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PDF file 96K, Pre-treatment of donor T-cells with a c-Rel antagonist promotes IL-2 secretion in vivo

PDF file 128KB, Treg depletion from c-Rel deficient donor T-cells results in exacerbated GVHD

PDF file 92K, Table 1. Characteristics of c-Rel inhibitor compounds; Table 2. Roles of c-Rel in T-cell responses and transplantation immunology

PDF file 135KB, Non-specific negative effect of DMSO on the GVT activity of T-cells

PDF file 128KB, c-Rel-deficient T-cells are polarized towards Th2 responses during GVHD

PDF file 191K, Recipients of c-Rel-deficient T-cells exhibit reduced damage of GVHD target organs

PDF file 121K, IL-2 secretion is enhanced in irradiated recipients transplanted with allogeneic T-cells

Supplementary S1: PLZF expression affects CD4+ driven alloreactivity. Supplementary S2: PLZF-TG T cells undergo allo-activation that is similar to donor B6-WT T cells. Supplementary S3: Fewer deaths from GVHD are seen in BMT recipients of PLZF-TG T cells compared to the BMT recipients of B6-WT T cells.

Background: The plantar plate is a major stabilizing structure of the metatarsophalangeal (MTP) joint with instability frequently occurring after a tear or attenuation of this structure. Commonly, a McGlamry elevator is used to strip the plantar plate from the plantar surface of the metatarsal to improve exposure of the MTP joint. The anatomy of the proximal plantar plate and vascular consequence of stripping the plantar plate from the metatarsal is not yet well understood. The purpose of this study is to describe the proximal attachment of the plantar plate anatomically and quantify the relative contribution of blood supply to the proximal plantar plate from both the metatarsal and the plantar fascia. Methods: For anatomic evaluation, 6 lower extremity cadaver specimens without any gross evidence of foot and ankle deformity were utilized. For imaging analysis, 16 fresh frozen human adult cadaveric lower extremity specimens were used for this study, resulting in 35 MTP joints without deformity and 11 lesser MTP joints with cockup and/or crossover deformities. The specimens were prepared as described previously by Finney et al.5 Results: From gross anatomic dissection, the plantar plate origin consists of a stout fibrous pedicle distinct from the surrounding synovial-type tissue that firmly anchors the plantar plate to the metatarsal. Based on nano–computed tomographic imaging, an average of 63.5% of the vascular supply to the proximal portion of the plantar plate entered from the metatarsal pedicle. The remaining 36.5% of the vascular supply entered from the plantar fascia. Conclusion: The proximal attachment of the plantar plate includes a stout fibrous pedicle anchoring the proximal portion of the plantar plate to the notch between the medial and lateral plantar condyles of the metatarsal head. The vascular supply of the proximal plantar plate is supplied from both the metatarsal pedicle and plantar fascia. Level of Evidence: Level III, retrospective comparative study.

SummaryAtg16L1 mediates the cellular degradative process of autophagy and is considered a critical regulator of inflammation based on its genetic association with inflammatory bowel disease. Here we find that Atg16L1 deficiency leads to an exacerbated graft-versus-host disease (GVHD) in a mouse model of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Atg16L1-deficient allo-HSCT recipients with GVHD displayed increased T cell proliferation due to increased dendritic cell (DC) numbers and costimulatory molecule expression. Reduced autophagy within DCs was associated with lysosomal abnormalities and decreased amounts of A20, a negative regulator of DC activation. These results broaden the function of Atg16L1 and the autophagy pathway to include a role in limiting a DC-mediated response during inflammatory disease, such as GVHD.

Preventing unfavorable GVHD without inducing broad suppression of the immune system presents a major challenge of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We developed a novel strategy to ameliorate GVHD while preserving graft-versus-tumor (GVT) activity by small molecule–based inhibition of the NF-κB family member c-Rel. Underlying mechanisms included reduced alloactivation, defective gut homing, and impaired negative feedback on interleukin (IL)-2 production, resulting in optimal IL-2 levels, which, in the absence of competition by effector T cells, translated into expansion of regulatory T cells. c-Rel activity was dispensable for antigen-specific T-cell receptor (TCR) activation, allowing c-Rel–deficient T cells to display normal GVT activity. In addition, inhibition of c-Rel activity reduced alloactivation without compromising antigen-specific cytotoxicity of human T cells. Finally, we were able to demonstrate the feasibility and efficacy of systemic c-Rel inhibitor administration. Our findings validate c-Rel as a promising target for immunomodulatory therapy and demonstrate the feasibility and efficacy of pharmaceutical inhibition of c-Rel activity. Significance: Chemical inhibition of c-Rel diminishes alloactivation while preserving antigen-specific TCR activation, revealing the redundancy of c-Rel in T cell–mediated antitumor activity of both mouse and human T cells. Our study provides a highly innovative immunomodulatory approach that has true potential for drug development and clinical application with broad therapeutic implications, including allo-tolerance induction after allo-HSCT, as well as antitumor therapies. Cancer Discov; 4(5); 578–91. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 495

Velardi et al. show that sex steroids regulate thymopoiesis by directly modulating Notch signaling, and provide a novel clinical strategy to boost immune regeneration., Paradoxical to its importance for generating a diverse T cell repertoire, thymic function progressively declines throughout life. This process has been at least partially attributed to the effects of sex steroids, and their removal promotes enhanced thymopoiesis and recovery from immune injury. We show that one mechanism by which sex steroids influence thymopoiesis is through direct inhibition in cortical thymic epithelial cells (cTECs) of Delta-like 4 (Dll4), a Notch ligand crucial for the commitment and differentiation of T cell progenitors in a dose-dependent manner. Consistent with this, sex steroid ablation (SSA) led to increased expression of Dll4 and its downstream targets. Importantly, SSA induced by luteinizing hormone-releasing hormone (LHRH) receptor antagonism bypassed the surge in sex steroids caused by LHRH agonists, the gold standard for clinical ablation of sex steroids, thereby facilitating increased Dll4 expression and more rapid promotion of thymopoiesis. Collectively, these findings not only reveal a novel mechanism underlying improved thymic regeneration upon SSA but also offer an improved clinical strategy for successfully boosting immune function.

Despite significant advances in prevention and management, graft versus host disease (GVHD) is still a leading complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Although skin, gut, liver, thymus, and lung are GVHD targets, neurological complications (NC) have also been reported following allo-HSCT. We demonstrate that the central nervous system (CNS) can be a direct target of alloreactive T cells following allo-HSCT in mice. We found significant infiltration of the CNS with donor T lymphocytes and cell death of neurons and neuroglia in allo-HSCT recipients with GVHD. We also found that allo-HSCT recipients with GVHD had deficits in spatial learning/memory and demonstrated increased anxious behavior. These findings highlight CNS sensitivity to damage caused by alloreactive donor T cells and represent the first characterization of target cell subsets and NC during GVHD. Therefore, these clinically relevant studies offer a novel and rational explanation for the well-described neurological symptoms observed after allo-HSCT.

Coordinating the balance between haematopoietic stem cell (HSC) quiescence and self-renewal is crucial for maintaining haematopoiesis lifelong. Equally important for haematopoietic function is modulating HSC localization within the bone marrow niches, as maintenance of HSC function is tightly controlled by a complex network of intrinsic molecular mechanisms and extrinsic signalling interactions with their surrounding microenvironment. In this study we demonstrate that nuclear factor erythroid 2-related factor 2 (Nfe2l2, or Nrf2), well established as a global regulator of the oxidative stress response, plays a regulatory role in several aspects of HSC homeostasis. Nrf2 deficiency results in an expansion of the haematopoietic stem and progenitor cell compartment due to cell-intrinsic hyperproliferation, which was accomplished at the expense of HSC quiescence and self-renewal. We further show that Nrf2 modulates both migration and retention of HSCs in their niche. Moreover, we identify a previously unrecognized link between Nrf2 and CXCR4, contributing, at least partially, to the maintenance of HSC function.

Despite a growing understanding of the link between intestinal inflammation and resident gut microbes, longitudinal studies of human flora before initial onset of intestinal inflammation have not been reported. Here, we demonstrate in murine and human recipients of allogeneic bone marrow transplantation (BMT) that intestinal inflammation secondary to graft-versus-host disease (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 before BMT aggravated GVHD, whereas reintroducing the predominant species of Lactobacillus mediated significant protection against GVHD. We then characterized gut flora of patients during onset of intestinal inflammation caused by GVHD and found patterns mirroring those in mice. We also identified increased microbial chaos early after allogeneic 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 allogeneic BMT recipients.

Efforts to limit GVHD mediated by alloreactive donor T cells after allogeneic bone marrow transplantation are limited by a concomitant decrease in graft-versus-tumor (GVT) activity and increased possibilities of tumor relapse. Using a novel approach, we adoptively transferred conventional T cells expressing the transcription factor promyelocytic leukemia zinc finger (PLZF), which confers effector properties resembling invariant natural killer T cells, such as copious production of cytokines under suboptimal stimulation. PLZF expression in T-cell allografts attenuates expansion of alloreactive T cells, leading to lower GVHD. Intact alloreactivity-driven antitumor cytokine responses result in preserved GVT effects, leading to improved survival. Our findings suggest that therapy with PLZF-overexpressing T cells would result in overall improved outcomes due to less GVHD and intact GVT effects. Cancer Res; 73(15); 4687–96. ©2013 AACR.

SummaryLittle is known about the maintenance of intestinal stem cells (ISCs) and progenitors during immune-mediated tissue damage or about the susceptibility of transplant recipients to tissue damage mediated by the donor immune system during graft versus host disease (GVHD). We demonstrate here that deficiency of recipient-derived IL-22 increased acute GVHD tissue damage and mortality, that ISCs were eliminated during GVHD, and that ISCs as well as their downstream progenitors expressed the IL-22 receptor. Intestinal IL-22 was produced after bone marrow transplant by IL-23-responsive innate lymphoid cells (ILCs) from the transplant recipients, and intestinal IL-22 increased in response to pretransplant conditioning. However, ILC frequency and IL-22 amounts were decreased by GVHD. Recipient IL-22 deficiency led to increased crypt apoptosis, depletion of ISCs, and loss of epithelial integrity. Our findings reveal IL-22 as a critical regulator of tissue sensitivity to GVHD and a protective factor for ISCs during inflammatory intestinal damage.

Thymopoiesis is a complex process dependent on precise signals from the supporting thymic stromal microenvironment that orchestrates the progression of precursor T cells through well-defined maturation stages. It is well documented that the decline in thymic size and function with age is in part correlated with an increase in sex steroids. This age-related decline in function can be detrimental to the recovery of the thymus in patients receiving radio or chemo-therapy with hematopoietic stem cell transplantation (HSCT). Delayed immune reconstitution, especially in the T cell lineage, is associated with an increased risk of opportunistic infections and malignant relapses. Therefore strategies to enhance thymic reconstitution has the potential to decrease the period of T cell lymphopenia and increase overall clinical outcome. In the process of evaluating the effects of sex steroids in the decline of the thymic function, we found a decrease in the expression of the key thymopoietic factors IL-7, CCL25 and Delta-like 4 (DLL4) by thymic stromal cells after testosterone treatment (Figure 1A). We then addressed if these transcriptional changes were the result of a direct regulation by the androgen receptor (AR). Using a computational approach, and subsequently confirmed by ChIP studies, we found that AR directly bound and negatively regulated the promoter of DLL4, a critical gene involved in T cell commitment and differentiation. We and others have previously shown that sex steroid ablation (SSA) can regenerate young and aged immune system by promoting bone marrow and thymic lymphopoiesis and promoting recovery from autologous and allogeneic HSCT. However the mechanisms underlying the sex steroid-mediate thymic involution and its regeneration after SSA are poorly understood. Moreover, one of the main drawbacks to standard clinical methods of sex steroid ablation using luteinizing hormone releasing hormone (LHRH) agonists (LHRH-Ag) is the initial surge in sex steroids they cause. To address this, we employed a novel class of LHRH-antagonists (LHRH-Ant) that rapidly block the secretion of sex steroids without causing their initial surge that can be even more detrimental to thymopoiesis. Mice treated with LHRH-Ant showed a significantly faster increase in thymic cellularity compared with LHRH-Ag treated mice (Figure 1B). Given the negative regulation of DLL4 by the AR, we hypothesized that DLL4 expression would conversely increase after SSA in vivo. Indeed, we found a significant increase in DLL4 expression after SSA and also an increase in genes downstream of DLL4, such as Ptcra, Hes1 and Cd25 (Figure 1C). We next evaluated if treatment with the LHRH-Ant would provide a faster immune recovery after injury to the immune system. We found that mice treated with LHRH-Ant showed a faster thymic regeneration after total body irradiation (TBI) compared to the control irradiated mice (Figure 1D) and enhanced viral clearance (Figure 1E). Finally, we also found that LHRH-Ant enhanced thymic and peripheral reconstitution up to 3 months after allo-HSCT (Figure 1F). In conclusion, we found that down-regulation of DLL4 may represent one of the mechanisms underlying the effects of sex steroids on thymic function. We demonstrate that SSA with a novel LHRH-Ant increases DLL4 expression and enhances thymic and peripheral T cell recovery and function after immune injury. These findings suggest that the employment of a LHRH-Ant, which is already in clinical use for prostate cancer patients, represents a novel therapeutic strategy to enhance immune recovery and function in immunocompromised patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 454 Allogeneic hematopoietic stem cell transplantation (HSCT) was initially developed as a rescue from therapy-related bone marrow failure following high dose chemo/radiation therapy to rescue from therapy-related bone marrow failure, the emphasis has shifted towards allogeneic HSCT as a strategy to facilitate graft-versus-tumor (GVT) activity. Strategies to suppress graft-versus-host diesase (GVHD) are often associated with broader suppression of the immune system leading to immune deficiency and compromised GVT activity. The aim of this study was to modulate T cell responses in the context of GVHD by targeting the NF-kappaB family member c-Rel, a transcription factor that upon antigen receptor triggering regulates lymphocyte survival and proliferation. In T cells, the main target gene of c-Rel is interleukin 2 (IL-2), a cytokine required for normal T cell proliferative and differentiative responses. The effect of c-Rel deficiency on T cell differentiation and function has been investigated previously demonstrating that c-Rel deficient T cells show reduced Th1, Treg and Th17 but normal Th2 responses. Inhibition of c-Rel is therefore expected to impair T cell activation, differentiation, and function. Using methods to inhibit the c-Rel pathway we developed a novel strategy to minimize the severity of GVHD while leaving GVT activity intact. We investigated the role of c-Rel during GVHD in MHC mismatched as well as MHC matched, minor antigen mismatched (more clinically relevant) murine HSCT models. c-Rel−/− T cells caused significantly less GVHD than normal T cells, as determined by survival (p We next evaluated if c-Rel−/− T cells were able to mediate antitumor activity. We challenged allogeneic HSCT recipients with either a liquid tumor (A20 lymphoma) or a solid tumor (RENCA-renal cell carcinoma) and found that GVT activity in c-Rel−/− T cells recipients was intact in the absence of GVHD, resulting in significantly improved survival compared to recipients of wildtype T cells (p The potential for clinical translation of our approach is highlighted by a series of experiments testing the efficacy of a recently developed Pyrimidinetrione-based small molecule c-Rel inhibitor compound. We found that incubation of T cells with this compound resulted in efficient downregulation of c-Rel expression as well as IL-2 expression in vitro. When we administered T cells that were pre-incubated with this c-Rel antagonizing small molecule, we were able to reproduce our above described findings regarding the effect of c-Rel deficient T cells on GVHD (Figure 1). Importantly, normal T cells pre-incubated with a c-Rel antagonist mediated intact GVT activity, indicating that they are viable and functional T cells. This strongly suggests that the diminished capacity of these cells to induce GVHD is due to the c-Rel inhibitory mechanism of the small molecule compound and not due to a non-specific cytotoxic effect of the in vitro manipulation procedure. Taken together, our findings identify c-Rel as a promising target for the development of a clinical strategy to prevent or ameliorate GVHD while preserving GVT activity. Disclosures: No relevant conflicts of interest to declare.

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.

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.

Abstract 1895 One of the major limitations of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is graft-versus-host disease (GVHD), which is a severe and common complication that impacts on multiple organ systems with varying degrees of severity. Neurological complications such as encephalopathy, meningoencephalitis, cerebrovascular disease, demyelination and in particular neurocognitive deficits, have been reported in patients after allo-HSCT but their etiology remains in most cases poorly understood. In this study we analyzed whether the central nervous system (CNS) is a target of acute GVHD. We used a murine MHC-disparate allo-HSCT model to study cerebral involvement in GVHD: C57BL/6 (B6) (H-2b) into lethally irradiated (850 cGy, split dose) BALB/c (H-2d). The control group received a syngeneic BMT (syn-HSCT) with BALB/c into BALB/c. CNS infiltrating lymphocytes were isolated from brains after complete perfusion of the animals. We found increasing numbers of CNS-infiltrating donor derived CD4 and CD8 alloreactive T cells on day 7, 14, and 21 (p In addition, in order to assess cytokines and homing molecules necessary for CNS infiltration in GVHD, we performed competitive BMTs by mixing control B6 (CD45.1) with either WT B6, IL17−/−, IL21R−/−, or L-selectin−/− (all CD45.2) in a 1:1 ratio. We found statistically significant and reproducibly decreased numbers of infiltrating T cells using IL21R−/− donor T cells, which we have recently reported to mediate decreased systemic GVHD and gastrointestinal infiltration as well. To confirm the cerebral involvement in situ, histological analysis and immunohistochemistry for CD3, CD4 and TUNEL (Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling) on coronal brain sections from perfused animals were performed. We observed infiltration of T cells in Cortex, Hippocampus, Midbrain (Basal ganglia, Thalamus, Hypothalamus), Cerebellum, and Medulla oblongata of allo-HSCT recipients, which was consistent with lymphocytic multifocal meningoencephalitis. Sections from syn-HSCT recipients showed no pathology and T cells were virtually undetectable (p To test whether these histopathological findings were associated with cognitive deficits, we performed behavioral tests at 14 to 20 days after HSCT: Locomotor activity was evaluated in the accelerated rotorod (latency to fall) and open field (total distance moved) tests; neuromuscular function and strength were tested in the grip test (latency to fall); anxiety-related behavior and exploratory activity were tested in the open field (distance moved/time spent in center) and in the elevated plus maze (time spent in open arm); and spatial learning and memory were analyzed in the Morris water maze. Although allo-HSCT recipients had developed mild to moderate GVHD in the first three weeks after transplant as measured by GVHD score and weight loss, we found no differences between allo-HSCT versus syn-HSCT recipients or non-transplanted mice in locomotor activity, motor coordination, strength and neuromuscular function. However, allo-HSCT recipients demonstrated increased anxious behavior and decreased exploratory activity in the open field test (p Taken together these findings demonstrate that the CNS can be a target organ of acute GVHD in allo-HSCT recipients, which is associated with early donor T cell infiltration, histopathology consistent with lymphocytic multifocal meningoencephalitis and specific cognitive defects (increased anxious behaviour and decreased spatial learning), which are not due to lack of mobility. Further studies, particularly in the clinical setting, will reveal the extent of GVHD-related cognitive defects and may lead to new approaches to diagnose and treat a new aspect of GVHD. Disclosures: No relevant conflicts of interest to declare.

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 In mucosal tissues the regulation of IL-22 production has been closely associated with IL-23 produced by dendritic cells (DCs) and ex vivo incubation of cells with IL-23 stimulates the production of IL-22. Following thymic insult there was a significant increase in the amount of IL-23 produced by DCs (Fig 1b) resulting in similar kinetics of intrathymic levels of IL-22 and IL-23. We identified a population of radio-resistant CD3−CD4+IL7Ra+RORg(t)+ thymic innate lymphoid cells (tILCs) that upregulate both their production of IL-22 (Fig 1c) and expression of the IL-23R (p=0.0006) upon exposure to TBI. This suggests that they are responsive to IL-23 produced by DCs in vivo following TBI and, in fact, in vitro stimulation of tILCs by IL-23 led to upregulation of Il-22 production by these cells (Fig 1d). We found expression of the IL-22Ra on cortical and medullary TECs (cTECs and mTECs, respectively), and uniform expression across both mature MHCIIhi mTEC (mTEChi) and immature MHCIIlo mTECs (mTEClo). However, in vitro stimulation of TECs with recombinant IL-22 led to enhanced TEC proliferation primarily in cTEC and mTEClo subsets (p=0.002 and 0.004 respectively). It is currently unclear if IL-22 acts as a maturation signal for mTECs, however, the uniform expression of IL-22Ra between immature mTEClo and mature Aire-expressing mTEChi, together with the preferential promotion of proliferation amongst mTEClo and cTEC seem to argue against IL-22 as a maturational signal but rather as promoter of proliferation, which ultimately leads to terminal differentiation of TECs. Of major clinical importance, administration of exogenous IL-22 led to enhanced thymic recovery (Fig. 1e) following TBI, primarily by promoting the proliferation of TECs. Consistent with this, the administration of IL-22 also led to significantly enhanced thymopoiesis following syngeneic BMT. Taken together these findings suggest that following thymic insult, and specifically the depletion of developing thymocytes, upregulation of IL-23 by DCs induces the production of IL-22 by tILCs and regeneration of the supporting microenvironment. This cascade of events ultimately leads to rejuvenation of the thymocyte pool (Fig. 1f). These studies not only reveal a novel pathway underlying endogenous thymic regeneration, but also identify a novel regenerative strategy for improving immune competence in patients whose thymus has been damaged from infection, age or cytoreductive conditioning required for successful hematopoietic stem cell transplantation. Finally, these findings may also provide an avenue of study to further understand the repair and regeneration of other epithelial tissues such as skin, lung and breast. Disclosures: No relevant conflicts of interest to declare.

Efforts to improve graft-versus-tumor (GVT) activity of alloreactive donor T cells are limited by a concomitant rise in graft-versus-host disease (GVHD). We employed experimental allogeneic bone marrow transplantation (allo-BMT) models to assess two novel strategies, in which T lineage cells were genetically engineered to enhance selective effector functions. In addition to the transgenic expression of these molecules on mature donor T cells, we also used genetically engineered precursor T cells as an “off the shelf” adoptive cell therapy. One strategy relies on T cell cytolytic molecule TNF-Related Apoptosis Inducing Ligand (TRAIL), which induces apoptosis through death receptors (DR) 4 and 5 (only DR5 in mice). Certain tumor cells express high levels of DR5 making TRAIL an attractive candidate for engineering T cells to augment GVT. Mature T cells were transduced with a lentiviral TRAIL expression vector and adoptively transferred into lethally irradiated allogeneic bone marrow transplantation (allo-BMT) recipients, bearing LB27.4 tumors (B6 into CBF1+LB27.4). TRAIL+ T cells had enhanced antitumor effect compared to control-transduced T cells in vitro and upon transfer into tumor-bearing allo-BMT recipients (p The second strategy explores the effect of overexpressing the transcription factor promyelocytic leukemia zinc finger (PLZF) in T cells. PLZF transgenic (tg) T cells have an effector/memory phenotype with innate-like characteristics. Following transfer, they cells display an altered cytokine profile with increased expression of TNF-alpha and decreased IFN-gamma. Using adoptive transfer of CFSE-labeled donor T cells we observed that fast proliferating allo-responsive PLZF+ T cells died after only a few cell cycles. Using PLZF Tg mice, we found that PLZF+ T cells cause less GVHD with intact GVT activity (p Our data suggest that adoptive therapy with genetically engineered TRAIL+ or PLZF+ T cells cause less GVHD while displaying intact or enhanced GVT activity. Furthermore, the “off the shelf” use of genetically enhanced preT cells represents a promising cell therapy strategy to enhance anti-tumor activity in both autologous and allo-BMT patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4743. doi:10.1158/1538-7445.AM2011-4743