ALBERT

Description: Abstract 817 Strategies to suppress GVHD are often associated with broader suppression of the immune system leading to a compromised GVT effect. Using experimental models, we have demonstrated a novel strategy to enhance GVT effects and explicitly suppress GVHD using genetically engineered T lineage cells over-expressing TNF-Related Apoptosis Inducing Ligand (TRAIL). TRAIL can induce apoptotic signals through death receptor (DR) 4 and 5 molecules (only DR5 in mice) expressed on target cells. Expression of DR5 is higher on certain tumors and can be enhanced on others using small molecules rendering them susceptible to TRAIL mediated killing. TRAIL is therefore an attractive candidate for genetic engineering of donor T cells to enhance their GVT potential. We evaluated the effect of TRAIL over-expression (TRAIL+) in donor T cells (mature and precursor) on GVHD and GVT. Mature T cells derived from donor B6 splenocytes were transduced with a lentiviral TRAIL expression vector. The transduced TRAIL+ T cells were adoptively transferred on day 0 into lethally irradiated CBF1 recipients of T cell depleted allografts and LB27.4 tumor (B6 ^ CBF1+LB27.4) to assess their GVHD and GVT activity. TRAIL+ T cells displayed significantly enhanced antitumor immunity compared to T cells transduced with a control vector against LB27.4 tumor cell lines in vitro and upon transfer into tumor bearing allo-BMT recipients (p

Description: The success of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is limited by acute graft-versus-host disease (GVHD). Improving the procedure depends on identifying the mechanisms that contribute to this damaging T cell reactivity, while preserving graft-versus-leukemia (GVL) activity against hematopoietic malignancies. "Tonic" type I IFN signaling in BMT recipients and therapeutic application of recombinant IFN-α have been shown to play an important role in defining the balance between GVHD and GVL responses, but the molecular mechanisms inducing this protective response remains unknown. In this regard, pattern recognition receptors (PRRs) that detect cytosolic nucleic acids and lead to the production of large amounts of type I interferons (IFN-α/β) such as the family of RIG-I-like receptors (RLRs) are of particular interest. RLRs, a subfamily of the cytoplasmic DExD/H- box family of helicases, consist of three members: retinoic acid inducible gene I (RIG-I), melanoma differentiation factor 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2). RIG-I senses viral and bacterial RNA to induce the production of type I interferons, proinflammatory cytokines and inflammasome activation. Double-stranded RNA (dsRNA) carrying a 5'-triphosphate (3pRNA) has been identified as the natural ligand for RIG-I and serves as a selective trigger for RIG-I signaling. Although initially characterized as a main regulator for antiviral host defense, mice deficient in components of the RLR and type I IFN signaling pathway develop inflammatory bowel disease (IBD)-related pathologies. Furthermore, patients suffering from IBD show a highly significant downregulation of RIG-I in ileal epithelial cells and in a recent meta-analysis of genome-wide association studies (GWAS) data, IFNAR1 and MDA-5 were identified as primary candidate genes in susceptible loci for IBD. Together, these results indicate that RLRs and type I IFN signaling have important functions in the suppression of IBD by yet ill defined mechanisms. Given that the pathophysiology of GVHD shares several features with inflammatory bowel disease (IBD), we tested the role of the RIG-I pathway in the context of allo-HSCT. We utilized MAVS-deficient mice, which lack a common adapter for RIG-I signaling, as recipients in an MHC-disparate (BALB/c into B6) model of allo-HSCT. Compared to wild-type (WT) B6 mice, MAVS-KO mice receiving allogeneic BM + T cells displayed significantly worse GVHD mortality (Fig.1 A). Given the enhanced GVHD observed in the absence of RIG-I signaling, we hypothesized that selective engagement of RIG-I by 3pRNA (RIG-I ligand) in vivo would protect recipients from GVHD. Using a B6 into BALB/c model we observed that i.v. administration of a selective RIG-I ligand on d-1 significantly reduced mortality, weight loss and intestinal GVHD histopathology (Fig. 1B and data not shown). In addition, the translocation of LPS and microorganisms from the bowel lumen through the damaged intestinal mucosa to the systemic circulation during pre-transplant conditioning represents a crucial step in GVHD pathophysiology. We observed that administration of RIG-I ligand prior to allo-HSCT augmented intestinal barrier function measured by less fluorescence in the serum after FITC-dextran gavage compared to untreated WT recipients (Fig. 1C). Moreover, RIG-I stimulation augmented epithelial regeneration as determined by organoid formation from freshly isolated crypts (Fig. 1D) and inhibited activation of dendritic cells (DCs) during pre-transplant conditioning (Fig. 1E). To investigate the impact of 3pRNA administration during GVT, we used luciferase+ A20 bioluminescence in B6 into BALB/c tumor challenge recipients demonstrating that RIG-I ligands do not limit GVL (data not shown). Taken together, our results (i) uncover a previously unknown role of the RIG-I-MAVS signaling pathway in GVHD and (ii) offer a novel strategy to foster epithelial regeneration and inhibit antigen presentation during pre-transplant conditioning, while maintaining GVL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Graft versus host disease (GVHD) remains a major limitation of allogeneic hematopoietic stem cell transplantation (allo-HSCT), and gut GVHD specifically is a major cause of GVHD-related morbidity and mortality. Little is known about regulation of the intestinal stem cell (ISC) compartment in gut GVHD. We have found that Interleukin-22 (IL-22) produced by innate lymphoid cells is important for ISC recovery after transplant. However, the mechanism of action and specific cellular targets of IL-22 leading to ISC recovery are poorly understood. Using clinically modeled LP into C57BL/6 (B6) minor antigen-mismatched HSCT (H-2 into H-2b), we found that daily treatment with recombinant murine (rm)IL-22 (4ug, intraperitoneal injection) starting day seven after transplant led to reduced intestinal pathology from GVHD without altering alloreactive immunity. Both overall GVHD pathology and epithelial apoptosis scores were significantly lower three weeks post-BMT in rmIL-22-treated mice with GVHD compared to PBS-treated controls (p

Description: Highly diverse bacterial populations inhabit the gastrointestinal tract, modulate host inflammation and promote immune tolerance. In allogeneic hematopoietic stem cell transplantation (allo-HSCT), the gastrointestinal mucosa is damaged, and colonizing bacteria are impacted, leading to an impaired intestinal microbiota with reduced diversity. Because bacteremia is a frequent complication of allo-HSCT, we examined the microbiota of patients undergoing allo-HSCT and correlated microbial shifts with the risk of bacteremia. Fecal specimens were collected longitudinally from 94 patients undergoing allo-HSCT from pre-transplant until 35 days post-transplantation. The intestinal microbiota was characterized by 454 pyrosequencing of the V1-V3 region of bacterial 16S rRNA genes. Phylogenetic classification was obtained using the Ribosomal Database Project (RDP) classifier. Associations of the microbiota with clinical predictors and outcomes were evaluated. During allo-HSCT, patients developed reduced diversity, with marked shifts in bacterial populations inhabiting the gut. Intestinal domination, defined as occupation of at least 30% of the microbiota by a single predominating bacterial taxon, occurred frequently. Commonly encountered dominating organisms included Enterococcus, Streptococcus, and various Proteobacteria. Enterococcal domination was increased three-fold by metronidazole administration, while domination by Proteobacteria was reduced ten-fold by fluoroquinolone administration. As a predictor of outcomes, enterococcal domination increased the risk of vancomycin resistant enterococcus bacteremia nine-fold, and proteobacterial domination increased the risk of gram negative rod bacteremia five-fold. We also examined whether intestinal diversity was predictive of clinical outcomes following transplantation. Subjects were classified into high, intermediate, and low bacterial diversity groups, and assessed for up to three years for differences in overall survival and transplant-related mortality. Overall survival was worse in subjects with low intestinal diversity, compared with intermediate or high diversity (at three years, 36%, 60%, and 67%, respectively; log-rank P=0.019); similar results were observed for transplant-related mortality. Multivariate Cox hazards analysis demonstrated a persistent association after adjusting for other clinical covariates. In conclusion, during allo-HSCT, the diversity and stability of the intestinal flora are disrupted, resulting in domination by bacteria associated with subsequent bacteremia. Assessment of fecal microbiota identifies patients at highest risk for bloodstream infection during allo-HCST. Diversity of the intestinal microbiota is also an independent predictor of mortality in allo-HSCT recipients following engraftment. These results indicate that the intestinal microbiota is an important factor in the success or failure in allo-HSCT. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1043 Thymopoiesis is a highly complex process involving cross-talk interactions between developing thymocytes and the supporting non-hematopoietic stromal microenvironment, which includes highly specialized thymic epithelial cells (TECs). Paradoxical to its importance for continually generating a diverse repertoire for effective adaptive immunity, the thymus undergoes profound atrophy with age. Age-related thymic involution is characterized by severe structural dysregulation of the supporting epithelial microenvironment (and in humans linked to a buildup of fatty tissue), reduced thymopoiesis, and subsequently reduced export of na•ve lymphocytes into the periphery. Together this degeneration in thymic function significantly narrows the T cell receptor repertoire and can causally linked to increased infection, autoimmunity and malignancy. Moreover, progressive thymic involution can also be a considerable hindrance to the regeneration of adaptive immunity following cytoreductive treatments such as chemotherapy or the conditioning required for successful hematopoietic stem cell transplant. Despite considerable work, little is understood about the underlying causes of age-related thymic involution. We have recently demonstrated a novel role for interleukin-22 (IL-22), a recently identified cytokine predominantly associated with maintenance of barrier function at mucosal surfaces, in endogenous thymic regeneration from acute immune injury. Our studies suggested that 1) the depletion of DP thymocytes triggers, 2) upregulation of IL-23 by dendritic cells (DCs), which induces 3) the production of IL-22 by intrathymic innate lymphoid cells (ILCs). IL-22 promotes the proliferation and survival of TECs, therefore this cascade of events leads to regeneration of the supporting microenvironment and, ultimately, to rejuvenation of thymopoiesis. Given these recent findings demonstrating a role for IL-22 in endogenous thymic regeneration following acute immune injury, one hypothesis would be that a breakdown in the IL-22 pathway contributes towards chronic age-related thymus involution. However, in contrast to this initial hypothesis, our studies revealed that rather than being depleted with age, there was actually a significant increase in the level of intrathymic IL-22 in aged (18+ months old) compared to young (2 months old) mice (Figure 1a). These findings highlighted that, in addition to being triggered by the depletion of CD4+CD8+ double positive thymocytes during acute immune injury, the IL-22 regenerative pathway can also be activated by the chronic atrophy that is a hallmark of age-related thymic involution. Similar to our findings in models of thymic injury in young mice, we found that these increased levels of IL-22 with age were predicated on the increased production of IL-22 by thymic innate lymphoid cells (Figure 1b). Moreover, in keeping with our findings in young mice with acute thymic injury, intrathymic levels of IL-22 in aged mice correlated with those of IL-23 - production of which by dendritic cells was significantly increased with age (Figure 1c). As predicted by this increase in the production of IL-22 with age, TECs from aged mice displayed all the hallmarks of increased IL-22 signaling including increased expression of the IL-22 receptor (Figure 1d) as well as increased phosphorylation of STAT-3 (Y705) (Figure 1e). However, although in vitro incubation of aged TECs with IL-22 led to increased proliferation, consistent with our findings in young mice, in vivo analysis revealed significantly reduced proliferation among TECs in aged mice (Figure 1f), as has been previously reported. Given the role for inflammasome components in mediating thymic involution, it is possible that although endogenous regenerative pathways are triggered with age (in the case of IL-22 likely due to the depletion of DP thymocytes), these regular processes fail in the face of an overwhelming inflammatory milieu in the thymus with age. Although further studies need to elucidate the specific inhibitory interactions constraining thymic regeneration, it is clear that strategies harnessing these endogenous pathways for enhancing immunity in the aging thymus first need to overcome these negative stimuli for effective regeneration. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 143 Despite being exquisitely sensitive to insult, the thymus is remarkably resilient in young healthy animals. Endogenous regeneration of the thymus is a crucial function that allows for renewal of immune competence following infection or immunodepletion caused by cytoreductive chemotherapy or radiation. However, the mechanisms governing this regeneration remain poorly understood. Thymopoiesis is a highly complex process involving cross-talk between developing thymocytes and their supporting non-hematopoietic stromal microenvironment, which includes highly specialized thymic epithelial cells (TECs) that are crucial for T cell development. IL-22 is a recently identified cytokine predominantly associated with maintenance of barrier function at mucosal surfaces. Here we demonstrate for the first time a critical role for IL-22 in endogenous thymic repair. Comparing IL-22 KO and WT mice we observed that while IL-22 deficiency was redundant for steady-state thymopoiesis, it led to a pronounced and prolonged loss of thymus cellularity following sublethal total body irradiation (SL-TBI), which included depletion of both thymocytes (p=0.0001) and TECs (p=0.003). Strikingly, absolute levels of IL-22 were markedly increased following thymic insult (p

Description: Abstract 1468 Allogeneic hematopoietic stem cell transplant (allo-HSCT) is one of the only curative therapies for leukemia patients and others with hematological malignancies. Its use, however, is restricted by several major complications, including graft versus host disease (GVHD) and post-transplant immune deficiency. Previous studies have found that GVHD can cause significantly reduced hematopoiesis and significant loss of hematopoietic stem and progenitor cells within the BM niche. Moreover, post-transplant immune deficiency, which is especially devastating to the T cell compartment, occurs independently of GVHD in autologous HSCT as well as following immunodepletion from chemotherapy and/or radiation therapy. This profound immune deficiency may be long-lasting and further exacerbated by GVHD that may be targeting hematopoietic reconstitution. Here we demonstrate that in both minor and major-mismatched models of GVHD there is significant depletion in the BM of hematopoietic stem cells (p

Description: Abstract 451 Chimeric antigen receptors (CAR) represent a potent strategy to target T cells against selected tumor antigens. Ongoing clinical trials indicate that autologous T cells expressing CARs targeting CD19, a B cell-associated antigen, can induce complete remission and B cell aplasia in patients with B cell malignancies. Donor CD19-CAR+ T cells could potentially be used to treat recipients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT), but the risk of alloreactivity mediated by endogenous T cell receptors (TCR) triggering an acute GVHD is not known. This is partly due to the absence of in vivo models to study the relative effects of CAR and endogenous TCR signaling. For the first time, we have evaluated the relative effects of CD19-targeted donor T cells on the elimination of CD19+ B cells and endogenous TCR-mediated alloreactivity in mouse models of allo-HSCT. We generated a panel of retroviral vectors encoding mouse CD19-specific CARs: as a control, CD19-delta, a tail-less CAR lacking the CD3ζ signaling domain; CD19z1, which signals through its CD3ζ endodomain; and CD19-28z, which signals through CD28 and CD3ζ (Figure 1A). CD19z1+ and CD19-28z+ T cells mediated specific lysis of CD19-expressing tumors in vitro, while CD19-delta+ T cells did not. In order to assess the anti-tumor capacity of CD19-CAR+ T cells in vivo, we transferred the transduced B6 donor T cells into lethally irradiated BALB/c recipients that were administered T cell-depleted allografts and CD19+ lymphoma A20-TGL (B6–〉 BALB/c+A20-TGL). CD19-CAR+ T cells (CD19z1 and CD19-28z) mediated clearance of A20 tumor cells visualized by in vivo imaging of luciferase-expressing tumor cells (Figure 1B and data not shown) and significantly improved tumor free survival. CD19-CAR+ B6 T cells could sustain prolonged B cell hypoplasia when adoptively transferred into lethally irradiated haploidentical CBF1 recipients of T cell-depleted allografts (B6–〉 CBF1, Figure 1C). These data indicate that under alloreactive conditions, donor CD19-CAR+ T cell signaled through the CAR leading to specific elimination of CD19+ tumors and B lineage cells. In order to determine the risk of GVHD, we transferred the donor CD19-CAR+ T cells into haploidentical HSCT recipients. Interestingly, CD19-CAR+ T cells mediated significantly less acute GVHD, resulting in improved survival and lower GVHD scores (Figure 1D). Donor CD19-delta+ T cells however mediated lethal GVHD, indicating that the endogenous TCR mediated strong alloreactivity in the absence of CAR signaling. Similar results were obtained from experiments using MHC-mismatched (B6–〉 BALB/c) models. It is known that signaling through endogenous TCR is accompanied by down-regulation of surface TCR expression. We found significant decreases in surface CD3ϵ, TCRβ and CD90 expressions in donor CD19-delta+ T cells under alloreactive conditions. In contrast, donor CD1928z+ T cells failed to down-regulate surface TCR expression under similar conditions, suggesting that endogenous TCR function was altered in CAR-activated T cells. In the context of allo-HSCT, preferential CAR signaling at the expense of alloreactive endogenous TCR signaling may thus lead to reduced alloreactivity and attenuation of GVHD. These results provide the first pre-clinical evidence suggesting that CAR-modified, unselected donor T cells may be safely applied in an allogeneic context. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract SCI-51 Regulators of the intestinal flora include diet, antibiotics, and importantly, intestinal inflammation. As a result, the cause-effect relationships between intestinal inflammation and changes in microbiota have been difficult to define. The success of allogeneic bone marrow transplantation (allo BMT), a standard therapy for conditions including hematopoietic malignancies and inherited hematopoietic disorders, is limited by graft-versus-host disease (GVHD) morbidity and mortality. With GVHD, vigorous activation of donor immune cells, most importantly T cells, leads to damage of the skin, liver, hematopoietic system, and gut. The major sources of immune activation are histocompatibility complex differences between donor and recipient. Combinations of chemotherapy and radiation also contribute, as damage to the intestinal epithelium results in systemic exposure to microbial products normally sequestered in the intestinal lumen. We have demonstrated in murine and human recipients of allo BMT that intestinal inflammation secondary to GVHD is associated with major shifts in the composition of the intestinal microbiota. The microbiota, in turn, can modulate the severity of intestinal inflammation. In mouse models of GVHD we observed loss of overall diversity and expansion of Lactobacillales and loss of Clostridiales. Eliminating Lactobacillales from the flora of mice prior to BMT aggravated GVHD, while reintroducing the predominant species of Lactobacillus mediated significant protection against GVHD. We then characterized the gut flora of patients during onset of intestinal inflammation due to GVHD and found patterns mirroring those in mice. We also identified increased microbial chaos soon after allo BMT as a potential risk factor for subsequent GVHD. Together, these data demonstrate regulation of flora by intestinal inflammation, and suggest that flora manipulation may reduce intestinal inflammation and improve outcomes for allo BMT recipients. Disclosures: No relevant conflicts of interest to declare.

Description: A relationship between the microbiota and GVHD has long been suspected but is still not well understood. Recently, several studies have indicated that obligately anaerobic commensal intestinal bacteria may be beneficial for intestinal health. Recent studies have found that obligate anaerobes in the intestine, in particular Clostridial species, are important mediators of intestinal homeostasis and prevent inflammation by upregulating intestinal regulatory T cells (Tregs). In this study we sought to further characterize the relationship between the microbiota and GVHD, focusing on the effects of antibiotics (ABX) with anaerobic coverage. First, we treated healthy 129S1 mice with either ABX that included significant activity against anaerobic bacteria (piperacillin-tazobactam; pip-tazo, imipenem-cilastatin; imipenem or ones with reduced activity (cefepime, aztreonam). Mice were treated by subcutaneous injections of each antibiotic twice a day for two days (500 mg/kg for pip-tazo and 100 mg/kg for others) and stool samples were collected, followed by 16S rRNA gene amplification and sequence analysis. We found that treatment with pip-tazo and imipenem significantly reduced the abundance of anaerobic Clostridial species and increased that of Enterococcus, while treatment with cefepime and aztreonam spared Clostridiales (Figure 1A). We next investigated the effects of antibiotic treatment in a clinically relevant MHC matched, minor antigen mismatched murine allogeneic BMT model. Lethally irradiated 129S1 recipients were transplanted with C57BL/6 T cell depleted bone marrow cells and 2 × 106 C57BL/6 T cells. Recipients were either treated with imipenem or aztreonam subcutaneously beginning on day +10 after BMT. We observed significantly worsened GVHD survival in imipenem-treated recipients (Figure 1B, P