Yaping Sun

Histone deacetylase inhibition modulates indoleamine 2,3-dioxygenase–dependent DC functions and regulates experimental graft-versus-host disease in mice

Histone deacetylase (HDAC) inhibitors are antitumor agents that also have antiinflammatory properties. However, the mechanisms of their immunomodulatory functions are not known. We investigated the mechanisms of action of 2 HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA) and ITF 2357, on mouse DC responses. Pretreatment of DCs with HDAC inhibitors significantly reduced TLR-induced secretion of proinflammatory cytokines, suppressed the expression of CD40 and CD80, and reduced the in vitro and in vivo allostimulatory responses induced by the DCs. In addition, injection of DCs treated ex vivo with HDAC inhibitors reduced experimental graft-versus-host disease (GVHD) in a murine allogeneic BM transplantation model. Exposure of DCs to HDAC inhibitors increased expression of indoleamine 2,3-dioxygenase (IDO), a suppressor of DC function. Blockade of IDO in WT DCs with siRNA and with DCs from IDO-deficient animals caused substantial reversal of HDAC inhibition-induced in vitro suppression of DC-stimulated responses. Direct injection of HDAC inhibitors early after allogeneic BM transplantation to chimeric animals whose BM-derived cells lacked IDO failed to protect from GVHD, demonstrating an in vivo functional role for IDO. Together, these data show that HDAC inhibitors regulate multiple DC functions through the induction of IDO and suggest that they may represent a novel class of agents to treat immune-mediated diseases.

Gut microbiome–derived metabolites modulate intestinal epithelial cell damage and mitigate graft-versus-host disease

The effect of alterations in intestinal microbiota on microbial metabolites and on disease processes such as graft-versus-host disease (GVHD) is not known. Here we carried out an unbiased analysis to identify previously unidentified alterations in gastrointestinal microbiota–derived short-chain fatty acids (SCFAs) after allogeneic bone marrow transplant (allo-BMT). Alterations in the amount of only one SCFA, butyrate, were observed only in the intestinal tissue. The reduced butyrate in CD326+ intestinal epithelial cells (IECs) after allo-BMT resulted in decreased histone acetylation, which was restored after local administration of exogenous butyrate. Butyrate restoration improved IEC junctional integrity, decreased apoptosis and mitigated GVHD. Furthermore, alteration of the indigenous microbiota with 17 rationally selected strains of high butyrate–producing Clostridia also decreased GVHD. These data demonstrate a heretofore unrecognized role of microbial metabolites and suggest that local and specific alteration of microbial metabolites has direct salutary effects on GVHD target tissues and can mitigate disease severity.

Interleukin-6 modulates graft-versus-host responses after experimental allogeneic bone marrow transplantation

Purpose: The graft-versus-tumor (GVT) effect is a potent form of immunotherapy against many hematologic malignancies and some solid tumors. The beneficial GVT effect after allogeneic bone marrow transplantation (BMT) is tightly linked to its most significant complication, graft-versus-host disease (GVHD). The role of interleukin-6 (IL-6) after allogeneic BMT is not well understood. This study used a series of complementary knockout and antibody blockade strategies to analyze the impact of IL-6 in multiple clinically relevant murine models of GVHD and GVT. Experimental Design: We examined the effect of the source of IL-6 by analyzing the role IL-6 deficiency in donor T cells, donor bone marrow or in host tissues. We confirmed and extended the relevance of IL-6 deficiency on GVHD and GVT by treating BMT recipients with anti-mouse IL-6 receptor (IL-6R), MR16-1. Results: Deficiency of IL-6 in donor T cells led to prolongation of survival. Total inhibition of IL-6 with MR16-1 caused an even greater reduction in GVHD-induced mortality. The reduction in GVHD was independent of the direct effects on T effector cell expansion or donor regulatory T cells. GVT responses were preserved after treatment with MR16-1. Conclusion: MR16-1 treatment reduced GVHD and preserved sufficient GVT. Tocilizumab, a humanized anti–IL-6R monoclonal antibody (mAb), is approved in several countries including the United States and European Union for the treatment of rheumatoid arthritis and other inflammatory diseases. Blockade of IL-6 with anti–IL-6R mAb therapy may be testable in clinical trials as an adjunct to prevent GVHD in BMT patients without a significant loss of GVT. Clin Cancer Res; 17(1); 77–88. ©2010 AACR.

Cutting Edge: Negative Regulation of Dendritic Cells through Acetylation of the Nonhistone Protein STAT-3

Histone deacetylase (HDAC) inhibition modulates dendritic cell (DC) functions and regulates experimental graft-vs-host disease and other immune-mediated diseases. The mechanisms by which HDAC inhibition modulates immune responses remain largely unknown. STAT-3 is a transcription factor shown to negatively regulate DC functions. In this study we report that HDAC inhibition acetylates and activates STAT-3, which regulates DCs by promoting the transcription of IDO. These findings demonstrate a novel functional role for posttranslational modification of STAT-3 through acetylation and provide mechanistic insights into HDAC inhibition-mediated immunoregulation by induction of IDO.

Alpha-1-antitrypsin monotherapy reduces graft-versus-host disease after experimental allogeneic bone marrow transplantation

Acute graft-versus-host disease (GvHD) is a major complication that prevents successful outcomes after allogeneic bone marrow transplantation (BMT), an effective therapy for hematological malignancies. Several studies demonstrate that donor T cells and host antigen-presenting cells along with several proinflammatory cytokines are required for the induction of GvHD and contribute to its severity. Increasing evidence demonstrates that human serum-derived αalpha-1- anti-trypsin (AAT) reduces production of proinflammatory cytokines, induces anti-inflammatory cytokines, and interferes with maturation of dendritic cells. Using well-characterized mouse models of BMT, we have studied the effects of AAT on GvHD severity. Administration of AAT early after BMT decreased mortality in three models of GvHD and reduced serum levels of proinflammatory cytokines in the allogeneic recipients compared with vehicle (albumin) treated animals. AAT treatment reduced the expansion of alloreactive T effector cells but enhanced the recovery of T regulatory T cells, (Tregs) thus altering the ratio of donor T effector to T regulatory cells in favor of reducing the pathological process. However, despite altering the ratio in vivo, AAT had no direct effects on either the donor T effector cells or T regulatory cells Tregs in vitro. In contrast, AAT suppressed LPS-induced in vitro secretion of proinflammatory cytokines such as TNF-α and IL-1β, enhanced the production of the anti-inflammatory cytokine IL-10, and impaired NF-κB translocation in the host dendritic cells. In light of its long history of safety in humans, these findings suggest that administration of AAT represents a novel unique and viable strategy to mitigate clinical GvHD.

Vorinostat plus tacrolimus and mycophenolate to prevent graft-versus-host disease after related-donor reduced-intensity conditioning allogeneic haemopoietic stem-cell transplantation: a phase 1/2 trial

BACKGROUND Acute graft-versus-host disease (GVHD) remains a barrier to more widespread application of allogeneic haemopoietic stem-cell transplantation. Vorinostat is an inhibitor of histone deacetylases and was shown to attenuate GVHD in preclinical models. We aimed to study the safety and activity of vorinostat, in combination with standard immunoprophylaxis, for prevention of GVHD in patients undergoing related-donor reduced-intensity conditioning haemopoietic stem-cell transplantation. METHODS Between March 31, 2009, and Feb 8, 2013, we did a prospective, single-arm, phase 1/2 study at two centres in the USA. We recruited adults (aged ≥18 years) with high-risk haematological malignant diseases who were candidates for reduced-intensity conditioning haemopoietic stem-cell transplantation and had an available 8/8 or 7/8 HLA-matched related donor. All patients received a conditioning regimen of fludarabine (40 mg/m(2) daily for 4 days) and busulfan (3.2 mg/kg daily for 2 days) and GVHD immunoprophylaxis of mycophenolate mofetil (1 g three times a day, days 0-28) and tacrolimus (0.03 mg/kg a day, titrated to a goal level of 8-12 ng/mL, starting day -3 until day 180). Vorinostat (either 100 mg or 200 mg, twice a day) was initiated 10 days before haemopoietic stem-cell transplantation until day 100. The primary endpoint was the cumulative incidence of grade 2-4 acute GVHD by day 100. This trial is registered with ClinicalTrials.gov, number NCT00810602. FINDINGS 50 patients were assessable for both toxic effects and response; eight additional patients were included in the analysis of toxic effects. All patients engrafted neutrophils and platelets at expected times after haemopoietic stem-cell transplantation. The cumulative incidence of grade 2-4 acute GVHD by day 100 was 22% (95% CI 13-36). The most common non-haematological adverse events included electrolyte disturbances (n=15), hyperglycaemia (11), infections (six), mucositis (four), and increased activity of liver enzymes (three). Non-symptomatic thrombocytopenia after engraftment was the most common haematological grade 3-4 adverse event (nine) but was transient and all cases resolved swiftly. INTERPRETATION Administration of vorinostat in combination with standard GVHD prophylaxis after related-donor reduced-intensity conditioning haemopoietic stem-cell transplantation is safe and is associated with a lower than expected incidence of severe acute GVHD. Future studies are needed to assess the effect of vorinostat for prevention of GVHD in broader settings of haemopoietic stem-cell transplantation. FUNDING Merck, Leukemia and Lymphoma Society, National Institutes of Health, St Baldricks Foundation, Michigan Institute for Clinical and Health Research.

Induction of acute GVHD by sex-mismatched H-Y antigens in the absence of functional radiosensitive host hematopoietic-derived antigen-presenting cells.

It is currently thought that acute GVHD cannot be elicited in the absence of Ag presentation by radiosensitive host hematopoietic-derived APCs after allogeneic BM transplantation. Because clinical data suggest that sex-mismatched H-Y Ags may be important minor histocompatibility Ags for GVH responses, we directly tested their relevance and ability to initiate GVHD when presented by either the hematopoietic- (host or donor) or the nonhematopoietic-derived APCs. H-Y minor Ag incompatibility elicited both CD4(+) and CD8(+) T-cell driven GVHD lethality. Studies with various well-established BM chimera recipients, in contrast to the current views, have reported that in the absence of functional radiosensitive host hematopoietic-derived APCs, H-Y Ag presentation by either the donor hematopoietic-derived or the host nonhematopoietic-derived APCs is sufficient for inducing GVHD. Our data further suggest that infusion of sufficient numbers of alloreactive donor T cells will induce GVHD in the absence of radiosensitive host hematopoietic-derived APCs.

GVHD pathophysiology: is acute different from chronic?

Graft-versus-host disease (GVHD) is the major complication of allogeneic hematopoietic cell transplantation (HCT). GVHD occurs in acute and chronic forms. Acute GVHD usually manifests within 100 days following HSCT. It is induced by donor T cells responding to the mismatched host polymorphic histocompatibility antigens. Chronic GVHD generally manifests later (>100 days) and has some features of autoimmune diseases. It may develop either de novo or following resolution of - or as an extension of - acute GVHD. Chronic GVHD is also thought to be induced by donor T cells, but the nature of relevant antigens, the critical cellular subsets and the mechanisms of chronic GVHD remain less well understood. In this chapter we briefly discuss and contrast the pathophysiologies of acute and chronic GVHD.

Combined Th2 cytokine deficiency in donor T cells aggravates experimental acute graft-vs-host disease

The role of T helper (Th) 1 and Th2 polarization in acute graft-vs-host-disease (GVHD) is unclear. We investigated the role of Th2 cytokine secretion by utilizing donor T cells that cannot make interleukin (IL)-4, IL-5, IL-9, and IL-13 from quadruple cytokine-deficient (Quad-KO) animals in a well-characterized BALB/c-->C57BL/6 model of allogeneic bone marrow transplantation. B6 recipients of BALB/c Quad-KO T cells demonstrated greater clinical severity, target organ damage, and mortality from GVHD than recipients of BALB/c wild-type (WT) T cells. When compared with donor T cells that are deficient in signal transducers and activators of transcription 6 signaling or the signature Th2 cytokine, IL-4, Quad-KO T cells demonstrated greater GVHD mortality. Mechanistic studies demonstrated that Quad-KO T cells demonstrated enhanced T-cell proliferation than WT T cells when stimulated with either allogeneic antigen-presenting cells or with nonspecific stimuli, such as anti-CD3 monoclonal antibody. Quad-KO T cells also secreted greater amounts of Th1 cytokines and IL-17 compared to WT T cells. Deficiency of Th2 cytokines, however, did not alter the allospecific cytotoxic responses, the numbers of immunoregulatory CD4(+)CD25(+) Foxp3(+) T cells or their suppressive functions. Our data thus unequivocally demonstrate that deficiency of the four classical Th2 cytokine enhances T-cell proliferative responses and aggravates GVHD.

A Crucial Role for Host APCs in the Induction of Donor CD4+CD25+ Regulatory T Cell-Mediated Suppression of Experimental Graft-versus-Host Disease

Allogeneic bone marrow transplantation is an effective treatment for a number of malignant and nonmalignant diseases (Applebaum. 2001. Nature. 411: 385–389 and Copelan. 2006. N Engl J Med. 354: 1813–1826). However, the application of this therapeutic modality has been impeded by a number of confounding side effects, the most frequent and severe of which is the development of graft-versus-host disease (GVHD) (Copelan. 2006. N Engl J Med. 354: 1813–1826 and Blazar and Murphy. 2005. Philos Trans R Soc Lond B Biol Sci. 360: 1747–1767). Alloreactive donor T cells are critical for causing GVHD (Fowler. 2006. Crit Rev Oncol Hematol. 57: 225–244 and Ferrara and Reddy. 2006. Semin Hematol. 43: 3–10), whereas recent data demonstrated a significant role for the naturally occurring thymic-derived donor CD4+CD25+Foxp3+ regulatory T cells (Tregs) (Bluestone and Abbas. 2003. Nat Rev Immunol. 3: 253–257 and Shevach. 2006. Immunity. 25: 195–201) in suppressing experimental GVHD after bone marrow transplantation (Blazar and Taylor. 2005. Biol Blood Marrow Transpl. 11: 46–49 and Joffe and van Meerwijk. 2006. Semin Immunol. 18: 128–135) . Host APCs are required for induction of GVHD by the conventional donor T cells. However, it is not known whether they are also obligatory for donor Treg-mediated suppression of GVHD. Using multiple clinically relevant MHC-matched and -mismatched murine models of GVHD, we investigated the role of host APCs in the suppression of GVHD by donor Tregs. We found that alloantigen expression by the host APCs is necessary and sufficient for induction of GVHD protection by donor Tregs. This requirement was independent of their effect on the maintenance of Treg numbers and the production of IL-10 or IDO by the host APCs.