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
. 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.