Combination of isoform-selective histone/protein deacetylase inhibitors improves Foxp3+ T-regulatory cell function

Comment on: Beier UH, et al. Sci Signal 2012; 5:ra45.     

Programmed death-1 targeting can promote allograft survival

The recently identified CD28 homolog and costimulatory molecule programmed death-1 (PD-1) and its ligands, PD-L1 and PD-L2, which are homologs of B7, constitute an inhibitory regulatory pathway of potential therapeutic use in immune-mediated diseases. We examined the expression and functions of PD-1 and its ligands in experimental cardiac allograft rejection. In initial studies, we found that most normal tissues and cardiac isografts had minimal expression of PD-1, PD-L1, or PD-L2, but intragraft induction of all three molecules occurred during development of cardiac allograft rejection. Intragraft expression of all three genes was maintained despite therapy with cyclosporin A or rapamycin, but was prevented in the early posttransplant period by costimulation blockade using CD154 or anti-inducible costimulator mAb. We prepared PD-L1.Ig and PD-L2.Ig fusion proteins and showed that each bound to activated PD-1(+) T cells and inhibited T cell functions in vitro, thereby allowing us to test the effects of PD-1 targeting on allograft survival in vivo. Neither agent alone modulated allograft rejection in wild-type recipients. However, use of PD-L1.Ig administration in CD28(-/-) recipients, or in conjunction with immunosuppression in fully MHC-disparate combinations, markedly prolonged cardiac allograft survival, in some cases causing permanent engraftment, and was accompanied by reduced intragraft expression of IFN-gamma and IFN-gamma-induced chemokines. PD-L1.Ig use also prevented development of transplant arteriosclerosis post-CD154 mAb therapy. These data show that when combined with limited immunosuppression, or in the context of submaximal TCR or costimulatory signals, targeting of PD-1 can block allograft rejection and modulate T and B cell-dependent pathologic immune responses in vivo.     

Foxp3+ cell infiltration and granzyme B+/Foxp3+ cell ratio are associated with outcome in neoadjuvant chemotherapy-treated ovarian carcinoma

Preoperative neoadjuvant chemotherapy (NAC) can significantly reduce tumour burden in patients with primarily unresectable chemosensitive tumours, allowing a more complete cytoreduction during debulking surgery and facilitating evaluation of tumour chemosensitivity, identification of appropriate treatment options and improvement of intervention protocols. In this study, we investigate, using immunohistochemistry, the impact of platinum/taxane-based NAC (NAC) on tumour-infiltrating lymphocytes (TILs) in advanced epithelial ovarian cancer (EOC) and their relationship with clinical outcome. All patients had clinical response, as shown by ascites volume and CA125 levels compared to pre-treatment findings. NAC intervention significantly increased CD4⁺, CD8⁺ and granzyme B⁺ infiltration while Foxp3⁺ accumulation remained unaffected. TILs were prognostically neutral for both progression-free survival (PFS) and overall survival (OS) before NAC. In contrast, after NAC, elevated granzyme B⁺ infiltration displayed a tendency for improved PFS (log-rank 0.064). Further, low Foxp3⁺ cell density was associated with longer PFS, as compared with strong Foxp3⁺ infiltration (median 20.94 vs. 11.24 months; log-rank 0.0001) and with improved OS (median 30.75 vs. 16.04 months, respectively; log-rank 0.056), demonstrating clear prognostic significance for PFS. In addition, high granzyme B⁺/Foxp3⁺ ratio post-NAC strongly correlated with improved PFS compared to low granzyme B⁺/Foxp3⁺ cell ratio (median 17.88 vs. 11.24 months, respectively), and showed to be a favourable prognostic factor for PFS (log-rank 0.014). Our findings indicate that NAC elicited an immunologic profile in which low immunosuppressive Foxp3⁺ infiltration and elevated numbers of activated granzyme B⁺ cells were significantly associated with EOC-specific PFS, suggesting a contribution of immunologic effects to improved clinical outcome.     

Anti-Pim-1 mAb inhibits activation and proliferation of T lymphocytes and prolongs mouse skin allograft survival

Pim-1 is an important signaling molecule mediating cell proliferation and survival. Our previous study identified a Pim-1 specific monoclonal antibody, P9, with significant inhibitory effect on cell proliferation. Herein, we report that P9 inhibited the activation and proliferation of PHA-stimulated human PBMC and induced them to undergo apoptosis. In contrast, P9 showed little effect on freshly isolated human blood T lymphocytes which poorly expressed Pim-1. P9 also detected an up-regulation of Pim-1 in mouse lymphocytes after mitogen stimulation, and showed similar selective inhibition on stimulated cells as observed with hPBMC. Furthermore, P9 inhibited the in vitro mixed lymphocyte reaction and P9 treatment significantly prolonged the survival of mouse skin allografts (P < 0.001). It is concluded that Pim-1 expression correlates with lymphocyte proliferation and activation. P9 functions as a Pim-1 antagonist and is potential for immunosuppressive therapy.     

microRNA-150 promotes cervical cancer cell growth and survival by targeting FOXO4

Nuclear receptor family member, Estrogen related receptor β, and the Hedgehog signal transduction pathway are both reported to relate to tumorigenesis and induced pluripotent stem cell reprogramming. We hypothesize that Estrogen related receptor β can modulate the Hedgehog signaling pathway and affect Hedgehog driven downstream gene expression. We established an estrogen related receptor β-expressing Hedgehog-responsive NIH3T3 cell line by Esrrb transfection, and performed mRNA profiling using RNA-Seq after Hedgehog ligand conditioned medium treatment. Esrrb expression altered 171 genes, while Hedgehog signaling activation alone altered 339 genes. Additionally, estrogen related receptor β expression in combination with Hedgehog signaling activation affects a group of 109 Hedgehog responsive mRNAs, including Hsd11b1, Ogn, Smoc2, Igf1, Pdcd4, Igfbp4, Stmn1, Hp, Hoxd8, Top2a, Tubb4b, Sfrp2, Saa3, Prl2c3 and Dpt. We conclude that Estrogen related receptor β is capable of interacting with Hh-signaling downstream targets. Our results suggest a new level of regulation of Hedgehog signaling by Estrogen related receptor β, and indicate modulation of Estrogen related receptor β can be a new strategy to regulate various functions driven by the Hedgehog signaling pathway.     

Autophagic targeting of Src promotes cancer cell survival following reduced FAK signalling

Here we describe a mechanism that cancer cells use to survive when flux through the Src/FAK pathway is severely perturbed. Depletion of FAK, detachment of FAK-proficient cells or expression of non-phosphorylatable FAK proteins causes sequestration of active Src away from focal adhesions into intracellular puncta that co-stain with several autophagy regulators. Inhibition of autophagy results in restoration of active Src at peripheral adhesions, and this leads to cancer cell death. Autophagic targeting of active Src is associated with a Src-LC3B complex, and is mediated by c-Cbl. However, this is independent of c-Cbl E3 ligase activity, but is mediated by an LC3-interacting region. Thus, c-Cbl-mediated autophagic targeting of active Src can occur in cancer cells to maintain viability when flux through the integrin/Src/FAK pathway is disrupted. This exposes a previously unrecognized cancer cell vulnerability that may provide a new therapeutic opportunity.     

Histone deacetylase inhibitors promote mice corneal allograft survival through alteration of CD4+ effector T cells and induction of Foxp3+ regulatory T cells

Trichostatin A (TSA) is classical Histone deacetylase inhibitors (HDACIs) II which is used in treatment of advanced cutaneous T-cells lymphoma. Our works focused on the roles of TSA on immuno-modulatory. We found that the TSA could induce resting Teff cells into apoptotic cell death and inhibit Teff cells proliferation in a dose-dependent manner. We also observed down-regulation effects of various costimulatory/adhesion molecules on Teff cells and up-regulation of Foxp3 expression on CD4+ CD25+ T cells. Treatment with TSA could improve mice corneal allograft survival by promoting the proportions and allosuppressive function of CD4+ CD25+ regulatory T cells. Our findings suggest that the use of TSA allows the beneficial pharmacological effect on CD4+ CD25- T activation in vitro and enhancement of Foxp3+ Treg cells in vivo.     

Exogenous IL-10 overexpression reduces perforin production by activated allogenic CD8+ cells and prolongs cardiac allograft survival

Perforin is a cytolytic mediator produced by cytotoxic T cells (CD8(+) cells) and natural killer cells. We previously reported that ex vivo IL-10 gene therapy induced apoptosis of allogenic infiltrative CD8(+) cells and significantly prolonged cardiac allograft survival. To further test the hypothesis that localized IL-10 overexpression in cardiac allografts may also effect the alloreactive CD8(+) T cell function by downregulating its perforin production, we used a rabbit functional heterotopic allograft heart transplant model. Human recombinant IL-10 gene complexed with liposome was intracoronary delivered into the cardiac allografts ex vivo. The percentage of apoptotic infiltrative CD8(+) cells in cardiac allografts was increased 6-fold in the gene therapy group vs. the control group, whereas the percentage of perforin-positive CD8(+) cells was decreased 2.9-fold (P < 0.01). Perforin expression level in the allograft myocardium of the gene therapy group was deceased 3.2-fold (P < 0.01). The amount of infiltrative perforin-positive CD8(+) cells and perforin expression level were inversely correlated with IL-10 transgene and protein expression level in the myocardium of cardiac allografts (P < 0.01), the percentage of apoptotic cardiac myocytes (P < 0.01), and the peak left ventricular systolic pressure of cardiac allografts (P < 0.01) but significantly correlated with the infiltrative T cell cytotoxicity (P < 0.01) and allograft rejection score (P < 0.01). These results suggest that localized IL-10 gene therapy prolongs cardiac allograft survival, at least in part, through downregulation of perforin production by activated allogenic CD8(+) T cells. Reduction of cytolytic function of cytotoxic effector cells prevents the apoptosis of cardiac myocytes.     

Prevention of acute and chronic allograft rejection with CD4+CD25+Foxp3+ regulatory T lymphocytes

A major challenge in transplantation medicine is controlling the very strong immune responses to foreign antigens that are responsible for graft rejection. Although immunosuppressive drugs efficiently inhibit acute graft rejection, a substantial proportion of patients suffer chronic rejection that ultimately leads to functional loss of the graft. Induction of immunological tolerance to transplants would avoid rejection and the need for lifelong treatment with immunosuppressive drugs. Tolerance to self-antigens is ensured naturally by several mechanisms; one major mechanism depends on the activity of regulatory T lymphocytes. Here we show that in mice treated with clinically acceptable levels of irradiation, regulatory CD4+CD25+Foxp3+ T cells stimulated in vitro with alloantigens induced long-term tolerance to bone marrow and subsequent skin and cardiac allografts. Regulatory T cells specific for directly presented donor antigens prevented only acute rejection, despite hematopoietic chimerism. By contrast, regulatory T cells specific for both directly and indirectly presented alloantigens prevented both acute and chronic rejection. Our findings demonstrate the potential of appropriately stimulated regulatory T cells for future cell-based therapeutic approaches to induce lifelong immunological tolerance to allogeneic transplants.     

The Tim-3 ligand galectin-9 negatively regulates CD8+ alloreactive T cell and prolongs survival of skin graft

CD8+ alloreactive T cells are the key mediators of accelerated rejection. Vigorous CD8+ alloreactive T cells responses against alloantigens, which is the main effector mechanism in acute allograft rejection, has been well described. But the molecular mechanisms to dampen activated CD8+ T cells are largely unknown. On the other hand, Tim-3 is a molecule expressed on terminally differentiated CD4+ Th1 cells. Engaging Tim-3 with its ligand galectin-9 causes an inhibitory signal, resulting in apoptosis of Th1 cells and negatively regulates Th1 type immunity. However, the question whether CD8+ T cells express surface molecular Tim-3 has not been fully elucidated. In this study, we have investigated which CD8+ subset express molecular Tim-3 by flow cytometric assay. In addition, cytotoxic assay was applied to analyze whether CD8+ alloreactive T cells were sensitive to galectin-9 induced apoptosis. Here, our results demonstrated that Tim-3 was expressed on activated CD8+ alloreactive T cells (CD8+CD44highCD62Llow), but not expressed on na?ve CD8+ T cells. Furthermore, alloreactive CD8+ cytotoxic T cells were sensitive to galectin-9 induced apoptosis both in vitro and vivo, resulting in attenuation of CD8+ alloreactive T cells mediated cytotoxicity and prolonged survival of skin graft.     

CD4+Foxp3+ regulatory T cell differentiation mediated by endometrial stromal cell-derived TECK promotes the growth and invasion of endometriotic lesions

Endometriosis is associated with an abnormal immune response to endometrial cells, which can facilitate the implantation and proliferation of ectopic endometrial tissue. The proportion of CD4⁺Foxp3⁺ regulatory T cells (Tregs) is significantly increased in the peritoneal fluid of women with endometriosis. The thymus-expressed chemokine TECK/CCL25 directly promotes the invasiveness of endometrial stromal cells (ESCs). The aim of this study was to investigate the effects of ESC-derived TECK on the crosstalk between Tregs and ESCs in the progress of endometriosis. We determined that the percentage of Tregs and the concentration of TECK increased in the peritoneal fluid with the progression of endometriosis. The supernatant from co-cultured human ESCs and macrophages not only induced Treg differentiation and increased Treg expression of interleukin-10 (IL-10), transforming growth factor-β (TGF-β) and CD73 by activating the AKT/STAT3 signaling pathway but also repressed Treg apoptosis by downregulating Fas and FasL expression and enhanced the Treg-mediated suppression of CD4⁺CD25⁻ T cells. In addition, in vitro and in vivo trials confirmed that these effects could be inhibited by anti-TECK neutralizing Abs. The secretion of IL-10 and TGF-β by Tregs increased MMP2 expression and decreased TIMP1 expression and further stimulated the proliferation and invasion of ESCs and the growth of ectopic lesions. These results indicate that TECK derived from ESCs and macrophages upregulates the number and function of Tregs in the ectopic milieu, which contributes to endometriotic immunotolerance and high levels of ESC proliferation and invasion, thereby facilitating the progression of endometriosis.Endometriosis is one of the most common gynecological diseases in women with a prevalence rate of ~10%. It is characterized by the presence of endometrial glands and stroma at extrauterine sites and manifests with pelvic pain and infertility.¹ Despite decades of intensive investigation, little is known about the pathogenesis of endometriosis. The most widely accepted etiology is Sampson''s theory of retrograde menstruation where shed endometrial tissue is refluxed through the fallopian tubes and attaches and proliferates within the pelvis.² However, it is not fully understood why, even though the majority of women have retrograde menstruation, only about one in ten women develop endometriosis. This suggests that other factors may mediate the formation of endometriotic lesions.Several recent studies have focused on the importance of immunologic imbalances in women with endometriosis. In fact, a permissive peritoneal environment may be associated with a dysregulated immune response to endometrial cells. Instead of effectively removing endometrial fragments at pelvic cavity, this environment can facilitate the implantation, neo-angiogenesis and proliferation of ectopic endometrial tissue.^3,4 These conditions may include elevated levels of activated peritoneal macrophages, reduced natural killer cell activity and an abnormal T lymphocyte response. Recently, several groups have reported the presence of Tregs in eutopic and ectopic endometrial tissue from patients with endometriosis.^5,6 In addition, the number of Tregs is significantly increased in peritoneal fluid of women with endometriosis.^7,8 However, the mechanism behind the increase in the number of Tregs in the peritoneal fluid of women with endometriosis and the role of Tregs in the progression of endometriosis are unknown.Chemokines produced in the endometriotic milieu may contribute to a feed-forward cascade of events, which promotes the recruitment of leukocytes to the peritoneal cavity and regulates the proliferation and invasion of endometrial stromal cells (ESCs) in patients with endometriosis. These chemokines include Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES), monocyte chemotactic protein and interleukin-8 (IL-8).^9,10 The thymus-expressed chemokine (TECK/CCL25), which was initially reported to be produced by thymic cells, is highly expressed in endothelial cells and a subset of cells in the small intestine.¹¹ CC chemokine receptor 9 (CCR9), previously designated GPR-9-6, as a specific receptor for TECK, is expressed mainly in immature T cells such as double-positive T cells and gut-associated T cells.¹² The TECK–CCR9 interaction has an important role in regulating T-cell development and tissue-specific homing.¹² In addition, CCR9-mediated signaling is involved in anti-apoptotic signaling to the T cells.¹³ Interestingly, our previous study confirmed that TECK from a variety of cells in the endometriotic milieu (for example, ESCs, peritoneal mesothelial cells and macrophages) promotes ESC invasion in endometriosis by increasing the expression of metalloproteinase 2/9 (MMP2/9).¹⁴Therefore, the aim of this study was to investigate whether TECK in the endometriotic milieu regulates the Treg differentiation, apoptosis and function and to explore further the effect of these educated Tregs on the growth and invasion of ESCs in endometriosis.     

SEK1-dependent JNK1 activation prolongs cell survival during G-Rh2-induced apoptosis

We provide here evidence that c-Jun N-terminal protein kinase 1 (JNK1) activity is differentially up-regulated during apoptosis of SK-HEP-1 cells after treatment with ginsenoside Rh2 (G-Rh2). The G-Rh2-mediated JNK1 activation that occurred for the first 10-30min was associated with SEK1 activity, but thereafter, the sustained activation was associated not with SEK1 activity, but with proteolytic cleavage of JNK1-associated p21(WAF1/CIP1). Supporting this is that the expression of the dominant negative SEK1 mutant effectively blocked the early JNK1 activation phase but did not alter the sustained activation phase or apoptosis. Furthermore, expression of p21D112N, an uncleavable mutant of p21(WAF1/CIP1), suppressed the later JNK1 activation. Moreover, the stable overexpression of ectopic JNK1 suppressed apoptosis while expression of the dominant negative JNK1 mutant promoted it. We propose that the early SEK1-associated JNK1 activation phase acts to prolong cell survival in response to apoptosis-inducing agents, thereby serving as an intervening checkpoint prior to the commitment to apoptosis.     

IL-33 expands suppressive CD11b+ Gr-1(int) and regulatory T cells, including ST2L+ Foxp3+ cells, and mediates regulatory T cell-dependent promotion of cardiac allograft survival

IL-33 administration is associated with facilitation of Th2 responses and cardioprotective properties in rodent models. However, in heart transplantation, the mechanism by which IL-33, signaling through ST2L (the membrane-bound form of ST2), promotes transplant survival is unclear. We report that IL-33 administration, while facilitating Th2 responses, also increases immunoregulatory myeloid cells and CD4(+) Foxp3(+) regulatory T cells (Tregs) in mice. IL-33 expands functional myeloid-derived suppressor cells, CD11b(+) cells that exhibit intermediate (int) levels of Gr-1 and potent T cell suppressive function. Furthermore, IL-33 administration causes an St2-dependent expansion of suppressive CD4(+) Foxp3(+) Tregs, including an ST2L(+) population. IL-33 monotherapy after fully allogeneic mouse heart transplantation resulted in significant graft prolongation associated with increased Th2-type responses and decreased systemic CD8(+) IFN-γ(+) cells. Also, despite reducing overall CD3(+) cell infiltration of the graft, IL-33 administration markedly increased intragraft Foxp3(+) cells. Whereas control graft recipients displayed increases in systemic CD11b(+) Gr-1(hi) cells, IL-33-treated recipients exhibited increased CD11b(+) Gr-1(int) cells. Enhanced ST2 expression was observed in the myocardium and endothelium of rejecting allografts, however the therapeutic effect of IL-33 required recipient St2 expression and was dependent on Tregs. These findings reveal a new immunoregulatory property of IL-33. Specifically, in addition to supporting Th2 responses, IL-33 facilitates regulatory cells, particularly functional CD4(+) Foxp3(+) Tregs that underlie IL-33-mediated cardiac allograft survival.     

Histone deacetylase 6 and heat shock protein 90 control the functions of Foxp3(+) T-regulatory cells

Foxp3(+) T-regulatory cells (Tregs) are key to immune homeostasis such that their diminished numbers or function can cause autoimmunity and allograft rejection. Foxp3(+) Tregs express multiple histone/protein deacetylases (HDACs) that regulate chromatin remodeling, gene expression, and protein function. Pan-HDAC inhibitors developed for oncologic applications enhance Treg production and Treg suppression function but have limited nononcologic utility given their broad actions and various side effects. We show, using HDAC6-deficient mice and wild-type (WT) mice treated with HDAC6-specific inhibitors, that HDAC6 inhibition promotes Treg suppressive activity in models of inflammation and autoimmunity, including multiple forms of experimental colitis and fully major histocompatibility complex (MHC)-incompatible cardiac allograft rejection. Many of the beneficial effects of HDAC6 targeting are also achieved by inhibition of the HDAC6-regulated protein heat shock protein 90 (HSP90). Hence, selective targeting of a single HDAC isoform, HDAC6, or its downstream target, HSP90, can promote Treg-dependent suppression of autoimmunity and transplant rejection.     

TCR affinity promotes CD8+ T cell expansion by regulating survival

Ligation with high affinity ligands are known to induce T lymphocytes to become fully activated effector cells while ligation with low affinity ligands (or partial agonists) may result in a delayed or incomplete response. We have examined the quantitative features of CD8(+) T cell proliferation induced by peptides of different TCR affinities at a range of concentrations in the mouse OT-I model. Both the frequency of cells responding and the average time taken for cells to reach their first division are affected by peptide concentration and affinity. Consecutive division times, however, remained largely unaffected by these variables. Importantly, we identified affinity to be the sole regulator of cell death in subsequent division. These results suggest a mechanism whereby TCR affinity detection can modulate the subsequent rate of T cell growth and ensure the dominance of higher affinity clones over time.     

Nitric oxide modulates TGF-beta-directive signals to suppress Foxp3+ regulatory T cell differentiation and potentiate Th1 development

TGF-β can induce Foxp3(+) inducible regulatory T cells (Treg) and also synergize with IL-6 and IL-4 to induce Th17 and Th9 cells. We now report that NO modulates TGF-β activity away from Treg but toward the Th1 lineage. NO potentiated Th1 differentiation in the presence of TGF-β in both IL-12-independent and -dependent fashions by augmenting IFN-γ-activated STAT-1 and T-bet. Differentiation into Treg, Th1, and Th17 lineages could be modulated by NO competing with other cofactors, such as IL-6 and retinoic acid. NO antagonized IL-6 to block TGF-β-directed Th17 differentiation, and together with IL-6, NO suppressed Treg development induced by TGF-β and retinoic acid. Furthermore, we show that physiologically produced NO from TNF and inducible NO synthase-producing dendritic cells can contribute to Th1 development predominating over Treg development through a synergistic activity induced when these cells cocluster with conventional dendritic cells presenting Ag to naive Th cells. This illustrates that NO is another cofactor allowing TGF-β to participate in development of multiple Th lineages and suggests a new mechanism by which NO, which is associated with protection against intracellular pathogens, might maintain effective Th1 immunity.