Differential induction of donor-reactive Foxp3+ regulatory T cell via blockade of CD154 vs CD40.

Recently published studies in both murine models and a meta-analysis of non-human primate renal transplant studies showed that anti-CD154 reagents conferred a significant survival advantage over CD40 blockers in both animal models and across multiple organs. Here we sought to compare the induction of donor-reactive forkhead box P3+-induced regulatory T cells (Foxp3+ iTreg) in mice treated with anti-CD154 versus anti-CD40 monoclonal antibodies (mAbs). Results indicated that while treatment with anti-CD154 mAb resulted in a significant increase in the frequency of donor-reactive CD4+ Foxp3+ iTreg following transplantation, treatment with anti-CD40 or Cd40 deficiency failed to recapitulate this result. Because we recently identified CD11b as an alternate receptor for CD154 during alloimmunity, we interrogated the role of CD154:CD11b interactions in the generation of Foxp3+ iTreg and found that blockade of CD11b in Cd40-/- recipients resulted in increased donor-reactive Foxp3+ iTreg as compared with CD40 deficiency alone. Mechanistically, CD154:CD11b inhibition decreased interleukin (IL)-1ß from CD11b+ and CD11c+ dendritic cells, and blockade of IL-1ß synergized with CD40 deficiency to promote Foxp3+ iTreg induction and prolong allograft survival. Taken together, these data provide a mechanistic basis for the observed inferiority of anti-CD40 blockers as compared with anti-CD154 mAb and illuminate an IL-1ß-dependent mechanism by which CD154:CD11b interactions prevent the generation of donor-reactive Foxp3+ iTreg during transplantation.

Induction of transplantation tolerance through regulatory cells: from mice to men.

Organ transplantation results in the activation of both innate and adaptive immune responses to the foreign antigens. While these responses can be limited with the use of systemic immunosuppressants, the induction of regulatory cell populations may be a novel strategy for the maintenance of specific immunological unresponsiveness that can reduce the severity of the detrimental side effects of current therapies. Our group has extensively researched different regulatory T-cell induction protocols for use as cellular therapy in transplantation. In this review, we address the cellular and molecular mechanisms behind regulatory T-cell suppression and their stability following induction protocols. We further discuss the use of different hematopoietically derived regulatory cell populations, including regulatory B cells, regulatory macrophages, tolerogenic dendritic cells, and myeloid-derived suppressor cells, for the induction of transplantation tolerance in light of new clinical trials developing therapies with some of these populations.

Inhibition of CD8+ T cell-derived CD40 signals is necessary but not sufficient for Foxp3+ induced regulatory T cell generation in vivo.

Current models of CD4(+) T cell help suggest a major role for CD154 binding to CD40 expressed on dendritic cells, with a lesser role for direct T:T interactions via CD40 expressed on CD8(+) T cells. However, the contribution of CD8(+) T cell-derived CD40 signals during the donor-reactive T cell response to a transplant has never been studied. In this study, we examined the graft-rejection kinetics and CD4(+) and CD8(+) donor-reactive T cell responses under conditions in which CD40 was genetically ablated only on APC, as well as under conditions in which CD40 was genetically ablated only on donor-reactive CD8(+) T cells. Our results revealed a significant role for CD8(+) T cell-expressed CD40 in the augmentation of donor-reactive CD8(+) T cell responses following transplantation and showed that CD40 expressed on CD8(+) T cells must be inhibited to allow conversion of CD4(+) T cells into induced regulatory T cells. Thus, this study identifies a major role for CD8(+) T cell-derived CD40 signals as a critical switch factor that both promotes optimal differentiation of cytokine-producing CD8(+) effector T cell responses and inhibits the differentiation of Ag-specific Foxp3(+) induced regulatory T cells in vivo.

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.

Antigen-specific induced Foxp3+ regulatory T cells are generated following CD40/CD154 blockade.

Blockade of the CD40/CD154 pathway potently attenuates T-cell responses in models of autoimmunity, inflammation, and transplantation. Indeed, CD40 pathway blockade remains one of the most powerful methods of prolonging graft survival in models of transplantation. But despite this effectiveness, the cellular and molecular mechanisms underlying the protective effects of CD40 pathway blockade are incompletely understood. Furthermore, the relative contributions of deletion, anergy, and regulation have not been measured in a model in which donor-reactive CD4(+) and CD8(+) T-cell responses can be assessed simultaneously. To investigate the impact of CD40/CD154 pathway blockade on graft-specific T-cell responses, a transgenic mouse model was used in which recipients containing ovalbumin-specific CD4(+) and CD8(+) TCR transgenic T cells were grafted with skin expressing ovalbumin in the presence or absence of anti-CD154 and donor-specific transfusion. The results indicated that CD154 blockade altered the kinetics of donor-reactive CD8(+) T-cell expansion, delaying differentiation into IFN-γ(+) TNF(+) multifunctional cytokine producers. The eventual differentiation of cytokine-producing effectors in tolerant animals coincided with the emergence of an antigen-specific CD4(+) CD25(hi) Foxp3(+) T-cell population, which did not arise from endogenous natural T(reg) but rather were peripherally generated from naïve Foxp3(-) precursors.

Cutting edge: Rapamycin augments pathogen-specific but not graft-reactive CD8+ T cell responses.

Recent evidence demonstrating that exposure to rapamycin during viral infection increased the quantity and quality of Ag-specific T cells poses an intriguing paradox, because rapamycin is used in transplantation to dampen, rather than enhance, donor-reactive T cell responses. In this report, we compared the effects of rapamycin on the Ag-specific T cell response to a bacterial infection versus a transplant. Using a transgenic system in which the Ag and the responding T cell population were identical in both cases, we observed that treatment with rapamycin augmented the Ag-specific T cell response to a pathogen, whereas it failed to do so when the Ag was presented in the context of a transplant. These results suggest that the environment in which an Ag is presented alters the influence of rapamycin on Ag-specific T cell expansion and highlights a fundamental difference between Ag presented by an infectious agent as compared with an allograft.

Loss of T cell tolerance in the skin following immunopathology is linked to failed restoration of the dermal niche by recruited macrophages.

T cell pathology in the skin leads to monocyte influx, but we have little understanding of the fate of recruited cells within the diseased niche, or the long-term impact on cutaneous immune homeostasis. By combining a murine model of acute graft-versus-host disease (aGVHD) with analysis of patient samples, we demonstrate that pathology initiates dermis-specific macrophage differentiation and show that aGVHD-primed macrophages continue to dominate the dermal compartment at the relative expense of quiescent MHCIIint cells. Exposure of the altered dermal niche to topical haptens after disease resolution results in hyper-activation of regulatory T cells (Treg), but local breakdown in tolerance. Disease-imprinted macrophages express increased IL-1ß and are predicted to elicit altered TNF superfamily interactions with cutaneous Treg, and we demonstrate the direct loss of T cell regulation within the resolved skin. Thus, T cell pathology leaves an immunological scar in the skin marked by failure to re-set immune homeostasis.

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.

Graft-versus-host disease reduces lymph node display of tissue-restricted self-antigens and promotes autoimmunity.

Acute graft-versus-host disease (GVHD) is initially triggered by alloreactive T cells, which damage peripheral tissues and lymphoid organs. Subsequent transition to chronic GVHD involves the emergence of autoimmunity, although the underlying mechanisms driving this process are unclear. Here, we tested the hypothesis that acute GVHD blocks peripheral tolerance of autoreactive T cells by impairing lymph node (LN) display of peripheral tissue-restricted antigens (PTAs). At the initiation of GVHD, LN fibroblastic reticular cells (FRCs) rapidly reduced expression of genes regulated by DEAF1, an autoimmune regulator-like transcription factor required for intranodal expression of PTAs. Subsequently, GVHD led to the selective elimination of the FRC population, and blocked the repair pathways required for its regeneration. We used a transgenic mouse model to show that the loss of presentation of an intestinal PTA by FRCs during GVHD resulted in the activation of autoaggressive T cells and gut injury. Finally, we show that FRCs normally expressed a unique PTA gene signature that was highly enriched for genes expressed in the target organs affected by chronic GVHD. In conclusion, acute GVHD damages and prevents repair of the FRC network, thus disabling an essential platform for purging autoreactive T cells from the repertoire.

A wave of monocytes is recruited to replenish the long-term Langerhans cell network after immune injury.

A dense population of embryo-derived Langerhans cells (eLCs) is maintained within the sealed epidermis without contribution from circulating cells. When this network is perturbed by transient exposure to ultraviolet light, short-term LCs are temporarily reconstituted from an initial wave of monocytes but thought to be superseded by more permanent repopulation with undefined LC precursors. However, the extent to which this process is relevant to immunopathological processes that damage LC population integrity is not known. Using a model of allogeneic hematopoietic stem cell transplantation, where alloreactive T cells directly target eLCs, we have asked whether and how the original LC network is ultimately restored. We find that donor monocytes, but not dendritic cells, are the precursors of long-term LCs in this context. Destruction of eLCs leads to recruitment of a wave of monocytes that engraft in the epidermis and undergo a sequential pathway of differentiation via transcriptionally distinct EpCAM+ precursors. Monocyte-derived LCs acquire the capacity of self-renewal, and proliferation in the epidermis matched that of steady-state eLCs. However, we identified a bottleneck in the differentiation and survival of epidermal monocytes, which, together with the slow rate of renewal of mature LCs, limits repair of the network. Furthermore, replenishment of the LC network leads to constitutive entry of cells into the epidermal compartment. Thus, immune injury triggers functional adaptation of mechanisms used to maintain tissue-resident macrophages at other sites, but this process is highly inefficient in the skin.

Peripheral tissues reprogram CD8+ T cells for pathogenicity during graft-versus-host disease.

Graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic stem cell transplantation induced by the influx of donor-derived effector T cells (TE) into peripheral tissues. Current treatment strategies rely on targeting systemic T cells; however, the precise location and nature of instructions that program TE to become pathogenic and trigger injury are unknown. We therefore used weighted gene coexpression network analysis to construct an unbiased spatial map of TE differentiation during the evolution of GVHD and identified wide variation in effector programs in mice and humans according to location. Idiosyncrasy of effector programming in affected organs did not result from variation in T cell receptor repertoire or the selection of optimally activated TE. Instead, TE were reprogrammed by tissue-autonomous mechanisms in target organs for site-specific proinflammatory functions that were highly divergent from those primed in lymph nodes. In the skin, we combined the correlation-based network with a module-based differential expression analysis and showed that Langerhans cells provided in situ instructions for a Notch-dependent T cell gene cluster critical for triggering local injury. Thus, the principal determinant of TE pathogenicity in GVHD is the final destination, highlighting the need for target organ-specific approaches to block immunopathology while avoiding global immune suppression.

CD154 blockade alters innate immune cell recruitment and programs alloreactive CD8+ T cells into KLRG-1(high) short-lived effector T cells.

CD154/CD40 blockade combined with donor specific transfusion remains one of the most effective therapies in prolonging allograft survival. Despite this, the mechanisms by which these pathways synergize to prevent rejection are not completely understood. Utilizing a BALB/c (H2-K(d)) to B6 (H2-K(b)) fully allogeneic skin transplant model system, we performed a detailed longitudinal analysis of the kinetics and magnitude of CD8(+) T cell expansion and differentiation in the presence of CD154/CD40 pathway blockade. Results demonstrated that treatment with anti-CD154 vs. DST had distinct and opposing effects on activated CD44(high) CD62L(low) CD8(+) T cells in skin graft recipients. Specifically, CD154 blockade delayed alloreactive CD8(+) T cell responses, while DST accelerated them. DST inhibited the differentiation of alloreactive CD8(+) T cells into multi-cytokine producing effectors, while CD40/CD154 blockade led to the diminution of the KLRG-1(low) long-lived memory precursor population compared with either untreated or DST treated animals. Moreover, only CD154 blockade effectively inhibited CXCL1 expression and neutrophil recruitment into the graft. When combined, anti-CD154 and DST acted synergistically to profoundly diminish the absolute number of IFN-γ producing alloreactive CD8(+) T cells, and intra-graft expression of inflammatory chemokines. These findings demonstrate that the previously described ability of anti-CD154 and DST to result in alloreactive T cell deletion involves both delayed kinetics of T cell expansion and differentiation and inhibited development of KLRG-1(low) memory precursor cells.