In this work we have demonstrated that PKC-θ is required for in vivo development of Th2 cell– but not Th1 cell–dependent immune responses. PKC-θ–deficient mice exhibited a highly impaired Th2 cell immune response against the helminth Nb, as shown by reduced IL-4–dependent IgE antibody production and IL-5–dependent blood and airway eosinophilia. In addition, and as expected from published in vitro observations, antigen-specific T cell proliferation was also significantly impaired after ex vivo restimulation. In support of these data, PKC-θ–deficient mice failed to develop allergic airway inflammation, IgE antibody responses, and AHR in a model of atopic asthma. A key finding was that although IL-4 production was selectively impaired in PKC-θ–deficient mice, a comparable number of antigen-specific CD4+ T cells from PKC-θ–deficient mice and wild-type controls produced IFN-γ. This finding was supported by the observation that the Th1 cell response against L. major infection was not affected by the absence of PKC-θ, although the Th2 cell response was compromised. Resolution of footpad swelling, parasite titer, IFN-γ production, and proliferation in response to L. major antigen were similar in PKC-θ–deficient and control C57BL/6 mice. Moreover, in BALB/c mice, which are normally susceptible to infection due to an early IL-4 response, resolution of footpad swelling was observed in animals lacking PKC-θ, further demonstrating the failure of a Th2 cell response to the parasite.
PKC-θ is required for activation of the transcription factors NF-κB and AP-1 after TCR ligation (10
). Both transcription factors have been implicated in Th cell development. NF-κB family members regulate IFN-γ and IL-12 as well as Th2 cell cytokine expression (12
), whereas AP-1, specifically Jun B, activates IL-4 expression in conjunction with c-MAF (30
). However, both transcription factors are also required for IL-2 expression, which is severely compromised in PKC-θ–deficient T cells (10
). Extensive evidence points to a role for IL-2 in regulation of Th2 cell cytokine responses. Le Gros et al. (29
) originally showed that IL-2 production enhances the development of Th2 cells. This result was consistent with the finding that in vivo blockade of IL-2 resulted in resistance to L. major
infection in normally susceptible BALB/c mice (31
). IL-2 is required for IL-4 expression in vitro (29
) and has recently been demonstrated to stabilize chromatin accessibility for STAT5 binding to the il4
gene, suggesting that it functions to maintain transcriptional activity at the il4
). Interestingly, IFN-γ expression appears to be less sensitive to the levels of IL-2, as mice treated with anti–IL-2 antibodies or lacking PKC-θ express sufficient levels of IFN-γ to combat L. major
infection ( and reference 31
). Anti–IL-2 treatment also inhibited IL-4 expression in DO11.10 TCR transgenic T cells stimulated in vivo (34
). Our in vitro T cell stimulation experiments demonstrate that IL-4 expression is partially rescued by the addition of exogenous IL-2 (). Therefore, failure to produce IL-2 might be a central mechanism underlying the impaired Th2 cell immune responses found in PKC-θ–deficient mice.
Because exogenous IL-2 failed to fully restore differentiation of PKC-θ–deficient Th2 cells, other pathways involved in Th1 cell differentiation may additionally be compromised in the mutant mice. In mice lacking the guanine nucleotide exchange factor Vav, which has been reported to interact with PKC-θ, there was also reduced IL-4 mRNA, but normal IFN-γ mRNA, in draining lymph nodes after immunization (35
). A similar phenotype to the one described in this work was found for Itk-deficient mice (36
), in which development of the Th2 cell immune responses against Nb and L. major
were impaired, whereas Th1 cell responses were normal. Although no direct association between Itk and PKC-θ has thus far been described, the strikingly similar phenotype of these mice suggests that these pathways of Th2 cell induction intersect. Moreover, Btk was reported to be associated with PKC-β in B cells, suggesting that Tec family kinases and PKCs may function in the same signaling pathway (37
). Therefore, the defect in the PKC-θ–deficient mice might be due to interruption of signals mediated through Itk and Vav after TCR and/or CD28 ligation. It is possible, however, that the impaired in vivo and in vitro Th2 cell responses of Itk-deficient mice may also be due to reduced levels of IL-2 (36
). In these mice, the reduced expression of IL-2 was shown to be due, at least in part, to defective activation of NFAT. Thus, the impaired Th2 cell development observed in both ITK and PKC-θ–deficient mice may derive from reduced IL-2 expression rather than from defects in PKC-θ–derived signals to activate the il4
PKC-θ and the CD28 costimulatory pathway closely interact in the activation of T cells. PKC-θ requires CD28 signaling to localize into the center of the mature immunological synapse (7
) and is thought to integrate TCR and CD28 signals, leading to complete T cell activation (38
). Ectopic expression of the serine-threonine kinase Akt in CD28-deficient mice has been shown to compensate for some of the costimulatory signal that is provided by CD28. Specifically, Kane et al. (39
) were able to reconstitute IFN-γ production, but not IL-4 or IL-5, by ectopic expression of Akt in CD28-deficient mice. These data are similar to the specific impairment of IL-4 and IL-5 production that we describe in this work, raising the possibility that although either Akt-mediated signaling or PKC-θ–mediated signaling might be sufficient to induce Th1 cell immune responses, Th2 cell immune responses require a productive interaction between the two molecules. Such ectopic expression of Akt may not have been adequate for the integration of PKC-θ into the immunological synapse, or its Lck-mediated activation, and consequently resulted in impaired production of Th2 cell cytokines. Indeed, PKC-θ and Akt have been reported to physically associate upon T cell activation and to activate the NF-κB signaling pathway (40
Whether loss of PKC-θ may directly affect Th2 cell cytokine gene expression remains unclear. Earlier studies have implicated members of the NF-κB and NFAT families of transcription factors in regulation of Th1 and Th2 cell differentiation. Although NF-κB family members have been reported to regulate the expression of both Th1 (15
) and Th2 cell cytokines (12
), IL-4 production and Th2 cell–dependent airway eosinophil responses were compromised in mice lacking expression of the p50 subunit of NF-κB (12
). In these mice, IFN-γ production was comparable to levels in wild-type animals (14
). Because PKC-θ is required for NF-κB activation after TCR ligation, impairment of this pathway in mutant mice may explain, at least in part, the defect in Th2 cell cytokine regulation. The role of NFAT signaling in the observed Th2 cell defect in the PKC-θ–deficient mice is more difficult to interpret. Although we did not observe a defect in NFAT activation in T cells from mutant mice, another group reported impaired nuclear NFAT DNA binding activity in a different strain of pkc
mice. Even if NFAT signaling were defective, the observed reduction in IL-4 levels cannot be readily explained by current literature, which implicates members of the NFAT family in both positive and negative regulation of expression of the cytokine. Moreover, NFAT proteins often associate with AP-1 heterodimers to bind to composite sites in regulatory regions of cytokine genes, and the defective AP-1 activation in PKC-θ–deficient mice may therefore contribute to the impaired Th2 cell polarization.
Although PKC-θ may play a synergistic role in the transduction of both Th1 and Th2 cell activation signals, our data indicate that it might be a central regulator of Th2 cell immune responses, whereas Th1 cell responses are maintained in its absence through alternate pathways. In purified PKC-θ–deficient T cells, induction of IFN-γ was enhanced even in the absence of proliferation, and after 2 h of anti-TCR stimulation we observed higher levels of IFN-γ mRNA in CD4+ cells from mutant versus control mice (unpublished data). These results suggest that the mutant T cells have an intrinsic propensity to express this Th1 cell cytokine. However, we did not observe enhanced Th1 cell responses in vivo in the mutant animals, and we cannot rule out that other factors, such as proinflammatory cytokines, sustain normal Th1 cell responses in the absence of PKC-θ. In an in vivo setting, the inflammatory cytokine milieu, strong costimulatory signals and activation of Toll-like receptor pathways may bypass the requirement for PKC-θ in T cell activation and differentiation during Th1 cell–inducing infections, whereas signals induced during helminth infection or allergic asthma might not be sufficient to bypass the requirement for PKC-θ in Th2 cell immune responses.
In summary, the experiments detailed in this work show that PKC-θ is critical for the development of in vivo Th2 cell immune responses. These data highlight PKC-θ as a potentially important target in therapy against Th2 cell–related diseases.