Quantifying antigen-specific CD4 T cells during a viral infection: CD4 T cell responses are larger than we think

The number of virus-specific CD8 T cells increases substantially during an acute infection. Up to 90% of CD8 T cells are virus specific following lymphocytic choriomeningitis virus (LCMV) infection. In contrast, studies identifying virus-specific CD4 T cell epitopes have indicated that CD4 T cells often recognize a broader array of Ags than CD8 T cells, consequently making it difficult to accurately quantify the total magnitude of pathogen-specific CD4 T cell responses. In this study, we show that CD4 T cells become CD11a(hi)CD49d(+) after LCMV infection and retain this expression pattern into memory. During the effector phase, all the LCMV-specific IFN-γ(+) CD4 T cells display a CD11a(hi)CD49d(+) cell surface expression phenotype. In addition, only memory CD11a(hi)CD49d(+) CD4 T cells make IFN-γ after stimulation. Furthermore, upon secondary LCMV challenge, only CD11a(hi)CD49d(+) memory CD4 T cells from LCMV-immune mice undergo proliferative expansion, demonstrating that CD11a(hi)CD49d(+) CD4 T cells are truly Ag specific. Using the combination of CD11a and CD49d, we demonstrate that up to 50% of the CD4 T cells are virus specific during the peak of the LCMV response. Our results indicate that the magnitude of the virus-specific CD4 T cell response is much greater than previously recognized.     

Differential requirements for Th1 and Th17 responses to a systemic self-antigen

T cell-APC interactions are essential for the initiation of effector responses against foreign and self-antigens, but the role of these interactions in generating different populations of effector T cells in vivo remains unclear. Using a model of CD4(+) T cell responses to a systemic self-antigen without adjuvants or infection, we demonstrate that activation of APCs augments Th17 responses much more than Th1 responses. Recognition of systemic Ag induces tolerance in self-reactive CD4(+) T cells, but induction of CD40 signaling, even under tolerogenic conditions, results in a strong, Ag-specific IL-17 response without large numbers of IFN-γ-producing cells. Transfer of the same CD4(+) T cells into lymphopenic recipients expressing the self-antigen results in uncontrolled production of IL-17, IFN-γ, and systemic inflammation. If the Ag-specific T cells lack CD40L, production of IL-17 but not IFN-γ is decreased, and the survival time of recipient mice is significantly increased. In addition, transient blockade of the initial MHC class II-dependent T cell-APC interaction results in a greater reduction of IL-17 than of IFN-γ production. These data suggest that Th17 differentiation is more sensitive to T cell interactions with APCs than is the Th1 response, and interrupting this interaction, specifically the CD40 pathway, may be key to controlling Th17-mediated autoimmunity.     

Functional Exhaustion of CD4+ T Lymphocytes during Primary Cytomegalovirus Infection

Human CMV establishes lifelong persistence after primary infection. Chronic CMV infection is associated with intermittent viral reactivation inducing high frequencies of CD4(+) T lymphocytes with potent antiviral and helper properties. Primary CMV infection is characterized by an intense viral replication lasting for several months. The impact of this prolonged exposure to high Ag loads on the functionality of CD4(+) T cells remains incompletely understood. In pregnant women with primary CMV infection, we observed that CMV-specific CD4(+) T lymphocytes had a decreased capacity to proliferate and to produce IL-2. A very large proportion of CMV-specific CD4(+) T cells had downregulated the expression of CD28, a costimulatory molecule centrally involved in the production of IL-2. Unexpectedly, both CD28(-) and CD28(+)CD4(+) T cells produced low levels of IL-2. This defective production of IL-2 was part of a larger downregulation of cytokine production. Indeed, CMV-specific CD4(+) T cells produced lower amounts of IFN-γ and TNF-α and showed lower functional avidity during primary as compared with chronic infection. Increased programmed death-1 expression was observed in CD28(+) CMV-specific CD4(+) T cells, and programmed death-1 inhibition increased proliferative responses. These results indicate that primary CMV infection is associated with the exhaustion of CMV-specific CD4(+) T cells displaying low functional avidity for viral Ags.     

Efficient lung recruitment of respiratory syncytial virus-specific Th1 cells induced by recombinant bacillus Calmette-Guérin promotes virus clearance and protects from infection

Infection by the respiratory syncytial virus (RSV) can cause extensive inflammation and lung damage in susceptible hosts due to a Th2-biased immune response. Such a deleterious inflammatory response can be enhanced by immunization with formalin- or UV-inactivated RSV, as well as with vaccinia virus expressing the RSV-G protein. Recently, we have shown that vaccination with rBCG-expressing RSV Ags can prevent the disease in the mouse. To further understand the immunological mechanisms responsible for protection against RSV, we have characterized the T cell populations contributing to virus clearance in mice immunized with this BCG-based vaccine. We found that both CD4(+) and CD8(+) T cells were recruited significantly earlier to the lungs of infected mice that were previously vaccinated. Furthermore, we observed that simultaneous adoptive transfer of CD8(+) and CD4(+) RSV-specific T cells from vaccinated mice was required to confer protection against virus infection in naive recipients. In addition, CD4(+) T cells induced by vaccination released IFN-γ after RSV challenge, indicating that protection is mediated by a Th1 immune response. These data suggest that vaccination with rBCG-expressing RSV Ags can induce a specific effector/memory Th1 immune response consisting on CD4(+) and CD8(+) T cells, both necessary for a fully protective response against RSV. These results support the notion that an effective induction of Th1 T cell immunity against RSV during childhood could counteract the unbalanced Th2-like immune response triggered by the natural RSV infection.     

Autocrine IFN-γ promotes naive CD8 T cell differentiation and synergizes with IFN-α to stimulate strong function

Autocrine IFN-γ signaling is important for CD4 differentiation to Th1 effector cells, but it has been unclear whether it contributes to CD8 T cell differentiation. We show in this paper that naive murine CD8 T cells rapidly and transiently produce low levels of IFN-γ upon stimulation with Ag and B7-1, with production peaking at ～8 h and declining by 24 h. The autocrine IFN-γ signals for upregulation of expression of T-bet and granzyme B and induces weak cytolytic activity and effector IFN-γ production. IFN-α acts synergistically with IFN-γ to support development of strong effector functions, whereas IL-12 induces high T-bet expression and strong function in the absence of IFN-γ signaling. Thus, IFN-γ is not only an important CD8 T cell effector cytokine, it is an autocrine/paracrine factor whose contributions to differentiation vary depending on whether the response is supported by IL-12 or type I IFN.     

TLR4 signaling via MyD88 and TRIF differentially shape the CD4+ T cell response to Porphyromonas gingivalis hemagglutinin B

Recombinant hemagglutinin B (rHagB), a virulence factor of the periodontal pathogen Porphyromonas gingivalis, has been shown to induce protective immunity against bacterial infection. Furthermore, we have demonstrated that rHagB is a TLR4 agonist for dendritic cells. However, it is not known how rHagB dendritic cell stimulation affects the activation and differentiation of T cells. Therefore, we undertook the present study to examine the role of TLR4 signaling in shaping the CD4(+) T cell response following immunization of mice with rHagB. Immunization with this Ag resulted in the induction of specific CD4(+) T cells and Ab responses. In TLR4(-/-) and MyD88(-/-) but not Toll/IL-1R domain-containing adapter inducing IFN-β-deficient (TRIF(Lps2)) mice, there was an increase in the Th2 CD4(+) T cell subset, a decrease in the Th1 subset, and higher serum IgG(1)/IgG(2) levels of HagB-specific Abs compared with those in wild-type mice. These finding were accompanied by increased GATA-3 and Foxp3 expression and a decrease in the activation of CD4(+) T cells isolated from TLR4(-/-) and MyD88(-/-) mice. Interestingly, TLR4(-/-) CD4(+) T cells showed an increase in IL-2/STAT5 signaling. Whereas TRIF deficiency had minimal effects on the CD4(+) T cell response, it resulted in increased IFN-γ and IL-17 production by memory CD4(+) T cells. To our knowledge, these results demonstrate for the first time that TLR4 signaling, via the downstream MyD88 and TRIF molecules, exerts a differential regulation on the CD4(+) T cell response to HagB Ag. The gained insight from the present work will aid in designing better therapeutic strategies against P. gingivalis infection.     

Human cord blood CD4+CD25hi regulatory T cells suppress prenatally acquired T cell responses to Plasmodium falciparum antigens

In malaria endemic regions, a fetus is often exposed in utero to Plasmodium falciparum blood-stage Ags. In some newborns, this can result in the induction of immune suppression. We have previously shown these modulated immune responses to persist postnatally, with a subsequent increase in a child's susceptibility to infection. To test the hypothesis that this immune suppression is partially mediated by malaria-specific regulatory T cells (T(regs)) in utero, cord blood mononuclear cells (CBMC) were obtained from 44 Kenyan newborns of women with and without malaria at delivery. CD4(+)CD25(lo) T cells and CD4(+)CD25(hi) FOXP3(+) cells (T(regs)) were enriched from CBMC. T(reg) frequency and HLA-DR expression on T(regs) were significantly greater for Kenyan as compared with North American CBMC (p < 0.01). CBMC/CD4(+) T cells cultured with P. falciparum blood-stage Ags induced production of IFN-γ, IL-13, IL-10, and/or IL-5 in 50% of samples. Partial depletion of CD25(hi) cells augmented the Ag-driven IFN-γ production in 69% of subjects with malaria-specific responses and revealed additional Ag-reactive lymphocytes in previously unresponsive individuals (n = 3). Addition of T(regs) to CD4(+)CD25(lo) cells suppressed spontaneous and malaria Ag-driven production of IFN-γ in a dose-dependent fashion, until production was completely inhibited in most subjects. In contrast, T(regs) only partially suppressed malaria-induced Th2 cytokines. IL-10 or TGF-β did not mediate this suppression. Thus, prenatal exposure to malaria blood-stage Ags induces T(regs) that primarily suppress Th1-type recall responses to P. falciparum blood-stage Ags. Persistence of these T(regs) postnatally could modify a child's susceptibility to malaria infection and disease.     

Cellular and molecular requirements for rejection of B16 melanoma in the setting of regulatory T cell depletion and homeostatic proliferation

We have recently demonstrated that adoptive transfer of regulatory T cell-depleted polyclonal T cells into lymphopenic mice leads to rejection of B16 melanoma, which generated an opportunity to study host requirements for tumor rejection when it effectively occurred. CD8(+) T cell priming and tumor rejection required tumor Ag cross-presentation, as evidenced by tumor outgrowth in Kb(-/-) bone marrow chimeric or B71/2(-/-) mice. CD4(+) T cells were additionally required for optimal tumor control, although not through classical CD4 "help," as the frequency of primed CD8(+) T cells was similar in the absence of CD4(+) T cells, and tumor rejection did not depend upon CD40-CD40L interactions or on IL-2 production by CD4(+) T cells. Rather, CD4(+) T cells appeared to act at the effector phase of tumor rejection and responded to B16-derived Ags in vitro. At the effector phase, IFN-γ production by transferred T cells, but not host cells, was necessary. IFN-γ acted either on host or tumor cells and was associated with reduced tumor vascularity. Finally, tumor rejection occurred after transfer of TNF-α, perforin, or FasL-deficient T cells. However, perforin/FasL double-knockout T cells failed to reject, arguing that the killing of B16 melanoma cells could occur either via the cytotoxic granule or Fas pathways. Collectively, these results support a model in which host tumor Ag cross-presentation primes adoptively transferred T cells, which remain functional in the setting of homeostatic proliferation and regulatory T cell depletion, and which promote tumor rejection via IFN-γ and lysis via cytotoxic granules and/or FasL.     

Negative Impact of IFN-γ on Early Host Immune Responses to Retroviral Infection

The immune system is tasked with defending against a myriad of microbial infections, and its response to a given infectious microbe may be strongly influenced by coinfection with another microbe. It was shown that infection of mice with lactate dehydrogenase-elevating virus (LDV) impairs early adaptive immune responses to Friend virus (FV) coinfection. To investigate the mechanism of this impairment, we examined LDV-induced innate immune responses and found LDV-specific induction of IFN-α and IFN-γ. LDV-induced IFN-α had little effect on FV infection or immune responses, but unexpectedly, LDV-induced IFN-γ production dampened Th1 adaptive immune responses and enhanced FV infection. Two distinct effects were identified. First, LDV-induced IFN-γ signaling indirectly modulated FV-specific CD8(+) T cell responses. Second, intrinsic IFN-γ signaling in B cells promoted polyclonal B cell activation and enhanced early FV infection, despite promotion of germinal center formation and neutralizing Ab production. Results from this model reveal that IFN-γ production can have detrimental effects on early adaptive immune responses and virus control.     

Macrophage migration inhibitory factor: a downregulator of early T cell-dependent IFN-gamma responses in Plasmodium chabaudi adami (556 KA)-infected mice

Neutralization of macrophage migration inhibitory factor (MIF) increases anti-tumor cytotoxic T cell responses in vivo and IFN-γ responses in vitro, suggesting a plausible regulatory role for MIF in T cell activation. Considering that IFN-γ production by CD4(+) T cells is pivotal to resolve murine malaria and that secretion of MIF is induced by Plasmodium chabaudi adami parasites, we investigated the effect of MIF deficiency on the infection with this pathogen. Infections with P. c. adami 556 KA parasites were more efficiently controlled in MIF-neutralized and MIF-deficient (knockout [KO]) BALB/c mice. The reduction in parasitemia was associated with reduced production of IL-4 by non-T/non-B cells throughout patent infection. At day 4 postinfection, higher numbers of activated CD4(+) cells were measured in MIF KO mice, which secreted more IFN-γ, less IL-4, and less IL-10 than did CD4(+) T cells from wild-type mice. Enhanced IFN-γ and decreased IL-4 responses also were measured in MIF KO CD4(+) T cells stimulated with or without IL-12 and anti-IL-4 blocking Ab to induce Th1 polarization. However, MIF KO CD4(+) T cells efficiently acquired a Th2 phenotype when stimulated in the presence of IL-4 and anti-IL-12 Ab, indicating normal responsiveness to IL-4/STAT6 signaling. These results suggest that by promoting IL-4 responses in cells other than T/B cells during early P. c. adami infection, MIF decreases IFN-γ secretion in CD4(+) T cells and, additionally, has the intrinsic ability to render CD4(+) T cells less capable of acquiring a robust Th1 phenotype when stimulated in the presence of IL-12.     

Independent regulation of lymphocytic choriomeningitis virus-specific T cell memory pools: relative stability of CD4 memory under conditions of CD8 memory T cell loss

Infection of mice with a series of heterologous viruses causes a reduction of memory CD8(+) T cells specific to viruses from earlier infections, but the fate of the virus-specific memory CD4(+) T cell pool following multiple virus infections has been unknown. We have previously reported that the virus-specific CD4(+) Th precursor (Thp) frequency remains stable into long-term immunity following lymphocytic choriomeningitis virus (LCMV) infection. In this study, we questioned whether heterologous virus infections or injection with soluble protein CD4 Ags would impact this stable LCMV-specific CD4(+) Thp memory pool. Limiting dilution analyses for IL-2-producing cells and intracellular cytokine staining for IFN-gamma revealed that the LCMV-specific CD4(+) Thp frequency remains relatively stable following multiple heterologous virus infections or protein Ag immunizations, even under conditions that dramatically reduce the LCMV-specific CD8(+) CTL precursor frequency. These data indicate that the CD4(+) and CD8(+) memory T cell pools are regulated independently and that the loss in CD8(+) T cell memory following heterologous virus infections is not a consequence of a parallel loss in the memory CD4(+) T cell population.     

CD4+ and CD8+ T cells exhibit differential requirements for CCR7-mediated antigen transport during influenza infection

Upon encounter of viral Ags in an inflammatory environment, dendritic cells up-regulate costimulatory molecules and the chemokine receptor CCR7, with the latter being pivotal for their migration to the lymph node. By utilizing mice deficient in CCR7, we have examined the requirement of dendritic cell-mediated Ag transport from the lung to the draining lymph node for the induction of anti-influenza immune responses in vivo. We found that CCR7-mediated migration of dendritic cells was more crucial for CD8(+) T cell than CD4(+) T cell responses. While no specific CD8(+) T cell response could be detected in the airways or lymphoid tissues during the primary infection, prolonged infection in CCR7-deficient mice did result in a sustained inflammatory chemokine profile, which led to nonspecific CD8(+) T cell recruitment to the airways. The recruitment of influenza-specific CD4(+) T cells to the airways was also below levels of detection in the absence of CCR7 signaling, although a small influenza-specific CD4(+) T cell population was detectable in the draining lymph node, which was sufficient for the generation of class-switched anti-influenza Abs and a normal CD4(+) T cell memory population. Overall, our data show that CCR7-mediated active Ag transport is differentially required for CD4(+) and CD8(+) T cell expansion during influenza infection.     

4-1BB triggering ameliorates experimental autoimmune encephalomyelitis by modulating the balance between Th17 and regulatory T cells

Agonistic anti-4-1BB Ab is known to ameliorate experimental autoimmune encephalomyelitis. 4-1BB triggering typically leads to the expansion of CD8(+) T cells, which produce abundant IFN-γ, and this in turn results in IDO-dependent suppression of autoimmune responses. However, because neutralization of IFN-γ or depletion of CD8(+) T cell only partially abrogates the effect of 4-1BB triggering, we sought to identify an additional mechanism of 4-1BB-triggered suppression of autoimmune responses using IFN-γ- or IFN-γR-deficient mice. 4-1BB triggering inhibited the generation of Th17 cells that is responsible for experimental autoimmune encephalomyelitis induction and progression, and increased Foxp3(+)CD4(+) regulatory T (Treg) cells, particularly among CD4(+) T cells. This was not due to a direct effect of 4-1BB signaling on CD4(+) T cell differentiation: 4-1BB signaling not only reduced Th17 cells and increased Treg cells in wild-type mice, which could be due to IFN-γ production by the CD8(+) T cells, but also did so in IFN-γ-deficient mice, in that case by downregulating IL-6 production. These results show that although secondary suppressive mechanisms evoked by 4-1BB triggering are usually masked by the strong effects of IFN-γ, 4-1BB signaling seems to modulate autoimmune responses by a number of mechanisms, and modulation of the Th17 versus Treg cell balance is one of those mechanisms.     

Heterogeneous and tissue-specific regulation of effector T cell responses by IFN-gamma during Plasmodium berghei ANKA infection

IFN-γ and T cells are both required for the development of experimental cerebral malaria during Plasmodium berghei ANKA infection. Surprisingly, however, the role of IFN-γ in shaping the effector CD4(+) and CD8(+) T cell response during this infection has not been examined in detail. To address this, we have compared the effector T cell responses in wild-type and IFN-γ(-/-) mice during P. berghei ANKA infection. The expansion of splenic CD4(+) and CD8(+) T cells during P. berghei ANKA infection was unaffected by the absence of IFN-γ, but the contraction phase of the T cell response was significantly attenuated. Splenic T cell activation and effector function were essentially normal in IFN-γ(-/-) mice; however, the migration to, and accumulation of, effector CD4(+) and CD8(+) T cells in the lung, liver, and brain was altered in IFN-γ(-/-) mice. Interestingly, activation and accumulation of T cells in various nonlymphoid organs was differently affected by lack of IFN-γ, suggesting that IFN-γ influences T cell effector function to varying levels in different anatomical locations. Importantly, control of splenic T cell numbers during P. berghei ANKA infection depended on active IFN-γ-dependent environmental signals--leading to T cell apoptosis--rather than upon intrinsic alterations in T cell programming. To our knowledge, this is the first study to fully investigate the role of IFN-γ in modulating T cell function during P. berghei ANKA infection and reveals that IFN-γ is required for efficient contraction of the pool of activated T cells.     

Stimulation of the glucocorticoid-induced TNF receptor family-related receptor on CD8 T cells induces protective and high-avidity T cell responses to tumor-specific antigens

Treatment of tumor-bearing mice with a stimulatory Ab to glucocorticoid-induced TNFR family-related receptor (GITR) has previously been shown to elicit protective T cell responses against poorly immunogenic tumors. However, the role of GITR stimulation on CD8 T cells and the nature of tumor rejection Ags have yet to be determined. In this study, we show that a stimulatory mAb to GITR (clone DTA-1) acts directly on CD8 T cells, but not on CD4(+)CD25(+) regulatory T (T(reg)) cells, in B16 tumor-bearing mice to induce concomitant immunity against secondary B16 tumors, as well as protective memory following surgical excision of the primary tumor. Melanoma growth itself induced GITR expression on tumor-specific CD8 T cells, providing a mechanism whereby these cells may respond to stimulatory anti-GITR. Unexpectedly, in contrast to T(reg) cell depletion therapy with anti-CD4, GITR stimulation induced very weak CD8 T cell responses to melanocyte differentiation Ags expressed by the tumor, and did not induce autoimmune vitiligo. Accordingly, GITR-stimulated hosts that were primed with B16 melanoma rejected B16, but not the unrelated JBRH melanoma, indicating that tumor rejection Ags are tumor-specific rather than shared. In support of this, we show that GITR stimulation induces CD8 T cell responses to a tumor-specific Ag, and that these responses are of higher functional avidity compared with those induced by T(reg) cell depletion. We conclude that stimulation of GITR on effector CD8 T cells results in high-avidity T cell responses to tumor-specific Ags, thereby inducing potent antitumor immunity in the absence of autoimmunity.     

Outer membrane protein A (OmpA) of Shigella flexneri 2a links innate and adaptive immunity in a TLR2-dependent manner and involvement of IL-12 and nitric oxide

We determine that OmpA of Shigella flexneri 2a is recognized by TLR2 and consequently mediates the release of proinflammatory cytokines and activates NF-κB in HEK 293 cells transfected with TLR2. We also observe that in RAW macrophages TLR2 is essential to instigate the early immune response to OmpA via NF-κB activation and secretion of cytokines and NO. Consistent with these results, TLR2 knockdown using siRNA abolishes the initiation of immune responses. Processing and presentation of OmpA depend on TLR2; MHCII presentation of the processed antigen and expression of CD80 significantly attenuated in TLR2 knockdown macrophages. The optimum production of IFN-γ by the macrophages:CD4(+) T cells co-culture depends on both TLR2 activation and antigen presentation. So, TLR2 is clearly recognized as a decisive factor in initiating host innate immune response to OmpA for the development of CD4(+) T cell adaptive response. Furthermore, we demonstrate in vivo that intranasal immunization of mice with OmpA selectively enhances the release of IFN-γ and IL-2 by CD4(+) T cells. Importantly, OmpA increases the level of IFN-γ production in Ag-primed splenocytes. The addition of neutralizing anti-IL-12p70 mAb to cell cultures results in the decreased release of OmpA-enhanced IFN-γ by Ag-primed splenocytes. Moreover, coincubation with OmpA-pretreated macrophages enhances the production of IFN-γ by OmpA-primed CD4(+) T cells, representing that OmpA may enhance IFN-γ expression in CD4(+) T cells through the induction of IL-12 production in macrophages. These results demonstrate that S. flexneri 2a OmpA may play a critical role in the development of Th1 skewed adaptive immune response.     

Evidence for human CD4+ T cells in the CD1-restricted repertoire: derivation of mycobacteria-reactive T cells from leprosy lesions

Both the CD4-CD8- (double negative) and CD4-CD8+ T cell lineages have been shown to contain T cells which recognize microbial lipid and glycolipid Ags in the context of human CD1 molecules. To determine whether T cells expressing the CD4 coreceptor could recognize Ag in the context of CD1, we derived CD4+ T cell lines from the lesions of leprosy patients. We identified three CD4+ Mycobacterium leprae-reactive, CD1-restricted T cell lines: two CD1b restricted and one CD1c restricted. These T cell lines recognize mycobacterial Ags, one of which has not been previously described for CD1-restricted T cells. The response of CD4+ CD1-restricted T cells, unlike MHC class II-restricted T cells, was not inhibited by anti-CD4 mAb, suggesting that the CD4 coreceptor does not impact positive or negative selection of CD1-restricted T cells. The CD4+ CD1-restricted T cell lines produced IFN-gamma and GM-CSF, the Th1 pattern of cytokines required for cell-mediated immunity against intracellular pathogens, but no detectable IL-4. The existence of CD4+ CD1-restricted T cells that produce a Th1 cytokine pattern suggests a contributory role in immunity to mycobacterial infection.     

MEKK3 regulates IFN-gamma production in T cells through the Rac1/2-dependent MAPK cascades

MEKK3 is a conserved Ser/Thr protein kinase belonging to the MAPK kinase kinase (MAP3K) family. MEKK3 is constitutively expressed in T cells, but its function in T cell immunity has not been fully elucidated. Using Mekk3 T cell conditional knockout (T-cKO) mice, we show that MEKK3 is required for T cell immunity in vivo. Mekk3 T-cKO mice had reduced T cell response to bacterial infection and were defective in clearing bacterial infections. The Ag-induced cytokine production, especially IFN-γ production, was impaired in Mekk3-deficient CD4 T cells. The TCR-induced ERK1/2, JNK, and p38 MAPKs activation was also defective in Mekk3-deficient CD4 T cells. In vitro, MEKK3 is not required for Th1 and Th2 cell differentiation. Notably, under a nonpolarizing condition (Th0), Mekk3 deficiency led to a significant reduction of IFN-γ production in CD4 T cells. Furthermore, the IL-12/IL-18-driven IFN-γ production and MAPK activation in Mekk3-deficient T cells was not affected suggesting that MEKK3 may selectively mediate the TCR-induced MAPK signals for IFN-γ production. Finally, we found that MEKK3 activation by TCR stimulation requires Rac1/2. Taken together, our study reveals a specific role of MEKK3 in mediating the TCR signals for IFN-γ production.