Evidence for a critical role for IL-2 in CD40-mediated activation of naive B cells by primary CD4 T cells

The interaction of CD40 on B cells with the CD40 ligand (CD40L) on preactivated CD4 T cells is critical for the initiation of T-dependent Ab responses. It is believed that signals via CD40 synergize with cytokines (e.g., IL-4 and IL-5) to drive B cell activation. However, primary T cells preactivated via CD3 alone cannot induce B cell proliferation; we have shown previously that costimulation of T cells via CD3 and CD28 stabilizes the expression of the CD40L, which we propose contributes to their capacity to act as competent helper-effector cells. Here we show that an additional, critical reason why CD3-stimulated CD40L-bearing T cells are incompetent helper cells is because they secrete insufficient IL-2. In contrast, CD28/CD3-activated T cells induce B cells to become IL-2 responsive via a combination of CD40L and IL-2-mediated signals, and these two stimuli subsequently drive B cell proliferation and IgM secretion. We therefore propose that T cells must first encounter Ag in conjunction with CD80/86 on APCs. This leads to the stable expression of CD40L and maximal secretion of IL-2, which together render primary T cells competent to activate B cells in an IL-2-dependent fashion.     

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.     

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.     

Regulation of C-C chemokine production by murine T cells by CD28/B7 costimulation

C-C chemokines play an important role in recruitment of T lymphocytes to inflammatory sites. T lymphocytes secrete chemokines, but the activation requirements for chemokine production by T cells are uncertain. We studied the regulation of C-C chemokine production by CD28 costimulatory signals by murine T lymphocytes. Splenocytes from BALB/c mice cultured with anti-CD3 mAb expressed macrophage-inflammatory protein (MIP)-1alpha mRNA and secreted MIP-1alpha, which was inhibited by anti-B7-1 plus anti-B7-2 mAbs. MIP-1alpha production by Ag-stimulated T cells from DO.11.10 TCR transgenic mice was augmented by anti-CD28 mAb and increased compared with DO.11.10/CD28(-/-) cells. When T cell costimulation was provided by IL-2, MIP-1alpha was not enhanced. Studies with IL-2, IL-4, STAT4, and STAT6 knock-out mice suggested that chemokine production is controlled by pathways different from those regulating T cell differentiation. Thus, CD28 costimulation may amplify an immune response by stimulating T cell survival, proliferation, and production of chemokines that recruit T cells to inflammatory sites.     

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.     

CD11b+CD28-CD4+ human T cells: activation requirements and association with HLA-DR alleles

Engagement of CD28 induces a major costimulatory pathway required by many CD4+ T cells in addition to activation via the TCR. In the absence of signals provided by CD28, ligation of the TCR alone can induce anergy or apoptosis in CD28+ cells. However, we report here characterization of a distinct subset of CD4+ T cells that are CD28-. Three autoreactive CD4+ human T cell clones that could be activated to produce IL-2 and proliferate by anti-CD3 alone were found to lack expression of CD28. CD28- clones that were activated with anti-CD3 alone were not anergic to restimulation via CD3. The presence of CD28-CD4+ T cells was verified in peripheral blood, and their frequency ranged from 0% to >22% of CD4+ T cells in different individuals. The percentage of CD28-CD4+ T cells in the peripheral blood of 57 individuals was significantly correlated with specific class II MHC alleles. Persons with HLA-DRB1*0401 and DR1 alleles had significantly higher numbers of CD28- T cells, while individuals with HLA-DR2(15) had significantly fewer CD28-CD4+ T cells than the mean. Like the CD28- clones, CD28-CD4+ T cells isolated from peripheral blood proliferated upon CD3 cross-linking in the absence of costimulation. The finding that CD28-CD4+ T cells resist induction of anergy following engagement of the TCR in the absence of conventional costimulation demonstrates one mechanism by which autoreactive T cells can escape processes that censor self-reactivity. The MHC associations observed suggest a relationship with autoimmunity and loss of self-tolerance.     

Differential effects of CD28 engagement and IL-12 on T cell activation by altered peptide ligands

To futher our understanding of the mechanisms underlying the diverse effects of altered peptide ligands (APL) on T cell activation, we used a population of nonactivated spleen cells from mice that expressed a transgenic TCR specific for myelin basic protein Ac1-11 and peptide analogues that display either enhanced or decreased affinities for TCR/MHC to address the question whether APL-induced signaling through the TCR can regulate the capability of APC to activate T cells. We demonstrate that weak agonists APL are poor inducers of all aspects of the activation of both the responder T cells and the APC. Enhancement of the antigenic signal by augmenting the binding of the weak agonists to MHC reversed their defective activating capacity. Enhancement of costimulation by engagement of CD28 only resulted in augmentation of the capacity of the weak agonist APL to induce proliferation and IL-2/IL-3 production, but not CD40L or IL-12Rbeta2 chain expression on T cells, CD80/CD86 expression on APC, IL-12 secretion, or IFN-gamma production. Exogenous IL-12 promoted IFN-gamma production in the presence of the weak agonists. These studies demonstrate that there is a critical threshold of antigenic signal required for full activation of the T cell-APC interactions needed for the differentiation of Th1 cells. The provision of excess costimulation can overcome some of the defects in T cell activation by weak agonists, but is insufficient to induce a sufficient level of CD40L expression needed for engagement of CD40 on APC with subsequent IL-12 production and induction of IL-12Rbeta2 chain expression.     

IL-10 impacts autoimmune diabetes via a CD8+ T cell pathway circumventing the requirement for CD4+ T and B lymphocytes

IL-10 is essential for an early phase of diabetes in nonobese diabetic (NOD) mice, but later becomes protective against its development. The mechanism by which IL-10 mediates the pathway to diabetes in these mice is unknown. Herein, we dissected the cellular and costimulation requirements for diabetes in transgenic (tg) NOD mice that expressed IL-10 in their pancreatic islets (IL-10-NOD mice). We found that IL-10 alone did not cause diabetes because the offspring (IL-10-NOD-scid mice) from back-crosses of IL-10-NOD mice with NOD-scid mice had no diabetes. Moreover, these IL-10-NOD-scid mice were free of lymphocytic infiltration. Treatment of IL-10-NOD mice with depleting anti-CD4 mAb or control mAb had no effect on diabetes. Surprisingly, depletion of CD8+ T cells by treatment with the corresponding mAb inhibited diabetes without attenuating insulitis, demonstrating a critical role for CD8+ T cells in the disease process. Interestingly, B cell-deficient IL-10-NOD mice readily developed diabetes with kinetics and incidence similar to those observed in wild-type mice, demonstrating that B lymphocytes as APCs were not required in the disease process. Administration of anti-CD40 ligand (CD40L) mAb did not prevent disease, indicating that CD40/CD40L costimulation is not required for diabetes in IL-10-NOD mice. Immunization of IL-10-NOD mice with CFA or heat-shock protein 65, known to block diabetes in NOD mice, had no effect on their diabetes. We demonstrate that IL-10 contributes early to the pathology of diabetes via a CD8+ T cell pathway, eliminating the requirement for B lymphocytes and CD40-CD40L costimulation. Our findings provide a mechanism for the participation of IL-10 in the early development of diabetes.     

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.     

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.     

Differential expression of CD40 ligand on T cell subsets. Implications for different roles of CD45RA+ and CD45RO+ cells in IgE production

Physical contact between human T lymphocytes and B lymphocytes is required for the induction of IgE production. In the present study, we examined the abilities of CD45RA+ and CD45RO+ human T cell subsets to provide help for IgE production by human peripheral blood B cells in the presence of IL-4. Purified peripheral CD45RA+ T cells are much better inducers of IgE synthesis than are CD45RO+ T cells. Activation of CD45RA+ T cells, but not CD45RO+ T cells, via the TCR/CD3 complex is sufficient to confer the ability to provide IgE help, suggesting that an inducible T cell surface molecule plays an important role in this system. The CD40 ligand, an inducible T cell surface molecule, is expressed at higher levels on CD45RA+ T cells as compared with CD45RO+ T cells following CD3-stimulation. Blocking of the CD40-CD40 ligand interaction in vitro by the addition of a soluble form of B cell CD40 Ag completely blocks IgE production induced by CD45RA+ T cells. Finally, the in vitro conversion of CD45RA+ T cells to the CD45RO+ phenotype is accompanied by a loss in the ability of these cells to express the CD40 ligand in response to anti-CD3 stimulation as well as a loss in their ability to provide IgE help. These results suggest that both CD45 subsets may play significant and distinct roles in the induction of IgE production under physiologic conditions: CD45RO+ T cells provide IL-4 and the CD45RA+ subset provides the second signal via the CD40 ligand.     

Processing and release of IL-16 from CD4+ but not CD8+ T cells is activation dependent

IL-16 is synthesized as a precursor molecule of 68 kDa (pro-IL-16) that is processed by caspase-3, a member of the IL-1 converting enzyme (ICE) family. This cleavage results in a 13-kDa carboxy terminal peptide, which constitutes the bioactive secreted form of IL-16. We have previously reported constitutive IL-16 mRNA expression and pro-IL-16 protein in CD4+ and CD8+ T cells. Although bioactive IL-16 protein is present in unstimulated CD8+ T cells, there is no bioactive IL-16 present in CD4+ T cells. Along these lines, unstimulated CD8+ T cells contain active caspase-3. In the current studies we investigated the regulation of IL-16 protein and mRNA expression in CD4+ T cells and determined the kinetics of secretion following stimulation of the TCR. CD4+ T cells release IL-16 protein following antigenic stimulation, and this release is accelerated in time by costimulation via CD28. However, CD3/CD28 costimulation did not alter IL-16 mRNA appearance or stability in either CD4+ or CD8+ T cells. The secretion of bioactive IL-16 from CD4+ T cells correlated with the appearance of cleavage of pro-caspase-3 into its 20-kDa active form. Thus, resting CD8+ T cells contain active caspase-3 that is capable of cleaving pro-IL-16, whereas CD4+ T cells require activation for the appearance of active caspase-3. The mechanism of release or secretion of bioactive IL-16 is currently unknown, but does not correlate with cellular apoptosis.     

Signaling through a CD3 gamma-deficient TCR/CD3 complex in immortalized mature CD4+ and CD8+ T lymphocytes

The biologic role of each CD3 chain and their relative contribution to the signals transduced through the TCR/CD3 complex and to downstream activation events are still controversial: they may be specialized or redundant. We have immortalized peripheral blood CD4+ and CD8+ T lymphocytes from a human selective CD3 gamma deficiency using Herpesvirus saimiri. The accessibility of the mutant TCR/CD3 complex to different Abs was consistently lower in immortalized CD8+ cells when compared with CD4+ cells, relative to their corresponding CD3 gamma-sufficient controls. Several TCR/CD3-induced downstream activation events, immediate (calcium flux), early (cytotoxicity and induction of surface CD69 or CD40L activation markers or intracellular TNF-alpha) and late (proliferation and secretion of TNF-alpha), were normal in gamma-deficient cells, despite the fact that their TCR/CD3 complexes were significantly less accessible than those of controls. In contrast, the accumulation of intracellular IL-2 or its secretion after CD3 triggering was severely impaired in gamma-deficient cells. The defect was upstream of protein kinase C activation because addition of transmembrane stimuli (PMA plus calcium ionophore) completely restored IL-2 secretion in gamma-deficient cells. These results suggest that the propagation of signals initiated at the TCR itself can result in a modified downstream signaling cascade with distinct functional consequences when gamma is absent. They also provide evidence for the specific participation of the CD3 gamma chain in the induction of certain cytokine genes in both CD4+ and CD8+ human mature T cells. These immortalized mutant cells may prove to be useful in isolating cytosolic signaling pathways emanating from the TCR/CD3 complex.     

The role of B7-1 and LFA-3 in costimulation of CD8+ T cells

This study compares the ability of LFA-3 (CD58) and B7-1 (CD80) ligands to provide costimulatory signals for superantigen (SAg)-stimulated CD8+ and CD4+ T cells. We show that B7-1 and LFA-3 costimulation activate CD8+ T cells to proliferation, cytokine production (IL-2, TNF, and IFN-gamma), and cytotoxicity. A long-lasting proliferative response was observed after combined DR/B7-1/LFA-3 costimulation. Detailed analysis of SEA-activated CD8+ T cells revealed that maximal production of IFN-gamma was seen in LFA-3-costimulated cells, while production of IL-2 was mainly induced after B7-1 costimulation. A fivefold increase in the IFN-gamma production was observed when activated CD8+ T cells were costimulated with Chinese hamster ovary (CHO)-DR/LFA-3 cells compared with the secretion induced by CHO-DR/B7-1. In contrast, SEA-treated CD4+ T cells costimulated with B7-1 or LFA-3 gave rise to a similar production of IFN-gamma, suggesting a preferential function for the CD2/LFA-3 pathway in the regulation of IFN-gamma in CD8+ T cells. Moreover, the generation of CTL was supported similarly by B7-1 and LFA-3 costimulation, but not by CHO-DR cells. We conclude that ligation of the CD28 and CD2 receptors mediate distinct effect on CD8+ and CD4+ T cell effector functions.     

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.     

Maturation of human neonatal CD4+ and CD8+ T lymphocytes into Th1/Th2 effectors

The increased susceptibility of neonates to infections has been ascribed to the immaturity of their immune system. More particularly, T cell-dependent responses were shown to be biased towards a Th2 phenotype. Our studies on the in vitro maturation of umbilical cord blood T cells suggest that the Th2 bias of neonatal response cannot be simply ascribed to intrinsic properties of neonatal T cells. Phenotypically, neonatal CD4+ T cells are more immature than their adult CD45RO-/RA+ naive counterparts and they contain a subset (10-20%) of CD45RO-/RA+ CD31- cells which is very low in adults and displays some unique functional features. The activation and maturation of neonatal CD4+ T cells is particularly dependent upon the strength of CD28-mediated cosignal which dictates not only the cytokine profile released upon primary activation but also the response to IL-12. Activation of adult as well as neonatal CD4+ T cells in the context of low CD28 costimulation yields to the production of low levels of only one cytokine, i.e. IL-2. In contrast, strong CD28 costimulation supports the production of high levels of type 1 (IL-2, IFN gamma and TNF beta) and low levels of type 2 (IL-4 and IL-13) cytokines by neonatal T cells. The low levels of naive T cell-derived IL-4 are sufficient to support their development into high IL-4/IL-5 producers by an autocrine pathway. The ability of IL-12 to prime neonatal CD4+ T cells for increased production of IL-4 (in addition to IFN gamma) is observed only when CD28 cosignal is minimal. Under optimal activation conditions (i.e. with anti-CD3/B7.1 or allogenic dendritic cells) the response and the maturation of neonatal and adult naive T cells are similar. Thus the Th2 bias of neonatal immune response cannot be simply ascribed to obvious intrinsic T cell defect but rather to particular conditions of Ag presentation at priming. Unlike CD4+ T cells, neonatal CD8+ T cells strictly require exogenous IL-4 to develop into IL-4/IL-5 producers. Most importantly, anti-CD3/B7-activated neonatal CD8 T cells coexpress CD4 as well as CCR5 and CXCR4 and are susceptible to HIV-1 infection in vitro.     

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.     

CD40 ligation prevents neonatal induction of transplantation tolerance

To investigate the consequences of CD40 engagement on the neonatal induction of transplantation tolerance, BALB/c mice were injected at birth with (A/J x BALB/c) F1 spleen cells together with activating anti-CD40 mAb and grafted 4 wk later with A/J skin. Whereas A/J allografts were accepted in mice neonatally injected with F1 cells and control Ab, they were acutely rejected in mice injected with F1 cells and anti-CD40 mAb. Neonatal administration of anti-CD40 mAb resulted in enhanced anti-A/J CTL activity, increased IFN-gamma, and decreased IL-4 production by donor-specific T cells in vitro. Experiments using anti-cytokine mAb and IFN-gamma-deficient mice demonstrated that CD40 ligation prevents neonatal allotolerance through an IFN-gamma- and IL-12-dependent pathway. Finally, we found that newborn T cells express less CD40L than adult T cells upon TCR engagement. Taken together these data indicate that insufficiency of CD40/CD40L interactions contribute to neonatal transplantation tolerance.     

Distinct regulation of T-cell death by CD28 depending on both its aggregation and T-cell receptor triggering: a role for Fas-FasL

CD28 is a major coreceptor that regulates cell proliferation, anergy, and viability of T cells. The negative selection by T-cell receptor (TCR)-induced cell death of immature thymocytes as well as of activated human antigen-specific T-cell clone, requires a costimulatory signal that can be provided by CD28. Conversely, CD28-mediated signals increase expression of Bcl-XL, a survival gene, and promote survival of naive T cells cultured in the absence of antigen or costimulation. Because CD28 appears to both protect from, or induce T-cell death, one important question is to define the activation and cellular parameters that dictate the differential role of CD28 in T-cell apoptosis. Here, we compared different CD28 ligands for their ability to regulate TCR-induced cell death of a murine T-cell hybridoma. In these cells, TCR triggering induced expression of Fas and FasL, and cell death was prevented by anti-Fas blocking monoclonal antibody (MoAb). When provided as a costimulus, both CD28 MoAb and the B7.1 and B7.2 counter receptors downregulated, yet did not completely abolish T-cell receptor-induced apoptosis. This CD28 cosignal resulted in both upregulation of Bcl-XL and prevention of FasL expression. In marked contrast, when given as a single signal, CD28 MoAb or B7.1 and B7.2 induced FasL expression and resulted in T-cell death by apoptosis, which was dependent on the level of CD28 ligation. Furthermore, triggering of CD28 upregulated FasL and induced a marked T-cell death of previously activated normal peripheral T cells. Our results identify Fas and FasL as crucial targets of CD28 in T-cell death regulation and show that within the same cell population, depending on its engagement as a single signal or as a costimulus together with the TCR, CD28 can either induce a dose-dependent death signal or protect from cell death, respectively. These data provide important insights into the role of CD28 in T-cell homeostasis and its possible implication in neoplastic disorders.