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Protein kinase C-θ has emerged as a central regulator of conventional effector T cell (Teff) and CD4+FoxP3+ regulatory T cell (Treg) function. In the years since this initial description PKC-θ regulation has taken on even greater complexity, as it has been implicated in negative regulation of immunological synapse stability and in mediating development and negative feedback regulation in natural Tregs. This review will examine current knowledge about PKC-θ in the immunological synapse, recent evidence for its distinct localization in Tregs and the therapeutic implications of targeting PKC-θ in Teffs and Tregs.
The protein kinase C (PKC) family of serine/threonine kinases has a critical role in immune regulation through signal transduction networks that coordinate practically every move and decision. There are three families of PKCs, the conventional, which are activated by Ca2+ and diacylglycerol, the novel, which are activated by diacylglycerol and the atypical, which are insensitive to both Ca2+ and diacylglycerol 1. In T cells, all sub-families are represented. PKC-θ is a novel PKC that is most abundant in hematopoietic cells 2. PKC-θ rose to prominence as the only PKC isoform that is selectively recruited to the conventional effector T cell (Teff) immunological synapse (IS) 3 (Figure 1). The IS is a stable cell-cell junction formed between a Teff and an antigen presenting cell (APC) and composed of a peripheral supramolecular activation cluster (pSMAC) and central supramolecular activation cluster (cSMAC) where LFA-1 and TCR microclusters converge, respectively 4–7 (Figure 1). Several groups have demonstrated that PKC-θ is recruited to the cSMAC in a CD28 co-stimulation dependent manner 3,8–10 . In addition, we have demonstrated that PKC-θ can be recruited to the IS in a multifocal pattern without CD28 engagement and negatively regulates IS stability by favoring symmetry breaking of the pSMAC in a manner counteracted by expression of Wiscott Aldrich Syndrome protein (WASp) 11. Furthermore, stabilization of the IS by PKC-θ inhibition can enhance killing by CD4+ cytotoxic T cells 12. The detailed mechanism by which PKC-θ stimulates symmetry breaking in Teffs is not known, but in other systems symmetry breaking involves cooperative myosin II activation 13.
CD4+ FoxP3+ regulatory T cells (Tregs) play a central role in suppressing inflammatory responses in TCR dependent manner 14. There is evidence for stable Treg-DC interactions in vivo that play an important role in Treg function 15–17, but the organization of the Treg IS has been less studied. In contrast to Teffs, PKC-θ is sequestered to the distal pole complex away from the Treg IS 18. Moreover, we found that Tregs form more stable IS than Teffs on supported planar bilayers presenting anti-CD3 antibodies and ICAM-1. This is consistent with the localization of PKC-θ at the distal pole of the IS, which may favor higher IS stability in Tregs. Thus, the location and regulation of PKC-θ is different in Teffs and Tregs.
PKC-θ plays a central role in controlling T cell function by regulation of signaling pathways leading to activation of transcription factors, i.e., nuclear factor κB (NF-κB), activation protein-1 (AP-1) and nuclear factor of T cells (NFAT) (Figure 2). PKC-θ activates NF-κB through phosphorylation and activation of the membrane associated guanylate kinase (MAGUK) Carma1 (also called Card11) 19. The MAGUK module consists of a PDZ domain, and SH3 domain and a non-enzymatically active guanylate kinase domain. The N-terminus of Carma1 contains a caspase recruitment domain (CARD) and a coiled-coil domain 20. Phosphoylation of Carma1 by PKC-θ initiates assembly of an oligomeric complex with CARD domain containing adapter Bcl10 and the death domain (DD) containing paracaspase Malt1 that mediates activation of NF-κB transcription factors 19. PKC-θ also phosphorylates the ste20-family kinase SPAK, which is important for AP-1 transcription factor activation 21. While one group found that Ca2+ and NFAT activation was relatively normal in PKC-θ deficient T cells in vitro 22, another group found that the Ca2+/NFAT pathway was also defective 23. The role of PKC-θ in NF-κB signaling is selective for the TCR/CD28 costimulation signaling pathways 24,25 as inflammatory cytokine mediated activation of NF-κB is PKC-θ independent 22. PKC-θ is essential for IL-2 production and downstream of TCR signals 23 and also appears to mediate the enhanced IL-2 production in response to CD28 co-stimulation 8,10. This pathway could be considered the canonical pathway for function of PKC-θ in T cell activation.
Reverse genetic studies in mice have revealed that the critical roles of PKC-θ in TCR signaling have variable penetrance in vivo. The development of conventional naïve CD8 and CD4 T cells appears grossly normal in PKC-θ-deficient mice 22,23. PKC-θ deficient mice have profound defects in generating Th2 responses to Nippostrongylus brasiliensis and are also resistant to induction of pulmonary allergic hypersensitivity responses to model antigens 26,27. In addition, PKC-θ deficient T cells had a reduced capacity to induce autoimmune colitis 28. In contrast, PKC-θ deficient mice can clear Listeria monocytogenes, Leishmania major, lymphocytic choroemeningitis virus (LCMV) and murine γ–herpesvirus 68 infections similarly to wild type mice 26,29–31. The surprising competence of PKC-θ deficient mice in combating bacteria and viral pathogens is explained at least in part by complementation of the TCR-dependent NF-κB activation defect by toll-like receptors responding to innate cues from microbial pathogens 32. Autoimmune and Th2 responses to multicellular parasites may lack of strong, NF-κB-activating innate signals leading to greater reliance on TCR signaling and a stronger phenotype in PKC-θ deficient mice.
Tregs are a subset of CD4+ T cells that are central in maintaining a balance between immune tolerance to self-antigens and anti-tumor responses 14. There are two developmentally distinct populations of CD4+ Treg, both of which depend upon the transcription factor FoxP3 33–35. Natural Treg (nTreg) are produced in the thymus and express that additional transcription factor Helios 36. Induced Treg are generated from naïve Teff in the periphery and do not express Helios 36. Foxp3 deficiency leads to defects in Treg function, which manifest as a multi-organ fatal inflammatory disease in mice 37,38. The unique feature of Tregs is their ability to suppress a large number of different target cell types that includes CD4+ and CD8+ subsets of T cells, B cells, NK cells and dendritic cells 14,39. The proposed mechanisms of Treg-mediated suppression include suppressor cytokine secretion, IL-2 consumption, target cell cytolysis and cell surface molecules 14,39.
In mice lacking PKC-θ PRKCQgene knockout), Treg development in the thymus is impaired, but the per cell function of peripheral Treg was intact 40. The requirement of PKC-θ for Treg development may relate to the stronger nature of the TCR ligands that drive differentiation of Tregs as compared to conventional T cells and the role of PKC-θ in augmenting signal tranduction in TCR ligated cells. For example, the PKC-θ downstream target cRel is critical for Treg development and expansion 41. Furthermore, PKC-θ deficiency impairs IL-2 production by Teffs as noted above, and Tregs need high levels of IL-2 to function, survive and proliferate 14,38,42. Therefore, reduced IL-2 in PKC-θ deficient mice may contribute to Treg defects 40,41.
Pre-treatment of mouse Tregs with PKC-θ inhibitor increases Treg engraftment measured one week after injection into TCR α−/−β−/− mice in presence of CD4+CD25-CD45RBhigh subset of Teff 18. Since Treg divided multiple times after introduction into lymphopenic mice 43, we can conclude that Tregs with inhibited PKC-θ show no major defect in proliferation in vivo, and may even proliferate more that Treg with fully active PKC-θ. In contrast, inhibition of PKC-θ abolished the ability of human Treg to proliferate in vitro upon anti-CD3/CD28 stimulation in presence of high levels of IL-2 18. This discrepancy between the role of PKC-θ in Treg expansion in vivo and in vitro might be explained by additional factors contributing to IL-2 production and potentially other growth factors present in vivo. Thus, in Tregs, PKC-θ is required for thymic development, but dispensable for in vivo homeostatic expansion and suppressive function.
TCR signals are required for Treg suppressive function and these signals can be received through a stable IS. Human Tregs form more stable IS compared to Teffs 18. This suggested a potential defect in PKC-θ signaling at the IS 11. Consistent with this, the Treg IS displays reduced markers of signaling down-stream of TCR including phosphorylated Src family kinases, PKC-θ and CARMA-1 (Figure 2). Furthermore, inactivation of PKC-θ in Treg with an irreversible small molecule inhibitor resulted in ~4-fold increase in Treg function 18. Knock-down of PKC-θ by siRNA generated a similar increase in Treg function. The mechanism by which PKC-θ signaling mediates negative feedback in Treg is not known, but may involve NF-κB mediated transcription in the presence of FoxP3 or other Treg associated transcription factors. Many questions remain about how the role of PKC-θ/NF-kB in Treg development in the thymus relates to the role of those molecules in maintenance of the Treg phenotype and function.
Tregs disfunction or insufficiency has been linked to autoimmune disease in human and animal models 44–46. We were able to fully reproduce the findings from the Lipsky and Shevach lab that Treg isolated from peripheral blood of patients with rheumatoid arthritis (RA) have reduced function due to negative regulation by tumor necrosis factor-α (TNF-α)18,46. Interestingly, we found that while conventional pathways for TNF-α signaling are not PKCθ dependent 22, TNFα treatment results in re-localization of PKC-θ in Treg to a more Teff like pattern 18. Moreover, PKC-θ inhibition protects Treg from TNF-a-mediated suppression and partially restores the diminished suppressive activity of Tregs purified from RA patients 18,46. Finally, PKC-θ inhibition in Treg significantly increases their potential to protect mice against inflammatory colitis in vivo 18,47. It was quite remarkable that pretreating Treg with a small molecule inhibitor, washing away the free drug in infusing the cells into a lymphopenic host would have a sufficiently durable effect to change the course of a disease that develops over a period of weeks. Since Treg expand rapidly in lymphopenic hosts and PKC-θ inhibition does not block this expansion, the drug is almost certainly diluted out. Thus, these effects my take place early and then set the stage for the development of disease, perhaps by determining the final ratio of Treg to Teff in the animal after reconstitution. Thus, PKC-θ suppressed, more potent Treg could change this ratio both by inhibition proliferation of the Teff and by proliferating faster.
The recent success of small molecule inhibitors of PKC-θ in phase I clinical trials for psoriasis raises the possibility of reducing Teff and increasing Treg function a single small molecule 48. It is useful to consider both the role of PKC-θ in Teff and Treg. PKC-θ deficient Teffs are more prone to apoptosis, a finding associated with reduced levels of BclxL expression. However, forced expression of BclxL in Treg did not improve their survival 40. The absence of PKC-θ results in a reduced survival of CD4+ and to a greater extent CD8+ T cells 49–51. These data predicted that PKC theta inhibition may be a useful approach to inhibit Teff responses such as those that would occur during allogeneic transplantation of solid or hematopoietic organs or autoimmunity (reviewed in 52). Thus, inhibiting PKC-θ would function to either reduce the number of activated Teffs by failing to protect Teffs from apoptosis and activation induced cell death or ablate CD28 signals, predisposing T cells to become anergic. Conversely, blocking CD28/B7 ligand interaction using an anti-CD28 monoclonal antibody results in PKC-θ and Jnk suppression, along with long-term cardiac allograft survival 53.
Regulation of IL-2 production and alloreactivity in vivo is influenced by both PKC-α and -θ, which affect NFAT translocation 54. Whereas PKC-α or -θ deficient recipients have only minimal prolongation of cardiac allograft survival, doubly deficient recipients have additive effects on allograft survival, indicating that PKC-α and -θ are non-redundant in controlling alloreactivity in vivo. PKC theta deficiency is partially compensated in PKC-θ deficient recipients as cardiac allograft rejection is delayed but not eliminated. The strength of the partial compensation is only modest since cardiac allografts in PKC-θ deficient recipients are hypersusceptible to blockade with anti-CD154 antibdy or CTLA4-Ig [Manicassamy, 2008 #41]. In T cell adoptive transfer experiments, PKC-θ deficient T cells fail to reject cardiac allografts unless Bclxl is expressed by transgenesis 50.
Consistent with the finding that PKC-θ regulates CD4+ and CD8+ T cell survival, alloreactivity and IL-2 production, graft-versus-host disease (GVHD) lethality in irradiated recipients of allogeneic CD8+ or CD4+ and CD8+ T cells was severely ameliorated 31. Despite the high degree of systemic inflammation and tissue destruction that is characteristic of GVHD, PKC-θ deficient T cells were hypoproliferative and had a higher frequency of annexin V staining after adoptive transfer in irradiated allogeneic recipients, giving rise to significantly fewer splenic CD4+ and CD8+ T cells early post-BMT transplant. Interestingly, neither TLR4 nor TLR9 ligation could overcome the profound defect of PKC-θ deficient T cells in GVHD induction. This contrasts with the situation in control of bacterial and viral infection in which TLR stimulation could compensate for PKC-θ deficiency. This is important because it suggests that PKC-θ inhibition may effectively block GVHD without eliminating anti-microbial defenses. Moreover, the presence of strong proinflammatory signals such as those derived from a mitogenic anti-CD40 antibody or lipopolysaccharide enhanced, but did not fully restore, the in vivo expansion and cytotoxicity of TCR transgenic CD8+ T cells administered to recipients expressing the relevant high affinity ligand. Skewing of cytokine responses away from a Th2 26, which have been associated with decreased GVHD in some models, was not evident. Intriguingly despite these profound defects in CD8+ T cell proliferation and cytotoxicity, clearance of a viral pathogen and leukemia cell line that are controlled by CD8+ T cells was intact. Relevant to bone marrow transplantation, Ca2+ signaling via calcineurin augments PKC-θ expression (Ref). Thus, calcineurin inhibitors may operate to reduce GVHD in part via downregulation of PKC-θ expression in donor T cells. Reduced PKC-θ expression may lead to lower FasL expression levels 55 that may also contribute to the lower GVHD lethality seen in recipients given PKC-θ deficient vs sufficient T cells. Taken together, these results suggest that the sterile inflammatory signaling driving GVHD may be insufficient activators of NF-κB expression in donor T cells early post-transplant. In contrast, the innate sensing mechanisms in graft-versus-leukemia may be better activators of NF-κB, resulting in complementation of the PKC-θ defect in TCR signaling. Alternatively, the reduced T cell expansion and cytotoxicity of PKC-θ deficient T cells may be insufficient to cause GVHD lethality due to absolute cell number, tissue location, or persistence of cells but sufficient to reduce viral load and tumor burden. Regardless of the mechanism, pharmacological targeting of PKC-θ may be an ideal approach to decrease GVHD lethality while preserving anti-infection and anti-tumor T cell responses post-transplant.
With respect to autoimmune diseases, PKC-θ deficient recipients are resistant to the development of experimental autoimmune encephalomyelitis 56–58 and myosin-induced autoimmune myocarditis, associated with reduced IL-17 secretion 59. Under these conditions, TLR9 signaling can act directly on PKC-θ ko T cells to activate NF-kB, resulting in near normal T cell expansion under Th1 polarizing conditions, restoring bclxL expression and promoting the generation of autoimmune myocarditis 59. However, PKC-θ inhibition may prove to be useful in preventing autoimmune colitis as was discussed above. 18,28. Thus, PKC-θ inhibition could be highly useful in preventing and treating autoimmune diseases via targeting Teffs and Tregs to reduce and conversely increase their in vivo survival and function.
Our attention was initially called to PKC-θ by its unique localization in the Teff cell immunological synapse 3,4. A dozen years later application of a second-generation technology to study the immunological synapse revealed that PKC-θ is excluded from the Treg synapse18 (Figure 3). These studies led to our current understanding that PKC-θ has dual roles in Teff and Treg cells that can be targeted to reduce inflammation in the context of alloreactivity and autoimmunity. In Teffs PKC-θ promotes inflammation by increasing activation of the NF-κB transcription factors at the immunological synapse to promote growth and survival 22,52. In Tregs, PKC-θ is sequestered to distal complex away from the immunological synapse and mediates a negative feedback on Treg suppressive function 18. The dual role of PKC-θ in controlling T cell function represents a therapeutic potential of PKC-θ inhibition as a powerful tool to block undesired inflammatory responses.