Rongxiang Han

Chemokines and their receptors as markers of allograft rejection and targets for immunosuppression

Although almost every known chemokine and chemokine receptor is expressed at some stage during development of allograft rejection, mechanistic studies indicate that the actual key effector mechanisms are rather few. Thus, in vivo studies have alleviated concerns regarding possible biological redundancy and the pleiotropic effects of these molecules, and have resulted in a focus on CXCR3, CCR5 and their respective ligands as key mediators of host alloresponses, especially in acute rejection. Data are also accruing regarding the importance of chemokine/chemokine receptor pathways in ischemia/reperfusion, chronic rejection and tolerance induction following co-stimulation blockade, providing new targets for immune monitoring and therapeutic intervention.

BAFF binding to T cell-expressed BAFF-R costimulates T cell proliferation and alloresponses

Binding of the TNF family member, B cell activating factor (BAFF), to its receptor (BAFF‐R, TNFRSF13C) is required for generation and maintenance of mature B cells, but there are no data as to any role for the BAFF/BAFF‐R pathway in T cell functions. We report that the binding of BAFF to BAFF‐R expressed by a subset of primarily CD4+ T cells costimulates T cell activation and allo‐proliferation in vitro and in vivo, and that mice with a mutation in the BAFF‐R, or with a targeted deletion of BAFF, show prolonged cardiac allograft survival as compared to wild‐type or transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI)–/– controls. Taken together, these data indicate the BAFF/BAFF‐R pathway contributes to both T and B cell responses and may be an attractive target for control of acute and chronic allograft rejection.

Sirtuin-1 Targeting Promotes Foxp3+ T-Regulatory Cell Function and Prolongs Allograft Survival

ABSTRACT Sirtuin 1 (Sirt1), a class III histone/protein deacetylase, is central to cellular metabolism, stress responses, and aging, but its contributions to various host immune functions have been little investigated. To study the role of Sirt1 in T cell functions, we undertook targeted deletions by mating mice with a floxed Sirt1 gene to mice expressing CD4-cre or Foxp3-cre recombinase, respectively. We found that Sirt1 deletion left conventional T-effector cell activation, proliferation, and cytokine production largely unaltered. However, Sirt1 targeting promoted the expression of Foxp3, a key transcription factor in T-regulatory (Treg) cells, and increased Treg suppressive functions in vitro and in vivo. Consistent with these data, mice with targeted deletions of Sirt1 in either CD4+ T cells or Foxp3+ Treg cells exhibited prolonged survival of major histocompatibility complex (MHC)-mismatched cardiac allografts. Allografts in Sirt1-targeted recipients showed long-term preservation of myocardial histology and infiltration by Foxp3+ Treg cells. Comparable results were seen in wild-type allograft recipients treated with Sirt1 inhibitors, such as EX-527 and splitomicin. Hence, Sirt1 may inhibit Treg functions, and its targeting may have therapeutic value in autoimmunity and transplantation.

Histone Deacetylases 6 and 9 and Sirtuin-1 Control Foxp3+ Regulatory T Cell Function Through Shared and Isoform-Specific Mechanisms

Combined inhibition of distinct histone deacetylases enhances the suppressive effects of regulatory T cells. Enhancing Regulatory T Cell Function Some therapies designed to help patients suffering from autoimmune diseases or recovering from organ transplant involve the removal of the patient’s regulatory T cells (Tregs), which suppress the undesirable proinflammatory actions of effector T cells, so that they can be expanded in number ex vivo before being put back into the patient. One problem associated with this approach is that the phenotype of the administered Tregs is unstable, and so their suppressive function is lost over time. Previous studies have shown that inhibition of certain members of the family of histone deacetylases (HDACs) enhances the function of Tregs. Through experiments with mice deficient in distinct HDACs and treated with HDAC-specific inhibitors, Beier et al. have demonstrated the mechanisms by which the simultaneous inhibition of combinations of distinct HDACs improved the suppressive function of Tregs and enhanced their stability, which suggests that such an approach may be of benefit to patients reliant on such therapies. Therapeutic inhibition of the histone deacetylases HDAC6, HDAC9, or sirtuin-1 (Sirt1) augments the suppressive functions of regulatory T cells (Tregs) that contain the transcription factor Foxp3 (Forkhead box P3) and is useful in organ transplant patients or patients with autoimmune diseases. However, it is unclear whether distinct mechanisms are involved for each HDAC or whether combined inhibition of HDACs would be more effective. We compared the suppressive functions of Tregs from wild-type C57BL/6 mice with those from mice with either complete or cell-specific deletion of various HDACs, as well as with those of Tregs treated with isoform-selective HDAC inhibitors. The improvement of Treg suppressive function mediated by inhibition of HDAC6, but not Sirt1, required an intact heat shock response. Although HDAC6, HDAC9, and Sirt1 all deacetylated Foxp3, each protein had different effects on transcription factors that control expression of the gene encoding Foxp3. For example, loss of HDAC9, but not other HDACs, was associated with stabilization of the acetylated form of signal transducer and activator of transcription 5 (STAT5) and promoted its transcriptional activity. Thus, targeting different HDACs increased Treg function through multiple and additive mechanisms, which suggests the therapeutic potential for using combinations of HDAC inhibitors in the management of autoimmunity and organ transplantation.

Blocking the Monocyte Chemoattractant Protein-1/CCR2 Chemokine Pathway Induces Permanent Survival of Islet Allografts through a Programmed Death-1 Ligand-1-Dependent Mechanism

Islet allografts are subject to rapid rejection through host cellular immune responses involving mononuclear cell recruitment and tissue injury. Interruption of leukocyte recruitment through chemokine receptor targeting is of therapeutic benefit in various experimental models, but little is known about the contribution of chemokine pathways to islet allograft rejection. We found that murine islets produce monocyte chemoattractant protein-1 (MCP-1; CCL2) in vitro and that islet allograft rejection was associated with intragraft expression of MCP-1 and its receptor, CCR2. We therefore investigated whether MCP-1 and CCR2 are required for the rejection of fully MHC-disparate islet allografts. Wild-type mice treated with blocking anti-MCP-1 mAb plus a brief, subtherapeutic course of rapamycin had long-term islet allograft survival, in contrast to the effect of treatment with either mAb or rapamycin alone. CCR2−/− mice treated with rapamycin also maintained islet allografts long-term. Both MCP/CCR2- and rapamycin-sensitive signals were required for maximal proliferation of alloreactive T cells, suggesting that MCP-1/CCR2 induce rejection by promoting alloreactive T cell clonal expansion and homing and migration. Prolonged islet allograft survival achieved by blockade of the MCP-1/CCR2 pathway plus rapamycin therapy was accompanied by a mononuclear cell infiltrate expressing the inhibitory receptor, programmed death-1 (PD-1), and its ligand (PD-L1, B7-H1), and prolongation of islet allograft survival was abrogated by anti-PD-L1 mAb therapy. These data show that the blockade of MCP-1 binding to CCR2 in conjunction with subtherapeutic immunosuppression can have profound effects on islet allograft survival and implicate the expression of the PD-1/PD-L1 pathway in the regulation of physiologic responses in vivo.

Differential Effects of B and T Lymphocyte Attenuator and Programmed Death-1 on Acceptance of Partially versus Fully MHC-Mismatched Cardiac Allografts

Although fully MHC-mismatched murine cardiac allografts are rapidly rejected, allografts mismatched at only MHC class I or class II alleles survive long term; the immunologic basis for the long-term survival of MHC class I- or II-mismatched allografts is unknown. We examined the roles of two recently described inhibitory receptors, B and T lymphocyte attenuator (BTLA) and programmed death-1 (PD-1), in the survival of partially or fully MHC-mismatched allografts using gene-deficient recipients as well as through use of blocking mAbs in wild-type hosts. Partially MHC-mismatched allografts showed strong induction of BTLA, but not PD-1 mRNA and survived long term in wild-type recipients, whereas targeting of BTLA or its ligand, herpesvirus entry mediator, but not PD-1, prompted their rapid rejection. By contrast, fully MHC-mismatched cardiac allografts were acutely rejected in wild-type recipients despite the induction of both BTLA and PD-1. Targeting of PD-1 in several fully MHC-mismatched models accelerated rejection, whereas targeting of BTLA unexpectedly enhanced PD-1 induction by alloreactive CD4 and CD8 T cells and prolonged allograft survival. In vitro studies using allogeneic dendritic cells and T cells showed that at low levels of T cell activation, BTLA expression was primarily induced, but that with increasing degrees of T cell activation, the expression of PD-1 was strongly up-regulated. These data suggest that BTLA and PD-1 exert distinct inhibitory actions in vivo, with the BTLA/herpesvirus entry mediator pathway appearing to dominate in regulating responses against a restricted degree of allogeneic mismatch.

Inhibition of p300 impairs Foxp3 + T regulatory cell function and promotes antitumor immunity

Forkhead box P3 (Foxp3)+ T regulatory (Treg) cells maintain immune homeostasis and limit autoimmunity but can also curtail host immune responses to various types of tumors. Foxp3+ Treg cells are therefore considered promising targets to enhance antitumor immunity, and approaches for their therapeutic modulation are being developed. However, although studies showing that experimentally depleting Foxp3+ Treg cells can enhance antitumor responses provide proof of principle, these studies lack clear translational potential and have various shortcomings. Histone/protein acetyltransferases (HATs) promote chromatin accessibility, gene transcription and the function of multiple transcription factors and nonhistone proteins. We now report that conditional deletion or pharmacologic inhibition of one HAT, p300 (also known as Ep300 or KAT3B), in Foxp3+ Treg cells increased T cell receptor–induced apoptosis in Treg cells, impaired Treg cell suppressive function and peripheral Treg cell induction, and limited tumor growth in immunocompetent but not in immunodeficient mice. Our data thereby demonstrate that p300 is important for Foxp3+ Treg cell function and homeostasis in vivo and in vitro, and identify mechanisms by which appropriate small-molecule inhibitors can diminish Treg cell function without overtly impairing T effector cell responses or inducing autoimmunity. Collectively, these data suggest a new approach for cancer immunotherapy.

B7‐H3 promotes acute and chronic allograft rejection

The B7 homolog B7‐H3 is important for the regulation of immune responses though its functions in vivo are controversial. We report the first clinical and experimental data concerning expression and function of B7‐H3 in alloresponses. Immunohistological and molecular analyses showed B7‐H3 expression by cells mediating rejection of human and mouse allografts. To analyze the significance of B7‐H3 in rejecting allografts, we generated B7‐H3–/– mice and showed that targeting of B7‐H3 was synergistic with other forms of immune modulation; e.g. a regimen of rapamycin gave 12–14 days of survival in wild‐type controls but led to permanent cardiac and islet allograft survival in B7‐H3–/– mice. Cardiac allografts in treated B7‐H3–/– mice showed markedly decreased production of key cytokine, chemokine and chemokine receptor mRNA transcripts as compared to wild‐type controls. The incidence of chronic rejection in two different cardiac allograft models was also inhibited in B7‐H3–/– as compared to wild‐type recipients. Lastly, in addition to the expected antigen‐presenting cell expression of B7‐H3, CD4 and CD8 T cells showed B7‐H3 induction upon cell activation, and both dendritic cell‐ and T cell‐expressed B7‐H3 each enhanced T cell proliferation in vitro and in vivo. We conclude that B7‐H3 promotes T cell‐mediated immune responses and the development of acute and chronic allograft rejection.

Programmed cell death 1 (PD-1) and its ligand PD-l1 are required for allograft tolerance

Programmed cell death‐1 (PD‐1, CD279) and its widely expressed, inducible ligand, PD‐L1 (CD274), together dampen T cell activation, but whether they are essential for allograft tolerance is unknown. We show, using gene‐deficient mice and blocking mAbs in wild‐type mice, that costimulation blockade is ineffectual in PD‐1–/– or PD‐L1–/– allograft recipients, or in wild‐type allograft recipients treated with anti‐PD‐1 or anti‐PD‐L1 mAb. Alloreactive PD‐1–/– CD4 and CD8 T cells had enhanced proliferation and cytokine production compared to wild‐type controls, and anergy could not be induced in PD‐1‐deficient CD4 T cells. We conclude that without inhibitory signals from PD‐1 ligation, alloantigen‐induced T cell proliferation and expansion cannot be regulated by costimulation blockade, and peripheral tolerance induction cannot occur.

Essential role of mitochondrial energy metabolism in Foxp3+ T-regulatory cell function and allograft survival

Conventional T (Tcon) cells and Foxp3+ T‐regulatory (Treg) cells are thought to have differing metabolic requirements, but little is known of mitochondrial functions within these cell populations in vivo. In murine studies, we found that activation of both Tcon and Treg cells led to myocyte enhancer factor 2 (Mef2)‐induced expression of genes important to oxidative phosphorylation (OXPHOS). Inhibition of OXPHOS impaired both Tcon and Treg cell function compared to wild‐type cells but disproportionally affected Treg cells. Deletion of Pgc1α or Sirt3, which are key regulators of OXPHOS, abrogated Treg‐dependent suppressive function and impaired allograft survival. Mef2 is inhibited by histone/protein deacetylase‐9 (Hdac9), and Hdac9 deletion increased Treg suppressive function. Hdac9‐/‐ Treg showed increased expression of Pgc1α and Sirt3, and improved mitochondrial respiration, compared to wild‐type Treg cells. Our data show that key OXPHOS regulators are required for optimal Treg function and Treg‐dependent allograft acceptance. These findings provide a novel approach to increase Treg function and give insights into the fundamental mechanisms by which mitochondrial energy metabolism regulates immune cell functions in vivo.—Beier, U. H., Angelin, A., Akimova, T., Wang, L., Liu, Y., Xiao, H., Koike, M. A., Hancock, S. A., Bhatti, T. R., Han, R., Jiao, J., Veasey, S. C., Sims, C. A., Baur, J. A., Wallace, D. C., Hancock, W. W. Essential role of mitochondrial energy metabolism in Foxp3+ T‐regulatory cell function and allograft survival. FASEB J. 29, 2315‐2326 (2015). www.fasebj.org