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.     

Human endothelial cells effectively costimulate cytokine production by, but not differentiation of, naive CD4+ T cells

We compared costimulatory signals provided by human endothelial cells (ECs) to those provided by conventional bone marrow-derived APCs, i.e., peripheral blood-adherent mononuclear cells (PBAMCs), by measuring their effects on cytokine production by naive or memory CD4+ T cells stimulated by PHA. In these assays, ECs effectively costimulate secretion of IL-2, IFN-gamma, and IL-4 from both naive and memory CD4+ T cells, quantified by ELISA or intracellular cytokine staining. ECs, which lack B7 molecules, use predominantly leukocyte-function associated Ag 3 (LFA-3) to provide costimulation. ECs are comparable to or better than PBAMCs, which use both the LFA-3 and B7 molecules, at costimulating IL-2 and IL-4 production. ECs are less effective than PBAMCs at costimulating IFN-gamma production by naive T cells. ECs do not secrete IL-12, and addition of exogenous IL-12 enables ECs to costimulate IFN-gamma at a level comparable to that observed with PBAMCs. ECs do not promote differentiation of naive T cells to Th1-like cells, whereas PBAMCs do. Again, addition of exogenous IL-12 enables ECs to do so. Transfection of ECs to express B7-1 or B7-2 is less effective than IL-12 supplementation for restoring these responses. These experiments suggest that a deficiency in costimulation due to lack of B7 molecule expression does not fully explain the inability of ECs to activate resting naive CD4+ T cells.     

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.     

Cross-linking of the CD40 ligand on human CD4+ T lymphocytes generates a costimulatory signal that up-regulates IL-4 synthesis

Although there is good evidence that the induction of IL-4 synthesis in CD4+ T lymphocytes is favored by Ag presentation by B cells and not macrophages, the precise molecular signals provided by B cells to T cells that enhance IL-4 synthesis are not clear. To examine this issue, we established an APC-independent system to activate highly purified T cells and induce cytokine synthesis, using immobilized mAbs against several T cell surface molecules, including CD3, CD28, and the CD40 ligand (CD40L). The counter-receptors for all three of these molecules are expressed on B cells, and include CD40, which is expressed primarily on B cells, but also on dendritic cells and thymic epithelium. We found that IL-4 synthesis was greatly enhanced by triggering of CD40L on the T cell surface in conjunction with ligation of CD3/TCR and CD28, whereas ligation of CD3/TCR and CD28 in the absence of CD40L triggering resulted in little or no IL-4 synthesis. CD40L costimulation greatly enhanced IL-4 synthesis both in T cells from normal nonallergic adult subjects as well as in naive T cells from cord blood. Furthermore, we demonstrated that IL-4 synthesis was optimally enhanced when the strength of the CD3/TCR signal was limiting, while IL-4 synthesis was inhibited when CD3/TCR stimulation was maximal. These studies confirm that IL-4 synthesis can be induced in normal T lymphocytes in the absence of exogenous IL-4, and demonstrate that CD40L costimulation is of fundamental importance in regulation of IL-4 production. In addition, these findings provide a mechanism by which B cells preferentially enhance IL-4 synthesis in T cells at low Ag concentrations.     

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.     

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.     

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.     

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.     

Critical role of CD28/B7 costimulation in the development of human Th2 cytokine-producing cells

CD28 is a major costimulatory signal receptor for T cells. We have used human naive CD4+ cells from cord blood to analyze the effect of the CD28/B7 costimulatory pathway on development of T helper (Th) subsets. We show that CD28 costimulation is critical for development of the Th2 cytokine-producing cells and that in the absence of CD28 costimulation, cells are not primed to produce Th2 cytokines and consequently "default" to the Th1 subset, independent of the presence of exogenous cytokines. After CD28 costimulation, cells differentiate into a subset that produces Th2 cytokines. However, further CD28 costimulation is not required to maintain Th2 cytokine production. We conclude that D28 costimulation is critical for the development of Th0 and Th2 subsets, but not for the maintenance of cytokine production.     

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.     

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.     

Active Wegener's granulomatosis is associated with HLA-DR+ CD4+ T cells exhibiting an unbalanced Th1-type T cell cytokine pattern: reversal with IL-10

Wegener's granulomatosis (WG) is a granulomatous vasculitis that affects the upper respiratory tract, lung, and kidney. Since T cells make up a significant proportion of cells infiltrating granulomatous lesions in WG, we investigated the proliferative response and cytokine profile of T cells from these patients. PBMCs were isolated from 12 patients with active WG, 7 patients with inactive disease, and 12 healthy normal donors. PBMCs from clinically active WG patients exhibited increased proliferation following stimulation with either PMA/ionomycin or anti-CD2 and anti-CD28, when compared with normal donors. In addition, these PBMCs exhibited increased secretion of IFN-gamma, but not of IL-4, IL-5, or IL-10. Furthermore, TNF-alpha production from PBMCs and CD4+ T cells isolated from patients with WG was elevated, when compared with healthy donors. In further studies, we investigated the ability of WG patients' monocytes to produce IL-12 and showed that both inactive and active patients produced increased amounts of IL-12. Finally, the in vitro IFN-gamma production by WG PBMC is inhibited in a dose-dependent manner by exogenous IL-10. These data suggest that T cells from WG patients overproduce IFN-gamma and TNF-alpha, probably due to dysregulated IL-12 secretion, and that IL-10 may therefore have therapeutic implications for this disease.     

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.     

Mucosally induced systemic T cell unresponsiveness to ovalbumin requires CD40 ligand-CD40 interactions

CD40 ligand (CD40L) gene-disrupted (CD40L-/-) mice were employed to examine the role of costimulatory signals via CD40L-CD40 interactions in mucosally induced tolerance. CD40L-/- and control (CD40L+/+) mice of the same C57BL/6 x 129/J background were immunized orally with 25 mg of OVA before systemic challenge with OVA in CFA. While CD40L+/+ mice showed reductions in Ag-specific T cell responses including delayed-type hypersensitivity (DTH) and proliferative responses, CD40L-/- mice underwent normal T cell responses. Further, cytokine analysis of splenic CD4+ T cells showed that both Th1-type (e.g., IFN-gamma and IL-2) and Th2-type (e.g., IL-4, IL-5, IL-6, and IL-10) responses were maintained in CD40L-/- mice orally immunized with OVA, whereas these cytokine responses in CD40L+/+ mice were significantly reduced. In addition, splenic CD4+ T cells from CD40L-/- mice orally immunized with OVA provided B cell help in Ag-specific Ab-forming cells when the cells were cultured with naive B cells in the presence of Ag and CD40L-transfected cell lines. In contrast, an identical culture condition containing splenic CD4+ T cells from orally tolerized CD40L+/+ mice did not exhibit helper activity. Taken together, these findings indicate that CD40L and CD40 interactions are essential for the induction of systemic T cell unresponsiveness to orally administered Ag.     

Differential capacities of CD4+, CD8+, and CD4-CD8- T cell subsets to express IL-18 receptor and produce IFN-gamma in response to IL-18

IL-12 and IL-18 have the capacity to stimulate IFN-gamma production by T cells. Using a T cell clone, we reported that IL-18 responsiveness is generated only after exposure to IL-12. Here, we investigated the induction of IL-18 responsiveness in resting CD8+, CD4+, and CD4-CD8- T cells. Resting T cells respond to neither IL-12 nor IL-18. After stimulation with anti-CD3 plus anti-CD28 mAbs, CD8+, CD4+, and CD4-CD8- T cells expressed IL-12R, but not IL-18R, and produced IFN-gamma in response to IL-12. Cultures of T cells with anti-CD3/anti-CD28 in the presence of rIL-12 induced IL-18R expression and IL-18-stimulated IFN-gamma production, which reached higher levels than that induced by IL-12 stimulation. However, there was a substantial difference in the expression of IL-18R and IL-18-stimulated IFN-gamma production among T cell subsets. CD4+ cells expressed marginal levels of IL-18R and produced small amounts of IFN-gamma, whereas CD8+ cells expressed higher levels of IL-18R and produced more IFN-gamma than CD4+ cells. Moreover, CD4-CD8- cells expressed levels of IL-18R comparable to those for CD8+ cells but produced IFN-gamma one order higher than did CD8+ cells. These results indicate that the induction of IL-18R and IL-18 responsiveness by IL-12 represents a mechanism underlying enhanced IFN-gamma production by resting T cells, but the operation of this mechanism differs depending on the T cell subset stimulated.     

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.     

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.     

CD40 expressed on thymic epithelial cells provides costimulation for proliferation but not for apoptosis of human thymocytes

Human thymic epithelial cells express CD40, so we examined the possible role of CD40 in activation of thymocytes. We observed that both CD4+CD8- and CD4-CD8+ thymocytes proliferate after stimulation by anti-CD3 mAb in the presence of cultured thymic epithelial cells. Costimulation of CD4+ thymocytes by thymic epithelial cells is partly inhibited by an anti-CD40 mAb, but this mAb has no effect on costimulation of CD8+ thymocytes. The selective costimulatory ability of CD40 for CD4+ thymocytes was confirmed in experiments in which thymocytes were stimulated with anti-CD3 in the presence of murine P815 cells transfected with CD40 cDNA. The level of costimulation induced by P815-CD40 was comparable with that induced by P815 cells expressing CD80 (B7.1). Treatment of thymocytes with the Ca2+ ionophore ionomycin and the phorbol ester PMA or with anti-CD3 mAb resulted in up-regulation of the CD40 ligand, suggesting that this molecule is involved in CD40-mediated costimulation of human thymocytes. Costimulation of thymocytes by CD80 strongly increased anti-CD3-induced death of fetal thymocytes. In contrast, costimulation by CD40 did not increase anti-CD3-mediated apoptosis of these thymocytes. To confirm that CD40 does not affect anti-CD3-induced cell death, we established a variant of the Jurkat T leukemic cell line that constitutively expresses CD40L and analyzed the sensitivity of this cell line for activation-induced apoptosis. In contrast to CD80, CD40 failed to increase anti-CD3-mediated apoptosis in CD40L+ Jurkat cells, whereas both CD40 and CD80 strongly increased IL-2 production induced by anti-CD3. These findings suggest that costimulation by CD40 is involved in clonal expansion of CD4+ thymocytes but not in activation-induced cell death.     

CD40 ligand/trimer DNA enhances both humoral and cellular immune responses and induces protective immunity to infectious and tumor challenge

CD40/CD40 ligand interactions have a central role in the induction of both humoral and cellular immunity. In this study, we examined whether a plasmid expressing CD40 ligand/trimer (CD40LT) could enhance immune responses in vivo. BALB/c mice were injected with plasmid expressing beta-galactosidase DNA with or without CD40LT DNA or IL-12 DNA, and immune responses were assessed. Mice vaccinated with beta-gal DNA plus CD40LT DNA or IL-12 DNA had a striking increase in Ag-specific production of IFN-gamma, cytolytic T cell activity, and IgG2a Ab. The mechanism by which CD40LT DNA enhanced these responses was further assessed by treating vaccinated mice with anti-IL-12 mAb or CTLA-4 Ig (CTLA4Ig). Production of IFN-gamma and CTL activity was abrogated by these treatments, suggesting that CD40LT DNA was mediating its effects on IFN-gamma and CTL activity through induction of IL-12 and enhancement of B7 expression, respectively. Physiologic relevance for the ability of CD40LT DNA to enhance immune responses by the aforementioned pathways was shown in two in vivo models. First, with regard to CTL activity, mice vaccinated with CD40LT DNA did not develop metastatic tumor following challenge with lethal dose of tumor. Moreover, in a mouse model requiring IL-12-dependent production of IFN-gamma, mice vaccinated with soluble Leishmania Ag and CD40LT DNA were able to control infection with Leishmania major. These data suggest that CD40LT DNA could be a useful vaccine adjuvant for diseases requiring cellular and/or humoral immunity.