Corticosteroid resistance after acute graft-versus-host disease (SR-aGVHD) results in high morbidity and mortality after allogeneic hematopoietic cell transplantation. Current immunosuppressive therapies for SR-aGVHD provide marginal effectiveness because of poor response or excessive toxicity, primarily from infection. α-Antitrypsin (AAT), a naturally abundant serine protease inhibitor, is capable of suppressing experimental GVHD by downmodulating inflammation and increasing ratios of regulatory (T) to effector T cells (Ts). In this prospective multicenter clinical study, we sought to determine the safety and response rate of AAT administration in SR-aGVHD. Forty patients with a median age of 59 years received intravenous AAT twice weekly for 4 weeks as first-line treatment of SR-aGVHD. The primary end point was overall response rate (ORR), the proportion of patients with SR-aGVHD in complete (CR) or partial response by day 28 without addition of further immunosuppression. Treatment was well tolerated without drug-related adverse events. A significant increase in serum levels of AAT was observed after treatment. The ORR and CR rates by day 28 were 65% and 35%, respectively, and included responses in all aGVHD target organs. At day 60, responses were sustained in 73% of patients without intervening immunosuppression. Infectious mortality was 10% at 6 months and 2.5% within 30 days of last AAT infusion. Consistent with preclinical data, correlative samples showed an increase in ratio of activated Ts to Ts after AAT treatment. These data suggest that AAT is safe and may be potentially efficacious in treating SR-aGVHD. This trial was registered at www.clinicaltrials.gov as #NCT01700036.

No abstract

Recent studies have examined the role of metabolism in pathogenic T cells in various diseases. The metabolic and bioenergetic characteristics of cells targeted by pathogenic T cells, such as gastrointestinal (GI) epithelial cells during immune-mediated disease processes are not known. Herein we explored the role of intestinal epithelial cell (IEC) metabolism on the severity of T cell mediated colitis including graft-versus-host disease (GVHD). We first determined the bioenergetics of IECs following allogeneic hematopoietic cell transplantation (HCT). We utilized the well-characterized MHC disparate BALB/c→B6 GVHD model. Recipient B6 mice were lethally irradiated (10Gy), transplanted with 5×106 BM and 3x106 splenic T cells from either syngeneic B6 or allogeneic BALB/c donors. CD326+ IECs from recipient animals were harvested on day 7 and 21 post-HCT and assessed for their bioenegetics utilizing the Seahorse analyzer. The allogeneic IECs (allo-IECs) demonstrated dramatically reduced oxygen consumption rates (OCR) but similar extracellular acidification rates (ECAR), and increased OCR/ECAR ratios compared with syngeneic IECs (syn-IECs) (OCR; 44.70 vs 110 pmol/min, P<0.0001, ECAR; 34.43 vs 40.72 mpH/min, P=0.1832, OCR/ECAR; 1.12 vs 2.836, P<0.01). Because these data indicated profound defects in mitochondrial oxidative pathways, we next profiled mitochondrial tricarboxylic acid (TCA) cycle metabolite composition in IECs and kidney (as non-GVHD controls) harvested from naïve, syngeneic and allogeneic animals (day 7 and 21 post-HCT) in an unbiased and blinded manner with gas chromatography-mass spectrometry (GC-MS). Among the TCA cycle metabolites, only succinate significantly increased in IECs, but not kidneys, from allogeneic recipients (25.6 ± 5.65vs 0.00µM/µg protein, P<0.05). To determine the reasons for high levels of succinate, we next performed metabolic flux studies with 13C-glucose and 13C-glutamine, and found that the increased succinate levels were not secondary to anaplerosis. We therefore next hypothesized that the accumulated succinate in IECs was from reduced succinate dehydrogenase A (SDHA), a component of mitochondria complex II (MC II), that converts succinate to fumarate. Consistent with this hypothesis, SDHA was significantly decreased in allo-IECs compared to syn-IECs (P<0.05), which was additionally validated in a blinded manner by immunofluorescence (Figure), immunohistochemistry and immuno-gold staining of mitochondria by electron microscopy. Analysis of IECs harvested from recipients following non-irradiated B6→B6D2F1, chemotherapy conditioned BALB/c→B6, and irradiated MHC matched mHA mismatched C3H.SW→B6 GVHD models, as well as in a CD4+CD45RBhigh T-cell→RAG1−/− model of inflammatory bowel disease (IBD), and an anti-CTLA-4 antibody augmented DSS colitis model (checkpoint-inhibitor colitis), all demonstrated reduction of MC II, SDHA, and an increase in the level of succinate. We next explored the in vivo functional relevance of SDHA in GI GVHD utilizing multiple approaches. Specifically, when Sdhaf1−/− B6 (30% of SDHA activity, P<0.01), bone marrow chimera (WT B6→Sdhaf1−/−, P<0.05) or WT animals treated with multiple chemical SDH inhibitors from day 0 to 21 post allo-HCT (itaconate; 2.5g/kg, P<0.0001, malonate; 5g/kg, P<0.001 or Atpenin A5; 9µg/kg, P<0.05) were used as recipients, all demonstrated significantly greater GI GVHD and mortality compared with allo-control animals. We furthermore generated IEC specific SDHA KO mice (villin-Cre+SDHAfl/fl), which when utilized as allogeneic recipients, they demonstrated significantly greater mortality and GI GVHD (P<0.0001). Cellular and biochemical mechanistic studies demonstrated the requirement of T cell contact with IECs, and a critical role for perforin (Prf)/ granzyme B (GB) in the breakdown and reduction of SDHA. The mechanisms for SDHA reduction were validated in vivo by transferring Prf−/− T cells into allogeneic BALB/c (GVHD) or RAG1−/−B6 (IBD) mice. Our data demonstrate that MC II component SDHA in IECs is a critical metabolic checkpoint that regulates severity of intestinal colitis caused by pathogenic allo- or auto-reactive T cells and thus provide seminal insights into GI GVHD, IBD and checkpoint-inhibitor colitis. Disclosures No relevant conflicts of interest to declare.

T cells play critical roles in both protective and pathogenic adaptive immune responses. The release of cytokines by T cells is critical for their development, differentiation, and effector functions. However, significant gaps remain in our understanding of the fundamental cellular and molecular pathways that are critical for T cell cytokine release. Coat Protein Complex II (COPII) enables protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus. It consists of an outer layer that facilitates the budding of vesicles from the ER membrane, and an inner layer responsible for selecting cargo proteins that comprises the protein SEC23B. The critical ER-Golgi pathway in T cell cytokine release is not known. Therefore, to explore the role of COPII secretory pathways, we generated novel Sec23bfl/-Cd4Cre mice that will lack Sec23b in T cells. We hypothesized that SEC23B-mediated COPII deficiency will lead to abnormalities in T cell development given the critical roles of T cell-derived cytokines in their ontogeny. Contrary to our hypothesis, SEC23B-deficient T cells developed normally, and were present in similar numbers in the thymus (p=0.20), lymph nodes (p=0.69), and spleen (p=0.10) as WT T cells. They also showed normal levels of surface phenotypic markers (TCR, CD25, CD44, CD62L, CD28). To rule out potential Cre-recombinase driven artifacts we next generated Sec23bfl/-Vav1Cre mice and found that T cell development was not affected. We explored the functions of mature fully developed T cells by stimulating them with aCD3/aCD28 for 3 days. Compared to WT T cells, SEC23B-deficient T cells demonstrated decreased release of cytokines such as IL-2, IL-7, and IFNg (all p<0.01), but not others, such as IL-17 and IL-10. This selective reduction in cytokine secretion was not due to defective T cell signaling events (such as phosphorylation of ZAP-70, LCK, AKT, ERK1/2) or production of cytokines at the mRNA level (including IL-2, IFNg, IL-10). However, the altered cytokine levels in the supernatant was associated with decreased proliferation by SEC23B-deficient T cells compared to WT when measured by CFSE (p=0.0003). We therefore hypothesized that SEC23B-deficient T cells were able to produce cytokines, but unable to release them selectively. To test this, we used Brefeldin A (BFA), an inhibitor of anterograde transport from the ER to the Golgi apparatus. In the presence of BFA, intracellular levels of IL-2 and IL-17 were similar in WT and SEC23B-deficient T cells (p=0.86). Collectively, these data suggest SEC23B-deficient T cells exhibit secretory defects of select cytokines upon stimulation compared to WT T cells. We next explored the in vivo relevance of SEC23B-dependent COPII vesicle formation in two clinically relevant models. We first tested the impact of disrupting Sec23b on in vivo protective functions of T cells in a model of viral immunity by utilizing the well-characterized LCMV Armstrong model of acute infection. Sec23b WTor Sec23bfl/-Cd4Cre mice were infected with 2x105 p.f.u. of LCMV Armstrong i.p., and T cells were analyzed on day 8 post-infection. In contrast to WT mice, Sec23bfl/-Cd4Cre mice demonstrated impaired ability to clear the virus by day 8 (9.1x107 p.f.u/gm vs N.D., p=0.0008), reduced expansion of viral antigen-specific cells (p=0.0001 for gp33, gp66, p=0.0006 for gp276), and reduced numbers of IFNg (p=0.0004) and TNFa (p=0.0003) producing T cells on day 8 post-infection. We next determined the pathogenic capacity of SEC23B-deficient T cells by utilizing a well-established MHC-mismatched C57BL/6->BALB/c model of Graft-versus-Host Disease (GVHD). BALB/c and C57BL/6 recipients were lethally irradiated (8.5 Gy) and transplanted with 5x106 allogeneic C57BL/6 WT bone marrow cells, and either 2x106 C57BL/6 WT or SEC23B-deficient T cells. All syngeneic recipients survived. By contrast, 100% of the recipients of allogeneic WT T cells died by day 61 due to GVHD. However, only 6% of allogeneic recipients of SEC23B-deficient T cells died (n=18/group, p<0.0001) and all recipients showed lower GVHD clinical scores compared to recipients of WT T cells (p<0.01 to 0.001 over 6 weeks). Collectively, our data demonstrate a heretofore unrecognized but critical role for SEC23B-dependent COPII in T cell protective and pathogenic effector functions and thus provide fundamental insights into T cell-mediated immunity. Disclosures Ginsburg: Portola pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Shire plc: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

In immune mediated diseases such as graft-versus-host disease (GVHD), research has focused almost entirely on hematopoietic cells. It has recently been posited that mechanisms within target tissues also play a significant role in regulating the severity of disease. Autophagy protects cells from a variety of stressors and is known to affect immune cell responses after allogeneic bone marrow transplant (allo-BMT). However, a definitive and exclusive role of target tissue pathways on mitigating GVHD has heretofore not been demonstrated. Because GVHD target organs are under stress from alloreactive T cells, we sought to determine the role of target tissue autophagy in mitigating the severity of GVHD. We hypothesized that the stress from alloreactive T cells would result in upregulation of target tissue autophagy. To test this in vivo, we utilized LC3-GFP-RFP reporter mice as transplant recipients in the well-characterized MHC disparate GVHD murine model BALB/c→B6. In these reporter mice, autophagosomes are marked with a dual GFP-RFP tag, and can be assessed for in vivo induction of autophagy by confocal microscopy through quantification of red punctal abundance. The recipient B6 reporter mice were lethally irradiated with split doses of 5Gy and transplanted with 5x106 BM and 3x106 splenic T-cells from either syngeneic B6 (syn) or allogeneic BALB/c (allo) donors. We assessed for induction of autophagy in GVHD target organs (gut, liver) and non-target organs (kidney, heart) and observed that autophagy was induced to a greater extent in target organs. Because the primary driver of morbidity and mortality after allo-BMT is gastrointestinal (GI) GVHD, we next hypothesized that autophagy protects the GI epithelium from alloimmune damage. To determine the effects of autophagy exclusively in the GI tract, we generated mice that lack a macroautophagy protein, ATG5, in the GI epithelium only (Atg5fl/flVil1-Cre) and utilized them as recipients in BALB/c→B6 allo-BMT. As hypothesized, survival of allo Atg5fl/flVil1-Cre mice was significantly reduced (P<0.0001) compared to littermate controls. Importantly, all syn Atg5 KO mice survived the entire period of observation, demonstrating that protection from conditioning regimen toxicity did not require ATG5-dependent autophagy in the gut. We harvested both GVHD target and non-target organs and performed histopathological analysis at day 7 post-transplant and found that histological GVHD severity in the gut was significantly higher in allo Atg5fl/flVil1-Cre mice compared to Cre-null WT littermate controls (P=0.0063). All other organs were unaffected by loss of ATG5 in the GI epithelium, and we observed no difference between WT and Atg5 KO syn recipients in any organ. We harvested splenocytes and tissue-resident lymphocytes from the gut and found similar numbers of donor CD4 and CD8 T cells, activation markers CD69 and CD62L, and IFNγ and TNFα positive cells. Furthermore, similar levels of the proinflammatory serum cytokines IFNγ, TNFα, and IL-6 were observed in the sera of WT and Atg5 KO mice. We next determined whether autophagy is critical for protection in other GVHD target organs after allo-BMT. To this end, we generated Atg5fl/flAlb-Cre mice, which lack Atg5 expression in hepatocytes. Using a similar model of acute GVHD as above, we observed dramatically shorter survival in the Atg5fl/flAlb-Cre allo recipients (P=0.025) along with an increase in the hepatocyte damage markers bilirubin (P=0.0001), alanine aminotransferase (P=0.036), and alkaline phosphatase (P=0.0002) in Atg5 KO allo recipients. As before, there were similar numbers of splenic and liver-resident donor T cells, activation markers, and proinflammatory cytokines. To determine if autophagy is exclusively protective in GVHD target organs, we next generated Atg5fl/flMyh6-Cre mice, which lack Atg5 expression in cardiac myocytes, and Atg5fl/flAlb-Cre mice, which lack Atg5 expression in podocytes, as a control for lack of ATG5 in non-GVHD target organs. Using a similar GVHD model as above, we monitored these mice and littermate controls for survival and severity of GVHD symptoms. In contrast to our findings in the liver and gut, we observed similar survival (P=0.17, Myh6; P=0.49, Nphs2) and GVHD severity between allo recipients. Collectively, our results demonstrated that autophagy is a tissue-intrinsic protective response that regulates disease severity in GVHD target organs. Disclosures No relevant conflicts of interest to declare.