinsic activation-threshold heterogeneity that is "downstream" from the TCR. Thus, "effective" frequencies of Ag-specific CD4+ memory T cells appear to depend on the activation status of available APC, a dependence that would allow the immune system to rapidly adjust the number of functional Ag-specific memory T cells in a particular effector site according to local conditions.     

CD40 ligand/CD40 stimulation regulates the production of IFN-gamma from human peripheral blood mononuclear cells in an IL-12- and/or CD28-dependent manner

CD40 ligand (CD40L)/CD40 costimulation is an important regulator of Th1 responses. Two mechanisms by which CD40L/CD40 stimulation may enhance IFN-gamma are via direct induction of IL-12 and augmentation of the expression of costimulatory molecules such as B7 from APCs. We examined the ability of CD40L/CD40 stimulation to regulate the production of IFN-gamma through IL-12 and/or CD28 costimulation from human PBMCs stimulated with T cell-specific stimuli. The roles of exogenous and endogenous CD40L/CD40 stimulation were evaluated using a trimeric soluble CD40L agonist (CD40T) and an anti-CD40L Ab, respectively. The presence of CD40T in cultures increased the production of IL-12 and IFN-gamma from PBMCs stimulated with varying amounts of PHA. The mechanism, however, by which CD40T enhanced IFN-gamma varied according to the level of T cell activation. Under maximal stimulatory conditions (PHA, 1/100), an IL-12-dependent pathway was dominant. At relatively low levels of T cell stimulation (PHA, 1/500 and 1/1000), however, an additional IL-12-independent CD28-dependent pathway was elucidated. We further studied the role of exogenous CD28 stimulation in regulating the production of IFN-gamma. The enhancement of IFN-gamma production induced by direct CD28 stimulation was primarily dependent on endogenous IL-12 or CD40L/CD40 stimulation. Together, these data suggest that the production of IFN-gamma involves a complex interaction between two interdependent, yet distinct, costimulatory pathways and provide evidence that CD40T may be an effective adjuvant for the enhancement of responses.     

Studies using antigen-presenting cells lacking expression of both B7-1 (CD80) and B7-2 (CD86) show distinct requirements for B7 molecules during priming versus restimulation of Th2 but not Th1 cytokine production

The differentiation of CD4+ T cells into a Th1 vs Th2 phenotype profoundly influences the outcome of autoimmune and infectious diseases. B7 costimulation has been shown to affect the production of both Th1 and Th2 cytokines, depending on the system studied. There is, consequently, great interest in manipulating the B7 costimulatory signal for therapeutic purposes. To optimally manipulate this key immunoregulatory pathway, the contribution of B7 costimulation to cytokine production requires further clarification. We have compared the B7 requirement for cytokine production by naive vs previously activated T cells using DO11.10 TCR transgenic CD4+ T cells and splenic APCs from mice lacking B7 expression. Our data indicate that induction of IL-4 production and Th2 differentiation by naive T cells is highly dependent on B7 molecules, whereas IL-4 production by previously activated T cells is B7 independent. The predominant contribution of B7-mediated signals to Th1 cytokine production by both naive and primed T cells is upon IL-2 production (and expansion) rather than IFN-gamma (effector cytokine) production. Thus, our studies demonstrate that the antigenic experience of a T cell at the time of B7 blockade may determine whether blockade predominantly affects T cell expansion, differentiation, or effector cytokine production. These differential effects of B7 costimulation on IL-2 vs IFN-gamma production and on IL-4 production by naive vs primed T cells have important implications for understanding how B7:CD28/CTLA4 blockade can be effectively used to manipulate cytokine production in vivo.     

T cell-derived IL-4 and dendritic cell-derived IL-12 regulate the lymphokine-producing phenotype of alloantigen-primed naive human CD4 T cells

Previous studies on human Th subset development were restricted to the analysis of naive T cells activated with anti-CD3 mAb in the absence of physiologic APC. In this study, we have analyzed the role of cytokines and physiologic APC on T cell maturation in an Ag-specific system, in which naive neonatal CD4 T cells were primed with allogeneic dendritic cells (DC). We found that the cytokine profile of primed cells was dependent upon 1) the ratio between T cells and allogeneic DC and 2) the endogenous production of IL-4 and IL-12. Neutralization of IL-4 during primary MLR increased IFN-gamma production at priming and shifted the phenotype of primed cells from Th0 to Th1. These effects were IL-12 dependent, in that they were suppressed by anti-IL-12 Abs. The production of IL-12 in primary MLR was further evidenced by the presence of IL-12 p40 in the culture supernatant fluids. IL-12 production was suppressed by exogenous IL-4 and increased by anti-IL-4 blocking mAbs, indicating that endogenous IL-4 down-regulated IL-12 production by DC. Finally, IL-12 was produced as a result of T cell/DC interaction involving the CD40/CD40 ligand and CD28/B7 costimulation pathways, as revealed by the inhibitory effect of anti-CD40 ligand mAb and CTLA-4Ig. These observations suggest that in neutral conditions, Ag presentation by DC results in the coordinate production of naive T cell-derived IL-4 and DC-derived IL-12 that in concert shape the cytokine profile of Th cells.     

Partial agonism and independent modulation of T cell receptor and CD8 in hapten-specific cytotoxic T cells

We recently demonstrated antagonism for hapten-reactive T cells by altered hapten ligands. Here we investigated partial peptide- or hapten-agonism and effects of antigen stimulation on the expression of TCR and the CD8 coreceptor using a set of DNP- or TNP-peptide-induced, H-2Kb-restricted mouse CTL clones. Various Kb-binding TNP- and DNP-peptides acted as partial agonists, cross-reactively stimulating individual clones for cytotoxicity and IFN-gamma secretion, but failing to induce proliferation or TNF-alpha production. Full agonism, i.e. activation of all possible functions, was usually restricted to those hapten-peptide combinations used for the induction of the respective clones. Our data imply distinctive kinetic optima for TCR antigen contacts in the induction of the various T cell effector functions. Down-regulation of TCR was efficiently induced by full, but with one exception not by partial, agonists, indicating the independence of cytotoxicity or IFN-gamma secretion from TCR modulation. On the other hand, a reduction of TCR expression induced by full agonists was usually not accompanied by synchronous down-modulation of CD8 as reported by others for human T cells. In fact, three of four full agonists and all partial agonists markedly enhanced rather than reduced the expression of CD8. Increased CD8 surface levels enhanced cytolytic potential and increased cross-reactivity patterns of individual clones. Brefeldin A blocked this CD8 induction by partial agonists, and in the case of full agonists resulted in a parallel reduction of both, TCR and CD8. Thus, antigenic stimulation of mouse T cells initially down-modulates CD8 together with TCR, but the loss of coreceptor is over-compensated by a signal for increased CD8 export.     

Interferon-gamma and interleukin-4 regulate T cell interleukin-12 responsiveness through the differential modulation of high-affinity interleukin-12 receptor expression

Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) are mutually antagonistic cytokines that stimulate CD4+ T cells to develop into either Th1 or Th2 cells. One feature of Th2 differentiation in mice is the loss of IL-12-induced Jak2 and Stat4 activation, which is accompanied by the inability to produce IFN-gamma in response to IL-12. In this report, we show that freshly isolated human T cells activated with phytohemagglutinin (PHA) in the presence of IL-4 exhibit a greatly diminished response to IL-12, whereas the IL-12 response of T cells activated with PHA plus IFN-gamma is enhanced. Radiolabeled IL-12 binding studies demonstrate that the impairment of T cell IL-12 responsiveness by IL-4 is associated with the down-regulation of high-affinity IL-12 receptor expression. In contrast, the enhancement of IL-12 responsiveness by IFN-gamma is associated with the upregulation of high-affinity IL-12 receptor expression. Through the use of a newly synthesized neutralizing antibody to the low-affinity IL-12 receptor beta subunit (IL-12Rbeta), we show that neither IL-4 nor IFN-gamma affect the expression of IL-12Rbeta, which we determine to be one of at least two low-affinity subunits required for high-affinity IL-12 binding. These findings suggest that IL-4 and IFN-gamma exert opposite effects on T cell IL-12 responsiveness by differentially modulating the expression of low-affinity IL-12 receptor subunits that are distinct from IL-12Rbeta and required, together with IL-12Rbeta, for high-affinity IL-12 binding and IL-12 responsiveness. This provides a basis for understanding the interplay between different cytokines at the level of cytokine receptor expression, and offers insight into one of the mechanisms governing Th1 and Th2 development.     

Weak peptide agonists reveal functional differences in B7-1 and B7-2 costimulation of human T cell clones

The influence of costimulation on the T cell response to altered peptide ligands that act as either partial or weak agonists for human CD4+ T cell clones was examined. Using stable Chinese hamster ovary (CHO) cell transfectants expressing DR2 (DRB1*1501) and human B7-1 or B7-2 as APC, presentation of native myelin basic protein (MBP) p85-99 peptide Ag or a partial agonist of MBP p85-99 induced equivalent T cell activation as measured by [3H]TdR incorporation and cytokine secretion. In marked contrast, presentation of cross-reactive peptides of MBP p85-99 that act as weak agonists with B7-1, but not B7-2, costimulation resulted in significant T cell activation as measured by [3H]TdR incorporation and cytokine secretion. These data suggest that decreasing the strength of the signal provided to the TCR allows differences in B7-1 and B7-2 signaling to be observed. Thus, the costimulatory environment during T cell activation may be a mechanism of regulating T cell cross-reactivity in the periphery.     

Defective Th1 and Th2 cytokine synthesis in the T-T cell presentation model for lack of CD40/CD40 ligand interaction

In this study, T or NK cell clones used as antigen-presenting cells (T- or NK-APC) were shown to be significantly less efficient than professional APC in inducing Th1 and Th2 cytokines by antigen-specific T cell clones. This phenomenon was not related to a limited engagement of TCR by T-APC, since comparable thresholds of TCR down-regulation were shown when antigen was presented by either T-APC or professional APC. Rather, the stimulatory T-APC weakness was due to their inability, because they are CD40-, to provide the appropriate co-stimuli to responder T cells both indirectly via IL-12, and partially via direct CD40L triggering on T cells. Indeed, the simultaneous addition of IL-12 and reagents directly engaging CD40L on responder T cells restored T cell cytokine synthesis when antigen was presented by T-APC. In addition, either IL-12 production or blocking of T cell cytokine synthesis by anti-IL-12 p75 antibodies was evident only when professional APC were used in our antigen-specific system. The down-regulation of cytokine synthesis in the system of T-T cell presentation could represent a novel mechanism of immune regulation, which may intervene to switch off detrimental Th1- or Th2-mediated responses induced by antigen presentation among activated T cells infiltrating inflamed tissues.     

CD9-mediated costimulation of TCR-triggered naive T cells leads to activation followed by apoptosis

The induction of full activation or death in TCR-triggered T cells depends largely on whether appropriate costimulatory signals are provided. In this study, we show that the costimulation of CD9 on naive T cells during TCR stimulation results in transient, albeit potent, activation followed by apoptosis, rather than full activation. Anti-CD9 mAb synergized with suboptimal doses of anti-CD3 mAb in inducing T cell activation. [3H]TdR incorporation determined 2 days after CD9 costimulation was as potent as that induced by CD28 costimulation. In contrast to progressive T cell proliferation induced by CD28 costimulation, CD9 costimulation led to the induction of apoptosis of once-activated T cells. Although IL-2R expression was induced significantly earlier and to a greater degree after CD9 costimulation than after CD28 costimulation, CD9 costimulation only transiently produced a small amount of IL-2 and induced apparently low levels of bcl-xL compared with those observed in CD28 costimulation. Addition of rIL-2 to cultures of CD9 costimulation induced strikingly enhanced expression of bcl proteins, especially of bcl-xL, and protected TCR-stimulated T cells from apoptosis. These data indicate that CD9-mediated costimulation of TCR-triggered naive T cells leads to activation followed by apoptosis as the result of failure to generate a positive signal for sufficient levels of IL-2 production.     

Type I IFNs inhibit human dendritic cell IL-12 production and Th1 cell development

We have investigated the role of type I IFNs (IFN-alpha and -beta) in human T cell differentiation using anti-CD3 mAb and allogeneic, in vitro-derived dendritic cells (DC) as APCs. DC were very efficient activators of naive CD4+ T cells, providing necessary costimulation and soluble factors to support Th1 differentiation and expansion. Addition of IFN-alphabeta to DC/T cell cultures resulted in induction of T cell IL-10 production and inhibition of IFN-gamma, TNF-alpha, and LT secretion. Diminished T cell IFN-gamma production correlated with IFN-alphabeta-mediated inhibition of the p40 chain of the IL-12 heterodimer secreted by DC. Suppression of p40 IL-12 and IFN-gamma was not due to increased levels of IL-10 in these cultures, and production of IFN-gamma could be restored by exogenous IL-12. These data indicate that type I IFNs inhibit DC p40 IL-12 expression, which is required for development of IFN-gamma-producing CD4+ T cells. Furthermore, when T cells were restimulated without IFN-beta, these cells induced less p40 IL-12 from DC, suggesting that the functional properties of T cells may regulate DC function. Thus, IFN-alphabeta inhibits both IL-12-dependent and independent Th1 cytokine production and provides a mechanism for inhibition of IL-12-mediated immunity in viral infections.