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Graft-versus-host disease (GVHD) and posttransplant immunodeficiency are frequently related complications of allogeneic hematopoietic transplantation. Alloreactive donor T cells can damage thymic epithelium, thus limiting new T-cell development. Although the thymus has a remarkable capacity to regenerate after injury, endogenous thymic regeneration is impaired in GVHD. The mechanisms leading to this regenerative failure are largely unknown. Here we demonstrate in experimental mouse models that GVHD results in depletion of intrathymic group 3 innate lymphoid cells (ILC3s) necessary for thymic regeneration. Loss of thymic ILC3s resulted in deficiency of intrathymic interleukin-22 (IL-22) compared with transplant recipients without GVHD, thereby inhibiting IL-22-mediated protection of thymic epithelial cells (TECs) and impairing recovery of thymopoiesis. Conversely, abrogating IL-21 receptor signaling in donor T cells and inhibiting the elimination of thymic ILCs improved thymopoiesis in an IL-22-dependent fashion. We found that the thymopoietic impairment in GVHD associated with loss of ILCs could be improved by restoration of IL-22 signaling. Despite uninhibited alloreactivity, exogenous IL-22 administration posttransplant resulted in increased recovery of thymopoiesis and development of new thymus-derived peripheral T cells. Our study highlights the role of innate immune function in thymic regeneration and restoration of adaptive immunity posttransplant. Manipulation of the ILC-IL-22-TEC axis may be useful for augmenting immune reconstitution after clinical hematopoietic transplantation and other settings of T-cell deficiency.

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.

Keratinocyte growth factor (KGF), which is given exogenously to allogeneic bone marrow transplantation (allo-BMT) recipients, supports thymic epithelial cells and increases thymic output of naive T cells. Here, we demonstrate that this improved T-cell reconstitution leads to enhanced responses to DNA plasmid tumor vaccination. Tumor-bearing mice treated with KGF and DNA vaccination have improved long-term survival and decreased tumor burden after allo-BMT. When assayed before vaccination, KGF-treated allo-BMT recipients have increased numbers of peripheral T cells, including CD8+ T cells with vaccine-recognition potential. In response to vaccination, KGF-treated allo-BMT recipients, compared with control subjects, generate increased numbers of tumor-specific CD8+ cells, as well as increased numbers of CD8+ cells producing interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). We also found unanticipated benefits to antitumor immunity with the administration of KGF. KGF-treated allo-BMT recipients have an improved ratio of T effector cells to regulatory T cells, a larger fraction of effector cells that display a central memory phenotype, and effector cells that are derived from a broader T-cell–receptor repertoire. In conclusion, our data suggest that KGF can function as a potent vaccine adjuvant after allo-BMT through its effects on posttransplantation T-cell reconstitution.

CD4+ interleukin-17 (IL-17)+ T cells (Th17 cells) have been implicated in allograft rejection of solid organs and several autoimmune diseases. However, the functional role of Th17 cells in the development of acute graft-versus-host disease (GVHD) has not been well-characterized. We detected significant numbers of alloreactive CD4+ donor T cells expressing IL-17, IL-17F, or IL-22 in the lymphoid organs of recipients of an allogeneic bone marrow transplant. We found no differences in GVHD mortality or graft-versus-tumor (GVT) activity between wild type (WT) and IL-17−/− T-cell recipients. However, upon transfer of murine IL-17−/− CD4+ T cells in an allogeneic BMT model, GVHD development was significantly delayed behind recipients of WT CD4+ T cells, yet overall GVHD mortality was unaffected. Moreover, recipients of IL-17−/− CD4+ T cells had significantly fewer Th1 cells during the early stages of GVHD. Furthermore, we observed a decrease in the number of IFN-γ–secreting macrophages and granulocytes and decreased production of proinflammatory cytokines (interferon [IFN]-γ, IL-4, and IL-6) in recipients of IL-17−/− CD4+ T cells. We conclude that IL-17 is dispensable for GVHD and GVT activity by whole T cells, but contributes to the early development of CD4-mediated GVHD by promoting production of proinflammatory cytokines.

Delayed T-cell recovery is an important complication of allogeneic bone marrow transplantation (BMT). We demonstrate in murine models that donor BM-derived T cells display increased apoptosis in recipients of allogeneic BMT with or without GVHD. Although this apoptosis was associated with a loss of Bcl-2 and Bcl-XL expression, allogeneic recipients of donor BM deficient in Fas-, tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)- or Bax-, or BM-overexpressing Bcl-2 or Akt showed no decrease in apoptosis of peripheral donor-derived T cells. CD44 expression was associated with an increased percentage of BM-derived apoptotic CD4+ and CD8+ T cells. Transplantation of RAG-2-eGFP–transgenic BM revealed that proliferating eGFPloCD44hi donor BM-derived mature T cells were more likely to undergo to apoptosis than nondivided eGFPhiCD44lo recent thymic emigrants in the periphery. Finally, experiments using carboxyfluorescein succinimidyl ester–labeled T cells adoptively transferred into irradiated syngeneic hosts revealed that rapid spontaneous proliferation (as opposed to slow homeostatic proliferation) and acquisition of a CD44hi phenotype was associated with increased apoptosis in T cells. We conclude that apoptosis of newly generated donor-derived peripheral T cells after an allogeneic BMT contributes to delayed T-cell reconstitution and is associated with CD44 expression and rapid spontaneous proliferation by donor BM-derived T cells.

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor family that mediates epithelial cell proliferation and differentiation in a variety of tissues, including the thymus. We studied the role of KGF in T-cell development with KGF-/- mice and demonstrated that thymic cellularity and the distribution of thymocyte subsets among KGF-/-, wildtype (WT), and KGF+/- mice were similar. However, KGF-/- mice are more vulnerable to sublethal irradiation (450 cGy), and a significant decrease was found in thymic cellularity after irradiation. Defective thymopoiesis and peripheral T-cell reconstitution were found in KGF-/- recipients of syngeneic or allogeneic bone marrow transplant, but using KGF-/- mice as a donor did not affect T-cell development after transplantation. Despite causing an early developmental block in the thymus, administration of KGF to young and old mice enhanced thymopoiesis. Exogenous KGF also accelerated thymic recovery after irradiation, cyclophosphamide, and dexamethasone treatment. Finally, we found that administering KGF before bone marrow transplantation (BMT) resulted in enhanced thymopoiesis and peripheral T-cell numbers in middle-aged recipients of an allogeneic BM transplant. We conclude that KGF plays a critical role in postnatal thymic regeneration and may be useful in treating immune deficiency conditions. (Blood. 2006;107:2453-2460)

T-cell deficiency related to disease, medical treatment, or aging represents a major clinical challenge and is associated with significant morbidity and mortality in cancer and bone marrow transplantation recipients. This study describes several innovative and clinically relevant strategies to manipulate thymic function based on an interventional radiology technique for intrathymic injection of cells or drugs. We show that intrathymic injection of multipotent hematopoietic stem/progenitor cells into irradiated syngeneic or allogeneic young or aged recipients resulted in efficient and long-lasting generation of functional donor T cells. Persistence of intrathymic donor cells was associated with intrathymic presence of cells resembling long-term hematopoietic stem cells, suggesting a self-renewal capacity of the intrathymically injected cells. Furthermore, our approach enabled the induction of long-term antigen-specific T-cell–mediated antitumor immunity following intrathymic injection of progenitor cells harboring a transgenic T-cell receptor gene. The intrathymic injection of interleukin-7 prior to irradiation conferred radioprotection. In addition, thymopoiesis of aged mice improved with a single intrathymic administration of low-dose keratinocyte growth factor, an effect that was sustained even in the setting of radiation-induced injury. Taken together, we established a preclinical framework for the development of novel clinical protocols to establish lifelong antigen-specific T-cell immunity.

IL-21 is a proinflammatory cytokine produced by Th17 cells. Abrogation of IL-21 signaling has recently been shown to reduce GVHD while retaining graft-versus-leukemia/lymphoma (GVL) responses. However, the mechanisms by which IL-21 may lead to a separation of GVHD and GVL remain incompletely understood. In a murine MHC-mismatched BM transplantation model, we observed that IL-21 receptor knockout (IL-21R KO) donor T cells mediate decreased systemic and gastrointestinal GVHD in recipients of a transplant. This reduction in GVHD was associated with expansion of transplanted donor regulatory T cells and with tissue-specific modulation of Th-cell function. IL-21R KO and wild-type donor T cells showed equivalent alloactivation, but IL-21R KO T cells showed decreased infiltration and inflammatory cytokine production within the mesenteric lymph nodes. However, Th-cell cytokine production was maintained peripherally, and IL-21R KO T cells mediated equivalent immunity against A20 and P815 hematopoietic tumors. In summary, abrogation of IL-21 signaling in donor T cells leads to tissue-specific modulation of immunity, such that gastrointestinal GVHD is reduced, but peripheral T-cell function and GVL capacity are retained. IL-21 is thus an exciting target for therapeutic intervention and improvement of clinical transplantation outcomes.

Thymopoiesis is a complex process involving crosstalk between developing thymocytes and the non-hematopoietic stromal microenvironment, which includes thymic epithelial cells (TECs), fibroblasts and endothelial cells (ECs). Despite its importance, the thymus is exquisitely sensitive to cellular insults, including cytoreductive chemo- and radiation therapy required for successful hematopoietic stem cell transplantation; therefore identification of thymic repair mechanisms will offer promising therapeutic targets for immune regeneration. Recent studies in tissues such as liver, lung and bone marrow have revealed that ECs not only passively deliver oxygen and nutrients to tissues, but also actively produce distinct paracrine factors that can orchestrate their repair. The role of thymic ECs in thymopoiesis beyond their contribution of local circulation and the importation of lymphoid progenitors has not been comprehensively studied. In order to evaluate the role of ECs in thymic regeneration, we closely examined the kinetics of thymic recovery following total body irradiation (TBI, 550cGy) in young C57BL/6 mice. Although we observed a dramatic decline in total thymic cellularity, we found no significant change in the number of thymic ECs, suggesting they are extremely radio-resistant (Fig. 1A). Interestingly, although thymic ECs appeared to be resistant to the cytoreductive effects of TBI, we revealed an increase in their proliferation as measured by expression of Ki67 shortly after injury, indicating a role in the endogenous regeneration of the thymus (Fig. 1B). Given their radioresistance and cycling after TBI, we hypothesized that thymic ECs can provide instructive signals supporting thymic regeneration. To explore potential regenerative signals stemming from ECs during thymic regeneration, we performed a transcriptome analysis of highly purified ECs of untreated mice and at days 4 and 7 following TBI. Among the 288 genes that were altered in ECs after TBI (131 upregulated and 157 downregulated), we found significant upregulation in the expression of BMP4 (which has previously been implicated in thymic regeneration) and was validated using quantitative PCR (Fig 1C). In addition, we have developed a novel light sheet fluorescence microscopy approach to visualize the thymic vasculature in 3D at high resolution (Fig. 1D). Consistent with their potential role in aiding thymic regeneration, administration of ex vivo expanded thymic ECs (ex-EC) could significantly enhance thymic regeneration when given after TBI. Intriguingly, this regenerative effect of ex-ECs was tissue specific as ex-EC derived from heart or kidney did not exhibit a similar regenerative effect (Fig. 1E). Similar to our findings in the endogenous regeneration setting, we found that thymic ex-ECs demonstrated a significantly higher BMP4 expression compared to cardiac and kidney ex-ECs (Fig. 1F). Previous reports have suggested that BMP4 signaling is capable of directly regulating the key transcription factor of TEC function, FOXN1. We found that incubation of the TEC cell line C9 with thymic ex-EC conditioned medium induced an increase in FOXN1 expression but was abrogated in the presence of Noggin, a potent BMP signaling inhibitor (Fig. 1G). These findings support the hypothesis that BMP4 mediates the regenerative effect of ECs in thymic regeneration. In summary, we found that thymic ECs are capable of mediating endogenous thymic regeneration, likely via their expression of BMP4. Adoptive transfer of ex-ECs leads to enhanced thymic regeneration after immune injury. Thus, adoptive transfer of thymic ECs represents a novel strategy to improve immune function in immunocompromised patients. Figure 1 Figure 1. Disclosures Ginsberg: Angiocrine Bioscience: Employment. Rafii:Angiocrine Biosciences: Founder Other.

NF-kB plays important roles in immunity and oncogenesis, indicating that therapeutic targeting of this pathway could be beneficial in various clinical settings; however,an NF-kB-specific inhibitor does not exist in clinical practice to date. One approach toward development of such a compound is small-molecule-mediated direct inhibition of one or several members of the NF-kB family of transcription factors, a network that comprises five structurally related proteins including p50, p52, RelA, RelB and c-Rel. After screening of a library of 15,000 small molecules with a biochemical assay, we identified two scaffolds with inhibitory activity specific for the NF-kB subunit c-Rel. These scaffolds act as direct c-Rel inhibitors by modifying the conformation of the c-Rel protein, thus preventing DNA binding. We previously reported that in vitro treatment of T cells with the thiohydantoin IT-603 induces c-Rel deficiency, resulting in suppression of T cell alloactivation without compromising T cell activation triggered by recognition of tumor-associated or viral antigens (Shono et al., Cancer Discovery, 2014). Here, we for the first time demonstrate in vivo efficacy of a c-Rel inhibitor treatment regimen in mouse models of graft-versus-host disease (GVHD) and graft-versus-lymphoma (GVL), as well as xenograft models of human B cell lymphomas, revealing that inhibition of c-Rel activity allows not only for suppression of GVHD while retaining GVL activity, but it also mediates promising anti-lymphoma effects. We first show that the novel small molecule IT-901 is a more potent c-Rel inhibitor than IT-603 and has a superior pharmacokinetic profile. IT-901 displayed significantly improved in vivo efficacy, ameliorating GVHD while preserving the anti-lymphoma activity of T cells (Figure 1a,b). Recent genetic evidence has established a pathogenetic role for NF-kB signaling in lymphoid malignancies. We therefore sought to explore the potential of IT-901 for targeted therapy of human lymphomas. We analyzed six representative diffuse large B cell lymphoma (DLBCL) cell lines including activated B-like (ABC; HBL1, TMD8, U2932) and germinal center B-like (GCB; Ly19, SU-DHL4, SU-DHL8) cell lines for nuclear translocation of c-Rel and found that c-Rel was constitutively active in all cell lines. To examine if c-Rel inhibition with IT-901 alters cytokine production by DLBCL cells, we analyzed cytokine levels in the supernatant after in vitro incubation with IT-901. IT-901 treatment resulted in decreased levels of a wide range of cytokines in TMD8 cells, with the notable exceptions of interleukin 8 (IL-8), tumor necrosis factor (TNF)-α, and TNF-β (P We next analyzed primary lymphoma cells and found that the c-Rel gene is widely expressed in human B cell malignancies and frequently amplified in DLBCL and EBV-transformed B cells. Importantly, intranuclear analysis of the c-Rel protein demonstrated that this transcription factor can be constitutively active in a wide range of human lymphomas. IT-901 efficiently inhibited growth of EBV-transformed B cells in vitro, and mediated significant anti-lymphoma activity in a xenograft model of EBV-induced lymphoma (P In summary, our findings underscore multiple therapeutic benefits and great potential for clinical translation of a novel c-Rel inhibitor. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

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 To evaluate direct effects on epithelial regeneration, we performed intestinal organoid culture assays in the presence of IL-22. Organoids generated from SI and large intestine (LI) crypts of wild-type B6 mice demonstrated substantially increased size after seven days of culture with IL-22 (p To investigate the translational potential for use in humans, we tested a human IL-22 dimer/Fc fusion molecule (F-652, Generon Corp., Shanghai) on mouse intestinal crypts and found that F-652 significantly increased the size of SI and LI organoids. Using the LP into B6 allo-HSCT model described above, we found that every other day subcutaneous (SQ) treatment with 100 ug/kg F-652 starting day seven post-BMT led to significant improvement in both clinical GVHD score (P In summary, we found that IL-22 and innate lymphoid cells can bridge immune function and tissue regeneration by acting directly on epithelial stem cells. IL-22 and F-652 therapy may represent a novel approach to promote intestinal recovery in patients with GVHD without increasing post-transplant immunodeficiency. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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.

Despite increasing insights into its immunobiology, graft vs host disease (GVHD) remains a major obstacle for successful allogeneic hematopoietic stem/progenitor cell transplantation (allo-HCT). Separation of GVHD from graft vs. leukemia/lymphoma (GVL) responses also remains an elusive goal for allo-HSCT. Efforts to delineate the transcriptional networks regulating T cell differentiation post-HCT have suggested that multiple transcription factors may be involved in the regulation of alloreactive helper T (Th) cells and GVHD. However, conflicting data have emerged regarding the role of Th1 and Th17 pathways, and it remains unclear which transcription factors mediate the early activation of alloreactive T cells necessary for subsequent GVHD development. The T-box transcription factor eomesodermin (Eomes) cooperates with T-bet to regulate CD8 T cell cytotoxic function, IFNy production, and memory cell formation. Recently, a role for Eomes in CD4 Th cell polarization has been described as well. In order to evaluate the role of Eomes in T cell function in the context of allo-HCT, we used a MHC-disparate mouse model (C57BL/6 into BALB/c) with T cell depleted donor bone marrow (TCD-BM) and wild-type (WT) or Eomes knock out (KO) donor T cells. Recipients were conditioned with lethal total body irradiation. Eomes deficiency in donor T cells led to a significant reduction in GVHD mortality (Fig 1, p In order to determine if Eomes was important for T cell-mediated GVL responses, we performed allo-HCT in the presence of A20 lymphoma cells. Despite the reduction in GVHD mortality as described above, A20 tumor challenge led to increased mortality in recipients of Eomes KO T cells, indicating that Eomes was also critical for effective GVL function. Given the importance of Eomes in early alloactivation of CD4 T cells, we evaluated if the impaired GVL function was due to an intrinsic CD8 defect or lack of CD4 help. B6 TCD-BM was transplanted into BALB/c recipients along with either WT or Eomes KO CD4 or CD8 T cells. Eomes deficiency in both CD4 and CD8 T cells again led to significant mortality, but HCT with Eomes KO CD4 T cells and WT CD8 T cells led to the greatest survival due to less GVHD and intact GVL (Fig 3), suggesting that Eomes is essential for intrinsic CD8 function during GVL, but not for CD4 help. In summary, we identified distinct requirements for Eomes in CD4 versus CD8 T cells in the context of allo-HCT. Eomes regulated multiple aspects of CD4 T cell function following allo-HCT, including early activation, cytokine production, and gut trafficking. The multifacted functions of Eomes in CD4 T cells likely explain its requirement for GVHD. In contrast, Eomes deficiency in CD8 T cells led to impaired GVL, consistent with its established importance for cytotoxic CD8 T cell differentiation. To our knowledge, this is one of the first descriptions of a transcription factor necessary for effective GVL capacity. Our results suggest that selective manipulation of Eomes function in T cell subsets may be useful for both limiting GVHD and enhancing GVL. 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 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.

Abstract 309 Despite decades of intensive research, graft vs. host disease (GVHD) remains a major complication of allogeneic transplantation. Much progress has been made toward understanding GVHD pathophysiology and the mechanisms regulating the donor immune response, however no factors have been identified to regulate the response of transplant recipients to GVHD and its concomitant damage. Furthermore, virtually all strategies available to reduce clinical GVHD do so by limiting the donor immune system at the expense of therapeutic graft vs. leukemia/lymphoma (GVL) responses. IL-22 is a recently characterized cytokine that is produced by both helper T cells and innate lymphoid cells (ILC). Expression of its receptor (IL-22R) is generally limited to epithelial and other non-hematopoietic cells, and it has demonstrated a protective role for intestinal epithelium during experimental colitis, providing a signal for intestinal epithelial cell survival, proliferation, and wound healing. We investigated the role of IL-22 in GVHD during MHC-mismatched C57BL/6 (B6) into BALB/c murine bone marrow transplantation (BMT). Elimination of IL-22 with anti-IL-22 neutralizing antibody lead to increased GVHD mortality post-BMT in comparison to isotype control (p IL-22 ELISA on tissue homogenates following BMT with wild type donors and recipients indicated that IL-22 levels are increased three weeks post-BMT in both small and large intestine (p In summary, IL-22 is produced post-BMT by host ILC that are eliminated during GVHD, and IL-22 deficiency increases GVHD morbidity and mortality. While IL-22 deficiency did not significantly alter the donor immune response, it did lead to increased GVHD pathology, loss of epithelial integrity, and damage to the ISC niche. IL-22 is thus critical for protection of host epithelium during GVHD but is not involved in GVL. This may be exploited in the future to reduce clinical GVHD without limiting the curative potential of the transplant. Disclosures: Fouser: Pfizer: Employment.

Abstract 730 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 can induce apoptotic signals through death receptor (DR) 4 and 5 molecules (only DR5 in mice) expressed on target cells. Certain tumor cells are known to express high levels of surface DR5 making TRAIL an attractive candidate for genetic engineering of donor T cells to enhance GVT activity. Extending previous studies indicating that TRAIL expression in donor T cells is dispensable for the development of GVHD, we evaluated the effect of TRAIL over-expression 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 a T cell depleted allo-BMT, bearing LB27.4 tumors, (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 The second strategy explores the use of promyelocytic leukemia zinc finger (PLZF). PLZF is a member of the BTB-ZF (Broad complex, Tram track, Bric-^-brac-zinc finger) family of transcription factors. In NKT and NK cells, it controls key steps in development, activation and effector functions. Its ectopic expression on conventional T cell confers NKT and NK-like properties. Recent studies using transgenic mice have shown that T cells ectopically expressing PLZF spontaneously acquire an effector/memory phenotype and CD8+ T cells spontaneously express IFN-gamma. Using PLZF+ transgenic donor (B6) T cells in A20-tumor challenged B6 ^ BALB/c allo-BMT model, we found that PLZF+ T cells have less GVHD activity while GVT activity remained intact (p In conclusion, our data suggest that adoptive therapy with genetically engineered TRAIL or PLZF expressing donor T cells exert less GVHD activity while displaying intact or enhanced GVT activity. Furthermore, the “off the shelf” use of genetically enhanced precursor T cells indicates a promising cell therapy strategy to enhance anti-tumor activity in both autologous and allogeneic BMT patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 2453 Poster Board II-430 NOD2 polymorphisms are independent risk factors for Crohn's disease and graft-versus-host disease (GVHD). In Crohn's disease, the pro-inflammatory state resulting from NOD2 mutations have been associated with a loss of anti-bacterial function of enterocytes, such as paneth cells. NOD2 has not been studied in experimental allogeneic bone marrow transplantation (allo-BMT). We studied the role of NOD2 during inflammation in murine models of graft-versus-host disease (GVHD) and in experimental colitis. To investigate the role of NOD2 in regulating GVHD in allo-BMT recipients, we used MHC-matched as well as MHC-disparate allo-BMT models. We first assessed the role of NOD2 deficiency of the allo-BMT donor (either donor T cells or bone marrow) and found no significant impact on the development of GVHD. In contrast, we observed significantly more lethal GVHD in NOD2-/- allo-BMT recipients as compared with WT allo-BMT recipients. We next created chimeric mice, which were NOD2 deficient either in the hematopoietic or non-hematopoietic system. After three months, we performed allo-BMTs (B10BRþB6 and LPþB6) using either WT, NOD2-/- or chimeric recipients. We found that the NOD2 deficiency in the hematopoietic system of the recipient, as opposed to a NOD2 deficiency in the non-hematopoietic system, is responsible for the increased severity of GVHD (Fig. 1A). In NOD2-/- allo-BMT recipients we observed that the absolute number of donor T cells as well as their activation status was significantly increased. Next, we transferred CFSE labeled allogeneic WT T cells to NOD2-/- allo-BMT recipients and found increased proliferation and activation, suggesting that NOD2 plays a role in the regulation of host antigen presenting cells (APCs). We then quantified the expression of activation markers and co-stimulatory molecules on host dendritic cells (DCs): CD40, CD80 and CD86 were significantly up-regulated of on host NOD2-/- DCs as compared with WT DCs during GVHD. To study DC function we selected splenic DCs from WT and NOD2-/- allo-BMT recipients with GVHD and used them as stimulators in mixed leukocyte reactions (MLRs). NOD2-/- DCs had a significantly increased ability to induce proliferation of allogeneic T cells as compared with WT DCs. Finally, we used bone marrow chimeras in an experimental colitis model (which has not been done before) and observed again that NOD2 deficiency in the hematopoietic cells results in increased intestinal inflammation (Figure 1B). We conclude that NOD2 regulates the development of GVHD through its inhibitory effect on host APC function. Fig. 1 (A) NOD2 deficiency of the hematopoietic system aggravatges GVHD. Chemeric mice with NOD2 deficiency either in the hematopoietic system or in the non-hematopoietic system were created by syngeneic BMT (B6 WT → B6 NOD2-/- or B6 NOD2-/→ B6 WT). After 90 days, lethally irradiated (11 Gy) B6 WT versus B6 NOD2-/- versus chemeric allo-BMT recipients were transplanted with 5×106 B10BR TCD-BM or 5×106 LP TCD-BM + 2×106 B10BR T Cells or 3×106 LP T cells. combined date from two independent experiments are shown; n = 16/group. (B) NOD2 deficiency of the hematopoietic system aggravates experimental colitis. First, chemeric mice with NOD2 deficiency either in the hematopoietic system or in the non-hematopoietic system were created by syngeneic BMT (B6 WT→B6 NOD2-/- or B6 NOD2-/-→ B6 WT). After 90 days, TNBS colitis (5 mg TNBS in 50% ethanol) was induced; colons were harvested at day+3 after induction of colitis; combined data from two independent experiments are shown; n=8 / group. Fig. 1. (A) NOD2 deficiency of the hematopoietic system aggravatges GVHD. Chemeric mice with NOD2 deficiency either in the hematopoietic system or in the non-hematopoietic system were created by syngeneic BMT (B6 WT → B6 NOD2-/- or B6 NOD2-/→ B6 WT). After 90 days, lethally irradiated (11 Gy) B6 WT versus B6 NOD2-/- versus chemeric allo-BMT recipients were transplanted with 5×106 B10BR TCD-BM or 5×106 LP TCD-BM + 2×106 B10BR T Cells or 3×106 LP T cells. combined date from two independent experiments are shown; n = 16/group. (B) NOD2 deficiency of the hematopoietic system aggravates experimental colitis. First, chemeric mice with NOD2 deficiency either in the hematopoietic system or in the non-hematopoietic system were created by syngeneic BMT (B6 WT→B6 NOD2-/- or B6 NOD2-/-→ B6 WT). After 90 days, TNBS colitis (5 mg TNBS in 50% ethanol) was induced; colons were harvested at day+3 after induction of colitis; combined data from two independent experiments are shown; n=8 / group. Disclosures: No relevant conflicts of interest to declare.

234 Thymic GVHD (tGVHD) after allogeneic bone marrow transplantation (allo-BMT) is associated with prolonged immunodeficiency. We have previously shown that thymic output after allo-BMT is directly related to thymus size, and inversely related to donor T cell dose and GVHD severity. Additionally, radiation-containing preparative regimens upregulate the death receptors Fas and DR5 on thymic stroma (especially epithelium) while decreasing expression of the anti-apoptotic protein cFLIP, thereby sensitizing the thymus to GVHD. Moreover, small numbers of donor alloreactive T cells are sufficient to cause tGVHD, and they utilize the Fas/Fas ligand (FasL) and TRAIL/DR5 pathways to mediate damage thymic stroma, architecture and function. We performed experiments in both MHC-mismatched and MHC-matched minor antigen-disparate model systems, and demonstrated the exquisite sensitivity of the thymus to as few as 1–2.5×10 5 donor T cells, which mediated tGVHD without evidence of overt clinical disease or significant weight loss. Additionally, tGVHD is partially reversible in our model systems (contingent on a low T cell dose), such that mice with tGVHD exhibit a transient but partially reversible decrease in thymic cellularity when measured at days 28 vs. 60 post-transplant (Figure 1). To further study the role of TRAIL in tGVHD, we asked whether (1) alloreactive T cells and (2) the inflammation associated with conditioning and acute GVHD, were strictly required for TRAIL/DR5-mediated thymic damage. We treated recipients of T cell-depleted allo-BMT with the amDR5-1 agonistic antibody (0.2 mg i.p. per dose) either in the ‘early‘ peri-transplant period, or ‘late,‘ in the second week post-transplant. Allo-BMT recipients treated ‘early‘ with amDR5-1 had significantly decreased thymic cellularity and splenic BM-derived T cells as compared with controls. Furthermore, we observed similar BM cellularity and BM-derived lineage − sca-1 + ckit + (LSK), all in the absence of donor alloreactive T cells and GVHD (Figure 2). We observed similar results with mice treated ‘late‘ amDR5-1 using the later schedule, which indicates that the thymus has continued sensitivity to TRAIL throughout the post-transplant period, and that GVHD and/or conditioning-associated cytokines are not required to enable TRAIL-mediated damage to the thymus. We further assessed the expression of DR5 on donor BM-derived thymocytes to determine whether amDR5-1 acted directly on thymocytes. We observed that on day 28 after T cell-depleted allo-BMT, only 1-2% of donor thymocytes expressed DR5, suggesting that amDR5-1 (and potentially TRAIL) mediate their effects on thymic cellularity and function primarily via an indirect mechanism (Figure 3). These data suggest to us that significant damage to the thymus and thymopoiesis during allo-BMT: can be mediated by soluble TRAIL agonists, even without the presence of whole donor alloreactive T cells can occur, via the TRAIL pathway, without a full complement of the inflammatory processes typically associated with GVHD pathophysiology, including the cytokine storm, and can occur early and late post-transplant, and that early exposure of the thymus to TRAIL/DR5 signals are sufficient to cause a durable loss in thymic cellularity, donor CD4 + CD8 + thymocytes counts, and numbers of donor BM T cells exported to the periphery. ccurs via the TRAIL pathway, without significant direct damage (via TRAIL/DR5 interactions) to the bone marrow compartment, and thus by implication, thymic-seeding precursors derived from the BM. Together, our data in clinically-relevant mouse allo-BMT models suggests that the thymus is highly sensitive to GVHD and endures severe damage at relatively low levels of systemic GVHD. Moreover, post-transplant thymic atrophy is a partially-reversible process which depends on the T cell dose, and which occurs via the TRAIL pathway. Finally, we provide significant mechanistic insight which shows that TRAIL-mediated thymic damage can occur (1) throughout the early post-transplant period, and (2) does not strictly require alloreactive T cells, or the inflammatory processes associated with conditioning and GVHD. Disclosures: No relevant conflicts of interest to declare.

Although thymic graft-versus-host-disease (tGVHD) has been recognized as an important contributor to impaired T cell reconstitution, limited T cell repertoire and increased infection risk in patients with GVHD, the molecular basis of interactions between donor alloreactive T cells, donor bone marrow (BM)-derived thymocytes, and host hematopoietic and non-hematopoietic thymic stromal cells in GVHD has not been well-defined. Here we analyzed the role of molecules relevant for T cell trafficking, cytolytic function, and co-stimulation and co-inhibition of alloreactive T cells in tGVHD. We first demonstrated that thymic output (as measured by RAG2+ splenic recent thymic emigrants) as well as the thymic cellularity (especially of CD4+CD8+ thymocytes) were inversely proportional to numbers of mature donor T cells infused with the allograft, suggesting that tGVHD severity was inversely associated with thymic function. We then studied the migration of alloreactive donor T cells in vivo with bioluminescence imaging (BLI) and found that luciferase-expressing donor T cells infiltrated the thymus within one week after allogeneic bone marrow transplantation (BMT) (Fig. 1). Upon adoptive transfer of CFSE-labeled donor T cells we noted that thymus-infiltrating alloreactive donor T cells were largely fast-proliferating (CFSElo) and highly activated (CD25+ CD44+). We analyzed the importance of T cell trafficking molecules for tGVHD using mice deficient for certain trafficking molecules, and assessed tGVHD by loss of BM-derived CD4+CD8+ thymocytes. We found that CCR9, b7 integrin subunit, and PSGL-1 were all partially required for tGVHD, while L-selectin and aE integrin subunit may be dispensable (Fig. 2A). Similarly, we examined the role of T cell cytolytic pathways for tGVHD, and found that FasL and TRAIL were required for tGVHD, but that perforin and TNF were dispensable (Fig. 2B). Finally, we assessed the role of various T cell co-stimulatory and co-inhibitory molecules for tGVHD, and found that CEACAM1, OX40 and CTLA4 were required, while GITR was partially required and ICOS was dispensable (Fig. 2C). Upon further analysis of donor BM-derived thymocytes, we observed that Bcl-2 expression in donor BM-derived thymocytes was decreased in recipients with GVHD vs. those without GVHD, which suggests that survival of thymocytes is decreased during tGVHD. Hollander and others have previously demonstrated in non-irradiated GVH reaction models that host non-hematopoietic thymic stroma may be an important target for donor alloreactive T cells. We assessed the expression of the death receptors Fas and DR5 in thymic stroma from normal and irradiated (850 cGy) BALB/c mice. We observed that in particular, MHC class II-negative stroma (endothelial cells and fibroblasts), as well as a population of MHC class II-intermediate stroma (epithelial cells) upregulated the expression of both Fas and DR5 after irradiation. Our study defines the specific pathways for cytolysis, trafficking and immune modulation involved in tGVHD and suggests selective therapeutic targets to attenuate tGVHD and improve post-transplant T-cell reconstitution in patients with GVHD. Fig 1. BLI demonstrate a distinct distribution pattern for alloreactive donor T cells in allogeneic BMT recipients, Allogeneic Balb/c recipients show a strong signal on day 4 post-transparent after transfer of 10×108 luc+ splenocytes as measured by total body photon emission. Ex vivo imaging confirms the infiltration of luc+ splenocytes to the thymus. Fig 1. BLI demonstrate a distinct distribution pattern for alloreactive donor T cells in allogeneic BMT recipients, Allogeneic Balb/c recipients show a strong signal on day 4 post-transparent after transfer of 10×108 luc+ splenocytes as measured by total body photon emission. Ex vivo imaging confirms the infiltration of luc+ splenocytes to the thymus. Fig 2. We assessed the role of molecules relevant for T cell trafficking (A), cytolytic function (B), and co-stimulation, co-inhibition (C). Irradiated BALB/c mice received 5×106 T cell depleted C57BL/6 bone marrow + 0.25×106 purified splenic T cells. Absolute numbers of donor-BM-derived CD4+CD8+ thymocytes are shown. Black bars indicate means. p-values were calculated vs. recipients of WT T cells(*p

T cell deficiencies can occur in many settings, including aging, autoimmune and genetic disorders, hematological malignancies, infectious diseases, and exposure to cytotoxic/cytostatics agents. Notch-based culture systems can be utilized for ex vivo generation of large numbers of T lineage committed lymphoid precursor cells, and we recently reported that the adoptive transfer of T cell precursors significantly enhances T cell reconstitution and function after allogeneic T cell depleted HSCT. The aim of this study was to evaluate if allogeneic T cell precursors are safe and effective when used for adoptive transfer across MHC barriers in the absence of allogeneic HSCs to overcome radiation injury, enhance T cell function and improve anti-tumor activity in immunosuppressed recipients. We found that positive selection of adoptively transferred allogeneic (C57BL/6) precursor cells ± syngeneic (BALB/c) HSCs in irradiated hosts (BALB/c) is dependent on MHC molecules on non-hematopoietic host cells, resulting in the in vivo development of host-MHC restricted allogeneic T cells. We demonstrate that negative selection of these cells is mediated by donor-derived antigen presenting cells (APCs), resulting in host-tolerance. Adoptively transferred allogeneic T cell precursors significantly improved survival of BALB/c mice after irradiation (675 cGy) and enhanced anti-tumor activity against liquid (A20 lymphoma) and solid (renal cell carcinoma) tumors in syngeneic HSCT recipients. Furthermore, we demonstrate the feasibility of genetic engineering of antigen-specific T cell precursors, by transducing them to express a chimeric antigen receptor (CAR) targeting human CD19. Transduction efficiencies were routinely in the range of 50%–70%. Adoptive transfer of CAR-expressing T cell precursors resulted in the in vivo generation of high numbers of appropriately selected CAR-expressing T cells with significantly enhanced anti-tumor activity (compared with CAR-negative T cell precursors) against a CAR-sensitive tumor, but without any undesirable auto/alloreactivity. We conclude that adoptively transferred allogeneic T cell precursors develop into host-MHC restricted and host-tolerant T cells characterized by selection of a functional TCR repertoire even in a fully mismatched thymic epithelial MHC environment. This strategy overcomes important limitations of conventional adoptive T cell therapies: rejection, alloreactivity and impaired antigen recognition due to restriction to MHC disparate from the one expressed on APCs. The use of allogeneic instead of autologous cells eliminates the risk of contamination with residual malignant patient cells and allows the generation and storage of virtually unlimited quantities of precursor cells for ‘off-the-shelf’ immunotherapy. This procedure has not only substantial logistic advantages, but it facilitates ex vivo manipulation, in particular genetic engineering, to generate antigen-specific or otherwise enhanced designer cells. Adoptive transfer of MHC mismatched and genetically enhanced T cell precursors therefore represents a promising novel strategy for targeted ‘off-the-shelf’ immunotherapy in immunosuppressed patients.

KGF is a fibroblast growth factor family member (FGF-7) that mediates epithelial cell proliferation and differentiation in a variety of tissues, including thymic epithelial cells. Recent studies have demonstrated that KGF administration (before conditioning) to the recipients of allogeneic bone marrow transplantation (BMT) can enhance T cell and thymic reconstitution. Therefore, we studied the role of KGF on T cell development under normal and stress conditions (such as irradiation) by using KGF (−/−) mice in experimental murine models. Phenotypic analysis of KGF (−/−) mice at varying ages demonstrated that the bone marrow, thymus and lymph node cellularity and the cell distribution among KGF (−/−), wild type (WT) and KGF (+/−) mice (6–11 weeks of age) were similar. However, splenic cellularity and splenic T cell numbers were slightly lower than WT and littermate controls. KGF (−/−) mice are more vulnerable to sublethal irradiation (450–600 cGy) and a more than six fold decrease was found in thymic cellularity when analyzed on day 28 after irradiation, whereas there was no delay in the erythroid, myeloid and platelet recovery after irradiation. We used bone marrow transplantation models to assess the relative contribution of KGF produced by thymocytes or thymic epithelial cells during thymic regeneration after irradiation. T cell reconstitution was impaired in syngeneic or allogeneic KGF (−/−) BMT recipients in comparison to littermate controls but there was no difference in the distribution of thymocyte subsets or splenic T cell content. The recipients of KGF (−/−) bone marrow had decreased thymic and splenic cellularity after allogeneic BMT. These data demonstrate that both donor and host derived KGF play a role in thymic regeneration. Finally, KGF administration to young and old mice (10 month old) enhanced thymopoiesis when analyzed 28 days after KGF administration. Pre-BMT KGF administration to the recipients of syngeneic and allogeneic BMT also resulted in a 2–6 fold increase in the thymic cellularity compared to the control group. We conclude that KGF produced by both thymocytes and epithelial cells is not required for normal and post-natal thymic development, but plays a role in post-natal thymic regeneration after irradiation. These data support the potential use of KGF to protect or restore thymic damage after chemo/radiotherapy.