CD40/CD154 blockade inhibits dendritic cell expression of inflammatory cytokines but not costimulatory molecules.

Blockade of the CD40/CD154 pathway remains one of the most effective means of promoting graft survival following transplantation. However, the effects of CD40/CD154 antagonism on dendritic cell (DC) phenotype and functionality following transplantation remain incompletely understood. To dissect the effects of CD154/CD40 blockade on DC activation in vivo, we generated hematopoietic chimeras in mice that expressed a surrogate minor Ag (OVA). Adoptive transfer of OVA-specific CD4(+) and CD8(+) T cells led to chimerism rejection, which was inhibited by treatment with CD154 blockade. Surprisingly, CD154 antagonism did not alter the expression of MHC and costimulatory molecules on CD11c(+) DCs compared with untreated controls. However, DCs isolated from anti-CD154-treated animals exhibited a significant reduction in inflammatory cytokine secretion. Combined blockade of inflammatory cytokines IL-6 and IL-12p40 attenuated the expansion of Ag-specific CD4(+) and CD8(+) T cells and transiently inhibited the rejection of OVA-expressing cells. These results suggest that a major effect of CD154 antagonism in vivo is an impairment in the provision of signal three during donor-reactive T cell programming, as opposed to an impact on the provision of signal two. We conclude that therapies designed to target inflammatory cytokines during donor-reactive T cell activation may be beneficial in attenuating these responses and prolonging graft survival

PD-1-dependent mechanisms maintain peripheral tolerance of donor-reactive CD8+ T cells to transplanted tissue.

Peripheral mechanisms of self-tolerance often depend on the quiescent state of the immune system. To what degree such mechanisms can be engaged in the enhancement of allograft survival is unclear. To examine the role of the PD-1 pathway in the maintenance of graft survival following blockade of costimulatory pathways, we used a single-Ag mismatch model of graft rejection where we could track the donor-specific cells as they developed endogenously and emerged from the thymus. We found that graft-specific T cells arising under physiologic developmental conditions at low frequency were actively deleted at the time of transplantation under combined CD28/CD40L blockade. However, this deletion was incomplete, and donor-specific cells that failed to undergo deletion up-regulated expression of PD-1. Furthermore, blockade of PD-1 signaling on these cells via in vivo treatment with anti-PD-1 mAb resulted in rapid expansion of donor-specific T cells and graft loss. These results suggest that the PD-1 pathway was engaged in the continued regulation of the low-frequency graft-specific immune response and thus in maintenance of graft survival

PD-1-dependent mechanisms maintain peripheral tolerance of donor-reactive CD8+ T cells to transplanted tissue.

Peripheral mechanisms of self-tolerance often depend on the quiescent state of the immune system. To what degree such mechanisms can be engaged in the enhancement of allograft survival is unclear. To examine the role of the PD-1 pathway in the maintenance of graft survival following blockade of costimulatory pathways, we used a single-Ag mismatch model of graft rejection where we could track the donor-specific cells as they developed endogenously and emerged from the thymus. We found that graft-specific T cells arising under physiologic developmental conditions at low frequency were actively deleted at the time of transplantation under combined CD28/CD40L blockade. However, this deletion was incomplete, and donor-specific cells that failed to undergo deletion up-regulated expression of PD-1. Furthermore, blockade of PD-1 signaling on these cells via in vivo treatment with anti-PD-1 mAb resulted in rapid expansion of donor-specific T cells and graft loss. These results suggest that the PD-1 pathway was engaged in the continued regulation of the low-frequency graft-specific immune response and thus in maintenance of graft survival

Protein kinase C-theta interacts with mTORC2 and vimentin to limit regulatory T-cell function

Regulatory T-cells (Tregs) play a critical role in preventing autoimmune and alloimmune reactions, including graft-versus-host disease (GVHD). Two recent clinical trials demonstrated that in patients undergoing hematopoietic stem cell transplantation, adoptive transfer of Tregs significantly reduced the incidence of grades II-IV GVHD. While Tregs significantly reduced GVHD severity, they did not eliminate GVHD. One potential way to augment Treg-mediated inhibition of GVHD is to increase Treg suppressive potency. We showed previously that Tregspecific inhibition of protein kinase C-theta (PKC-θ) enhances Treg function (Science 328:372, 2010). However, it is unclear whether PKC-θinhibition can boost Treg function in a systemic inflammatory condition like GVHD. Furthermore, the mechanism by which PKC-θinhibition augments Treg function is unknown. In this study, we address these unanswered questions

Protein kinase C-theta interacts with mTORC2 and vimentin to limit regulatory T-cell function

Regulatory T-cells (Tregs) play a critical role in preventing autoimmune and alloimmune reactions, including graft-versus-host disease (GVHD). Two recent clinical trials demonstrated that in patients undergoing hematopoietic stem cell transplantation, adoptive transfer of Tregs significantly reduced the incidence of grades II-IV GVHD. While Tregs significantly reduced GVHD severity, they did not eliminate GVHD. One potential way to augment Treg-mediated inhibition of GVHD is to increase Treg suppressive potency. We showed previously that Tregspecific inhibition of protein kinase C-theta (PKC-θ) enhances Treg function (Science 328:372, 2010). However, it is unclear whether PKC-θinhibition can boost Treg function in a systemic inflammatory condition like GVHD. Furthermore, the mechanism by which PKC-θinhibition augments Treg function is unknown. In this study, we address these unanswered questions

Vimentin restrains regulatory T-cell suppression of graft-versus-host disease

Regulatory T-cells (Treg) are critical for maintaining immune homeostasis. However, current Treg immunotherapies do not optimally treat inflammatory diseases in patients. Understanding the cellular processes that control Treg function may allow for the augmentation of therapeutic efficacy. In contrast to activated conventional T-cells, where protein kinase C-T (PKC-T) localizes to the contact-point between T-cells and antigen-presenting cells, in Treg, PKC-T localizes to the opposite end of the cell in the distal pole complex (DPC). Here, using a phosphoproteomic screen, we identified the intermediate filament vimentin as a PKC-T phosphotarget and show that vimentin forms a DPC superstructure on which PKC-T accumulates. Treatment of Treg with either a clinically relevant PKC-T inhibitor or vimentin siRNA disrupted vimentin and enhanced Treg metabolic and suppressive activity. Moreover, vimentin-disrupted Treg were significantly better than controls in suppressing alloreactive T-cell priming in graftversus-host disease, and graft-versus-host disease lethality. Interestingly, vimentin disruption augmented suppressor function of PKC-T-deficient Treg. This suggests that enhanced Treg activity after PKC-T inhibition is secondary to effects on vimentin, not just PKC-T kinase activity inhibition. Our data demonstrated that vimentin is a key metabolic and functional controller of Treg activity, and provide proof-of-principle that disrupting vimentin is a feasible, translationally relevant method to enhance Treg potency