Mitochondrial Deacetylase SIRT3 Plays an Important Role in Donor T Cell Responses after Experimental Allogeneic Hematopoietic Transplantation.

Allogeneic hematopoietic cell transplantation (allo-HCT) through its graft-versus-tumor (GVT) effects is a curative therapy against many hematological malignancies. However, GVT is linked to harmful graft-versus-host disease (GVHD) after allo-HCT. Both GVT and GVHD require allogeneic T cell responses, which is an energetically costly process that causes oxidative stress. Sirtuin 3 (SIRT3), a mitochondrial histone deacetylase (HDAC), plays an important role in cellular processes through inhibition of reactive oxygen species (ROS). Nonmitochondrial class of HDACs regulate T cell responses, but the role of mitochondrial HDACs, specifically SIRT3, on donor T cell responses after allo-HCT remains unknown. In this study, we report that SIRT3-deficient (SIRT3-/-) donor T cells cause reduced GVHD severity in multiple clinically relevant murine models. The GVHD protective effect of allogeneic SIRT3-/- T cells was associated with a reduction in their activation, reduced CXCR3 expression, and no significant impact on cytokine secretion or cytotoxic functions. Intriguingly, the GVHD protective effect of SIRT3-/- T cells was associated with a reduction in ROS production, which is contrary to the effect of SIRT3 deficiency on ROS production in other cells/tissues and likely a consequence of their deficient activation. Notably, the reduction in GVHD in the gastrointestinal tract was not associated with a substantial reduction in the GVT effect. Collectively, these data reveal that SIRT3 activity promotes allogeneic donor T cell responses and ROS production without altering T cell cytokine or cytolytic functions and identify SIRT3 as a novel target on donor T cells to improve outcomes after allo-HCT.

SAG/Rbx2-Dependent Neddylation Regulates T-Cell Responses.

Neddylation is a crucial post-translational modification that depends on the E3 cullin ring ligase (CRL). The E2-adapter component of the CRL, sensitive to apoptosis gene (SAG), is critical for the function of CRL-mediated ubiquitination; thus, the deletion of SAG regulates neddylation. We examined the role of SAG-dependent neddylation in T-cell-mediated immunity using multiple approaches: a novel T-cell-specific, SAG genetic knockout (KO) and chemical inhibition with small-molecule MLN4924. The KO animals were viable and showed phenotypically normal mature T-cell development. However, in vitro stimulation of KO T cells revealed significantly decreased activation, proliferation, and T-effector cytokine release, compared with WT. Using in vivo clinically relevant models of allogeneic bone marrow transplantation also demonstrated reduced proliferation and effector cytokine secretion associated with markedly reduced graft-versus-host disease. Similar in vitro and in vivo results were observed with the small-molecule inhibitor of neddylation, MLN4924. Mechanistic studies demonstrated that SAG-mediated effects in T cells were concomitant with an increase in suppressor of cytokine signaling, but not NF-κB translocation. Our studies suggest that SAG is a novel molecular target that regulates T-cell responses and that inhibiting neddylation with the clinically available small-molecule MLN4924 may represent a novel strategy to mitigate T-cell-mediated immunopathologies, such as graft-versus-host disease.

Sigma 1 Receptor antagonist potentiates the anti-cancer effect of p53 by regulating ER stress, ROS production, Bax levels, and caspase-3 activation.

Over the last years, many improvements have been made in the treatment of breast cancer; however, novel and less toxic therapies are still needed, especially for relapsing and chemo-resistant patients. Here, we analyzed the therapeutic potential of p53 and Rimcazole, a Sigma 1 Receptor antagonist. Rimcazole and p53 are being evaluated in preclinical and clinical trials, respectively. While p53 is a promising antitumor therapeutic agent, antagonists of Sigma 1 Receptor also inhibit tumor cell survival and induce apoptosis. Our current study demonstrates for the first time the synergistic effect of p53 in combination with the Sigma 1 Receptor antagonist Rimcazole. Furthermore, we show that shRNA knockdown of Sigma 1 Receptor in combination with p53, lead to a similar synergistic effect, and that this synergistic effect, in breast cancer growth suppression occurs independent of p53 status. Furthermore, this combination treatment induced ER stress, p38 MAPK activation, ROS production, and proteins involved in apoptosis (caspases-3, Bax) in breast cancer cells. Combining these therapeutic anti-cancer molecules provides an innovative approach for potentially treating human breast cancer.

ATG5-Dependent Autophagy Uncouples T-cell Proliferative and Effector Functions and Separates Graft-versus-Host Disease from Graft-versus-Leukemia.

Autophagy is a vital cellular process whose role in T immune cells is poorly understood, specifically, in its regulation of allo-immunity. Stimulation of wild-type T cells in vitro and in vivo with allo-antigens enhances autophagy. To assess the relevance of autophagy to T-cell allo-immunity, we generated T-cell-specific Atg5 knock-out mice. Deficiency of ATG5-dependent autophagy reduced T-cell proliferation and increased apoptosis following in vitro and in vivo allo-stimulation. The absence of ATG5 in allo-stimulated T cells enhanced their ability to release effector cytokines and cytotoxic functions, uncoupling their proliferation and effector functions. Absence of autophagy reduced intracellular degradation of cytotoxic enzymes such as granzyme B, thus enhancing the cytotoxicity of T cells. In several in vivo models of allo-HSCT, ATG5-dependent dissociation of T-cell functions contributed to significant reduction in graft-versus-host disease (GVHD) but retained sufficient graft versus tumor (GVT) response. Our findings demonstrate that ATG5-dependent autophagy uncouples T-cell proliferation from its effector functions and offers a potential new strategy to enhance outcomes after allo-HSCT. SIGNIFICANCE: These findings demonstrate that induction of autophagy in donor T-cell promotes GVHD, while inhibition of T-cell autophagy mitigates GVHD without substantial loss of GVL responses.

RNA-seq of human T cells after hematopoietic stem cell transplantation identifies Linc00402 as a regulator of T cell alloimmunity.

Mechanisms governing allogeneic T cell responses after solid organ and allogeneic hematopoietic stem cell transplantation (HSCT) are incompletely understood. To identify lncRNAs that regulate human donor T cells after clinical HSCT, we performed RNA sequencing on T cells from healthy individuals and donor T cells from three different groups of HSCT recipients that differed in their degree of major histocompatibility complex (MHC) mismatch. We found that lncRNA differential expression was greatest in T cells after MHC-mismatched HSCT relative to T cells after either MHC-matched or autologous HSCT. Differential expression was validated in an independent patient cohort and in mixed lymphocyte reactions using ex vivo healthy human T cells. We identified Linc00402, an uncharacterized lncRNA, among the lncRNAs differentially expressed between the mismatched unrelated and matched unrelated donor T cells. We found that Linc00402 was conserved and exhibited an 88-fold increase in human T cells relative to all other samples in the FANTOM5 database. Linc00402 was also increased in donor T cells from patients who underwent allogeneic cardiac transplantation and in murine T cells. Linc00402 was reduced in patients who subsequently developed acute graft-versus-host disease. Linc00402 enhanced the activity of ERK1 and ERK2, increased FOS nuclear accumulation, and augmented expression of interleukin-2 and Egr-1 after T cell receptor engagement. Functionally, Linc00402 augmented the T cell proliferative response to an allogeneic stimulus but not to a nominal ovalbumin peptide antigen or polyclonal anti-CD3/CD28 stimulus. Thus, our studies identified Linc00402 as a regulator of allogeneic T cell function.

A Pipeline for Faecal Host DNA Analysis by Absolute Quantification of LINE-1 and Mitochondrial Genomic Elements Using ddPCR.

Stool contains DNA shed from cells of the gastrointestinal (GI) tract and has great potential as a bio-specimen for non-invasive, nucleic acid-based detection of GI diseases. Whereas methods for studying faecal microbiome DNA are plentiful, there is a lack of well-characterised procedures for stabilisation, isolation, and quantitative analysis of faecal host DNA. We report an optimised pipeline for faecal host DNA analysis from the point-of-collection to droplet digital PCR (ddPCR) absolute quantification of host-specific gene targets. We evaluated multiple methods for preservation and isolation of host DNA from stool to identify the highest performing methods. To quantify host DNA even if present in partially degraded form, we developed sensitive, human-specific short-amplicon ddPCR assays targeting repetitive nuclear genomic elements (LINE-1) and mitochondrial genes. We validated the ability of these optimised methods to perform absolute quantification of host DNA in 200 stool DNA extracts from samples that were serially collected from three healthy individuals and three hospitalised patients. These specimens allowed assessment of host DNA day-to-day variability in stool specimens with widely varying physical characteristics (i.e., Bristol scores). We further extended this approach to mouse stool analysis, to enable faecal host DNA studies in animal disease models as well.

SNARE protein SEC22B regulates early embryonic development.

The highly conserved SNARE protein SEC22B mediates diverse and critical functions, including phagocytosis, cell growth, autophagy, and protein secretion. However, these characterizations have thus far been limited to in vitro work. Here, we expand our understanding of the role Sec22b plays in vivo. We utilized Cre-Lox mice to delete Sec22b in three tissue compartments. With a germline deletion of Sec22b, we observed embryonic death at E8.5. Hematopoietic/endothelial cell deletion of Sec22b also resulted in in utero death. Notably, mice with Sec22b deletion in CD11c-expressing cells of the hematopoietic system survive to adulthood. These data demonstrate Sec22b contributes to early embryogenesis through activity both in hematopoietic/endothelial tissues as well as in other tissues yet to be defined.

miR-142 controls metabolic reprogramming that regulates dendritic cell activation.

DCs undergo metabolic reprogramming from a predominantly oxidative phosphorylation (OXPHOS) to glycolysis to mount an immunogenic response. The mechanism underpinning the metabolic reprogramming remains elusive. We demonstrate that miRNA-142 (miR-142) is pivotal for this shift in metabolism, which regulates the tolerogenic and immunogenic responses of DCs. In the absence of miR-142, DCs fail to switch from OXPHOS and show reduced production of proinflammatory cytokines and the ability to activate T cells in vitro and in in vivo models of sepsis and alloimmunity. Mechanistic studies demonstrate that miR-142 regulates fatty acid (FA) oxidation, which causes the failure to switch to glycolysis. Loss- and gain-of-function experiments identified carnitine palmitoyltransferase -1a (CPT1a), a key regulator of the FA pathway, as a direct target of miR-142 that is pivotal for the metabolic switch. Thus, our findings show that miR-142 is central to the metabolic reprogramming that specifically favors glycolysis and immunogenic response by DCs.

Host NLRP6 exacerbates graft-versus-host disease independent of gut microbial composition.

Host NOD-like receptor family pyrin domain-containing 6 (NLRP6) regulates innate immune responses and gastrointestinal homeostasis. Its protective role in intestinal colitis and tumorigenesis is dependent on the host microbiome. Host innate immunity and microbial diversity also play a role in the severity of allogeneic immune-mediated gastrointestinal graft-versus-host disease (GVHD), the principal toxicity after allogeneic haematopoietic cell transplantation. Here, we examined the role of host NLRP6 in multiple murine models of allogeneic bone marrow transplantation. In contrast to its role in intestinal colitis, host NLRP6 aggravated gastrointestinal GVHD. The impact of host NLRP6 deficiency in mitigating GVHD was observed regardless of co-housing, antibiotic treatment or colonizing littermate germ-free wild-type and NLRP6-deficient hosts with faecal microbial transplantation from specific pathogen-free wild-type and Nlrp6-/- animals. Chimaera studies were performed to assess the role of NLRP6 expression on host haematopoietic and non-haematopoietic cells. The allogeneic [B6Ly5.2 → Nlrp6-/-] animals demonstrated significantly improved survival compared to the allogeneic [B6Ly5.2 → B6] animals, but did not alter the therapeutic graft-versus-tumour effects after haematopoietic cell transplantation. Our results unveil an unexpected, pathogenic role for host NLRP6 in gastrointestinal GVHD that is independent of variations in the intestinal microbiome and in contrast to its well-appreciated microbiome-dependent protective role in intestinal colitis and tumorigenesis.

Murine Models of Steroid Refractory Graft-versus-Host Disease.

Corticosteroids are the first line therapy for acute graft-versus-host disease (GVHD). However, the outcome of steroid refractory GVHD (SR-GVHD) is poor due to a lack of effective treatments. The development of therapies for SR-GVHD is limited by an incomplete understanding of its pathophysiology partly because of the absence of clinically relevant animal models of SR-GVHD. Here we addressed the need for a SR-GVHD animal model by developing both MHC matched multiple minor histocompatibility antigens (miHAs) mismatched and MHC mismatched haploidentical murine models of SR-GVHD. We demonstrate that animals can develop SR-GVHD regardless of whether steroids are initiated early or late post allogeneic bone marrow transplantation (allo-BMT). In general, we observed increased GVHD specific histopathological damage of target organs in SR-GVHD animals relative to steroid responsive animals. Interestingly, we found no significant differences in donor T cell characteristics between steroid refractory and responsive animals suggesting that donor T cell independent mechanisms may play more prominent roles in the pathogenesis of SR-GVHD than was considered previously.

Microbial metabolite sensor GPR43 controls severity of experimental GVHD.

Microbiome-derived metabolites influence intestinal homeostasis and regulate graft-versus-host disease (GVHD), but the molecular mechanisms remain unknown. Here we show the metabolite sensor G-protein-coupled receptor 43 (GPR43) is important for attenuation of gastrointestinal GVHD in multiple clinically relevant murine models. GPR43 is critical for the protective effects of short-chain fatty acids (SCFAs), butyrate and propionate. Increased severity of GVHD in the absence of GPR43 is not due to baseline differences in the endogenous microbiota of the hosts. We confirm the ability of microbiome-derived metabolites to reduce GVHD by several methods, including co-housing, antibiotic treatment, and administration of exogenous SCFAs. The GVHD protective effect of SCFAs requires GPR43-mediated ERK phosphorylation and activation of the NLRP3 inflammasome in non-hematopoietic target tissues of the host. These data provide insight into mechanisms of microbial metabolite-mediated protection of target tissues from the damage caused allogeneic T cells.

IAPs protect host target tissues from graft-versus-host disease in mice.

Inhibitors of apoptosis proteins (IAPs) regulate apoptosis, but little is known about the role of IAPs in the regulation of immunity. Development of IAP inhibition by second mitochondria-derived activator of caspase (SMAC) mimetics is emerging as a novel therapeutic strategy to treat malignancies. We explored the role of IAPs in allogeneic immunity with 2 distinct yet complementary strategies, namely, chemical and genetic approaches, in clinically relevant models of experimental bone marrow transplantation (BMT). The small-molecule pan-IAP inhibitor SMAC mimetic AT-406 aggravated gastrointestinal graft-versus-host disease (GVHD) in multiple models. The role of specific IAPs in various host and donor cellular compartments was explored by utilizing X-linked IAP (XIAP)- and cellular IAP (cIAP)-deficient animals as donors or recipients. Donor T cells from C57BL/6 cIAP1-/- or XIAP-/- animals demonstrated equivalent GVHD severity and allogeneic responses, both in vivo and in vitro, when compared with B6 wild-type (B6-WT) T cells. By contrast, when used as recipient animals, both XIAP-/- and cIAP1-/- animals demonstrated increased mortality from GVHD when compared with B6-WT animals. BM chimera studies revealed that cIAP and XIAP deficiency in host nonhematopoietic target cells, but not in host hematopoietic-derived cells, is critical for exacerbation of GVHD. Intestinal epithelial cells from IAP-deficient animals showed reduced levels of antiapoptotic proteins as well as autophagy-related protein LC3 after allogeneic BMT. Collectively, our data highlight a novel immune cell-independent but target tissue-intrinsic role for IAPs in the regulation of gastrointestinal damage from GVHD.

A Critical Analysis of the Role of SNARE Protein SEC22B in Antigen Cross-Presentation.

Cross-presentation initiates immune responses against tumors and viral infections by presenting extracellular antigen on MHC I to activate CD8+ T cell-mediated cytotoxicity. In vitro studies in dendritic cells (DCs) established SNARE protein SEC22B as a specific regulator of cross-presentation. However, the in vivo contribution of SEC22B to cross-presentation has not been tested. To address this, we generated DC-specific Sec22b knockout (CD11c-Cre Sec22bfl/fl) mice. Contrary to the paradigm, SEC22B-deficient DCs efficiently cross-present both in vivo and in vitro. Although in vitro small hairpin RNA (shRNA)-mediated Sec22b silencing in bone-marrow-derived dendritic cells (BMDCs) reduced cross-presentation, treatment of SEC22B-deficient BMDCs with the same shRNA produced a similar defect, suggesting the Sec22b shRNA modulates cross-presentation through off-target effects. RNA sequencing of Sec22b shRNA-treated SEC22B-deficient BMDCs demonstrated several changes in the transcriptome. Our data demonstrate that contrary to the accepted model, SEC22B is not necessary for cross-presentation, cautioning against extrapolating phenotypes from knockdown studies alone.

Siglec-G represses DAMP-mediated effects on T cells.

The role of negative regulators or suppressors of the damage-associated molecular pattern-mediated (DAMP-mediated) stimulation of innate immune responses is being increasingly appreciated. However, the presence and function of suppressors of DAMP-mediated effects on T cells, and whether they can be targeted to mitigate T cell-dependent immunopathology remain unknown. Sialic acid-binding immunoglobulin-like lectin G (Siglec-G) is a negative regulator of DAMP-mediated responses in innate immune cells, but its T cell-autonomous role is unknown. Utilizing loss-of-function-based (genetic knockout) and gain-of-function-based (agonist) approaches, we demonstrate that in the presence of certain DAMPs, Siglec-G suppressed in vitro and in vivo T cell responses. We also demonstrate that its T cell-autonomous role is critical for modulating the severity of the T cell-mediated immunopathology, graft-versus-host disease (GVHD). Enhancing the Siglec-G signaling in donor T cells with its agonist, a CD24Fc fusion protein, ameliorated GVHD while preserving sufficient graft-versus-tumor (GVT) effects in vivo. Collectively, these data demonstrate that Siglec-G is a potentially novel negative regulator of T cell responses, which can be targeted to mitigate GVHD.

Genome-Wide STAT3 Binding Analysis after Histone Deacetylase Inhibition Reveals Novel Target Genes in Dendritic Cells.

STAT3 is a master transcriptional regulator that plays an important role in the induction of both immune activation and immune tolerance in dendritic cells (DCs). The transcriptional targets of STAT3 in promoting DC activation are becoming increasingly understood; however, the mechanisms underpinning its role in causing DC suppression remain largely unknown. To determine the functional gene targets of STAT3, we compared the genome-wide binding of STAT3 using ChIP sequencing coupled with gene expression microarrays to determine STAT3-dependent gene regulation in DCs after histone deacetylase (HDAC) inhibition. HDAC inhibition boosted the ability of STAT3 to bind to distinct DNA targets and regulate gene expression. Among the top 500 STAT3 binding sites, the frequency of canonical motifs was significantly higher than that of noncanonical motifs. Functional analysis revealed that after treatment with an HDAC inhibitor, the upregulated STAT3 target genes were those that were primarily the negative regulators of proinflammatory cytokines and those in the IL-10 signaling pathway. The downregulated STAT3-dependent targets were those involved in immune effector processes and antigen processing/presentation. The expression and functional relevance of these genes were validated. Specifically, functional studies confirmed that the upregulation of IL-10Ra by STAT3 contributed to the suppressive function of DCs following HDAC inhibition.