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T cells are widely used to promote engraftment of hematopoietic stem cells (HSCs) during an allogeneic hematopoietic cell transplantation. Their role in overcoming barriers to HSC engraftment is thought to be particularly critical when patients receive reduced doses of preparative chemotherapy and/or radiation compared with standard transplantations. In this study, we sought to delineate the effects CD4+ cells on engraftment and blood formation in a model that simulates clinical hematopoietic cell transplantation by transplanting MHC-matched, minor histocompatibility–mismatched grafts composed of purified HSCs, HSCs plus bulk T cells, or HSCs plus T cell subsets into mice conditioned with low-dose irradiation. Grafts containing conventional CD4+ T cells caused marrow inflammation and inhibited HSC engraftment and blood formation. Posttransplantation, the marrows of HSCs plus CD4+ cell recipients contained IL-12–secreting CD11c+ cells and IFN-γ–expressing donor Th1 cells. In this setting, host HSCs arrested at the short-term stem cell stage and remained in the marrow in a quiescent cell cycling state (G0). As a consequence, donor HSCs failed to engraft and hematopoiesis was suppressed. Our data show that Th1 cells included in a hematopoietic allograft can negatively impact HSC activity, blood reconstitution, and engraftment of donor HSCs. This potential negative effect of donor T cells is not considered in clinical transplantation in which bulk T cells are transplanted. Our findings shed new light on the effects of CD4+ T cells on HSC biology and are applicable to other pathogenic states in which immune activation in the bone marrow occurs such as aplastic anemia and certain infectious conditions.

Graft-versus-host disease (GVHD) remains a major complication of allogeneic hematopoietic cell transplantation. Acute GVHD (aGVHD) results from direct damage by donor T cells, whereas the biology of chronic GVHD (cGVHD) with its autoimmune-like manifestations remains poorly understood, mainly because of the paucity of representative preclinical models. We examined over an extended time period 7 MHC-matched, minor antigen–mismatched mouse models for development of cGVHD. Development and manifestations of cGVHD were determined by a combination of MHC allele type and recipient strain, with BALB recipients being the most susceptible. The C57BL/6 into BALB.B combination most closely modeled the human syndrome. In this strain combination moderate aGVHD was observed and BALB.B survivors developed overt cGVHD at 6 to 12 months affecting eyes, skin, and liver. Naive CD4+ cells caused this syndrome as no significant pathology was induced by grafts composed of purified hematopoietic stem cells (HSCs) or HSC plus effector memory CD4+ or CD8+ cells. Furthermore, co-transferred naive and effector memory CD4+ T cells demonstrated differential homing patterns and locations of persistence. No clear association with donor Th17 cells and the phenotype of aGVHD or cGVHD was observed in this model. Donor CD4+ cells caused injury to medullary thymic epithelial cells, a key population responsible for negative T cell selection, suggesting that impaired thymic selection was an underlying cause of the cGVHD syndrome. In conclusion, we report for the first time that the C57BL/6 into BALB.B combination is a representative model of cGVHD that evolves from immunologic events during the early post-transplant period.

Adoptive transfer of freshly isolated natural occurring CD4+CD25+Foxp3+ regulatory T cells (Treg) prevents graft-versus-host disease (GVHD) in several animal models and following hematopoietic cell transplantation (HCT) in clinical trials. Donor-derived Treg have been mainly used, as they share the same MHC with CD4+ and CD8+ conventional T cells (Tcon) that are primarily responsible for GVHD. Third party–derived Treg are a promising alternative for cellular therapy, as they can be prepared in advance, screened for pathogens and activity, and banked. We explored MHC disparities between Treg and Tcon in HCT to evaluate the impact of different Treg populations in GVHD prevention and survival. Third-party Treg and donor Treg are equally suppressive in ex vivo assays, whereas both donor and third-party but not host Treg protect from GVHD in allogeneic HCT, with donor Treg being the most effective. In an MHC minor mismatched transplantation model (C57BL/6 → BALB/b), donor and third-party Treg were equally effective in controlling GVHD. Furthermore, using an in vivo Treg depletion mouse model, we found that Treg exert their main suppressive activity in the first 2 d after transplantation. Third-party Treg survive for a shorter period of time after adoptive transfer, but despite the shorter survival, they control Tcon proliferation in the early phases of HCT. These studies provide relevant insights on the mechanisms of Treg-mediated protection from GVHD and support for the use of third-party Treg in clinical trials.

Summary Embryonic stem cells (ESCs) hold promise for the treatment of many medical conditions; however, their utility is limited by immune rejection. The objective of our study is to establish tolerance or promote engraftment of transplanted ESCs as well as mature cell populations derived from ESCs. Luciferase ( luc + )-expressing ESCs were utilized to monitor the survival of the ESCs and differentiated progeny in living recipients. Allogeneic recipients conditioned with fractioned total lymphoid irradiation (TLI) and anti-thymocyte serum (ATS) or TLI plus regulatory T cells (T reg ) promoted engraftment of ESC allografts after transplantation. Following these treatments, the engraftment of transplanted terminally differentiated endothelial cells derived from ESCs was also significantly enhanced. Our findings provide clinically translatable strategies of inducing tolerance to adoptively transferred ESCs for cell replacement therapy of medical disorders.

Natural killer (NK) cells are potent anti-viral and antitumor “first responders” endowed with natural cytotoxicity and cytokine production capabilities. To date, attempts to translate these promising biologic functions through the adoptive transfer of NK cells for the treatment of cancer have been of limited benefit. Here we trace the fate of adoptively transferred murine NK cells and make the surprising observation that NK cells traffic to tumor sites yet fail to control tumor growth or improve survival. This dysfunction is related to a rapid down-regulation of activating receptor expression and loss of important effector functions. Loss of interferon (IFN)γ production occurs early after transfer, whereas loss of cytotoxicity progresses with homeostatic proliferation and tumor exposure. The dysfunctional phenotype is accompanied by down-regulation of the transcription factors Eomesodermin and T-bet, and can be partially reversed by the forced overexpression of Eomesodermin. These results provide the first demonstration of NK-cell exhaustion and suggest that the NK-cell first-response capability is intrinsically limited. Further, novel approaches may be required to circumvent the described dysfunctional phenotype.

Because of the perception that depleting hematopoietic grafts of T cells will result in poorer immune recovery and in increased risk of graft rejection, pure hematopoietic stem cells (HSC), which avoid the potentially lethal complication of graft-versus-host disease (GVHD), have not been used for allogeneic hematopoietic cell transplantation (HCT) in humans. Ideal grafts should contain HSC plus mature cells that confer only the benefits of protection from pathogens and suppression of malignancies. This goal requires better understanding of the effects of each blood cell type and its interactions during engraftment and immune regeneration. Here, we studied hematopoietic reconstitution post-HCT, comparing grafts of purified HSC with grafts supplemented with T cells in a minor histocompatibility antigen (mHA)-mismatched mouse model. Cell counts, composition, and chimerism of blood and lymphoid organs were evaluated and followed intensively through the first month, and then subsequently for up to 1 yr. Throughout this period, recipients of pure HSC demonstrated superior total cell recovery and lymphoid reconstitution compared with recipients of T cell-containing grafts. In the latter, rapid expansion of T cells occurred, and suppression of hematopoiesis derived from donor HSC was observed. Our findings demonstrate that even early post-HCT, T cells retard donor HSC engraftment and immune recovery. These observations contradict the postulation that mature donor T cells provide important transient immunity and facilitate HSC engraftment.

The adoptive transfer of CD4+CD25+Foxp3+ regulatory T cells (Tregs) in murine models of allogeneic hematopoietic cell transplantation (HCT) has been shown to protect recipient mice from lethal acute graft-versus-host disease (GVHD) and this approach is being actively investigated in human clinical trials. Here, we examined the effects of cryopreservation on Tregs. We found that freeze and thaw of murine and human Tregs is associated with reduced expression of L-selectin (CD62L), which was previously established to be an important factor that contributes to the in vivo protective effects of Tregs. Frozen and thawed murine Tregs showed a reduced capacity to bind to the CD62L binding partner MADCAM1 in vitro as well as an impaired homing to secondary lymphoid organs in vivo. Upon adoptive transfer frozen and thawed Tregs failed to protect against lethal GVHD compared with fresh Tregs in a murine model of allogeneic HCT across major histocompatibility barriers. In summary, the direct administration of adoptively transferred frozen and thawed Tregs adversely affects their immunosuppressive potential which is an important factor to consider in the clinical implementation of Treg immunotherapies.

Acute graft-versus-host disease (GVHD) is induced by alloreactivity of donor T cells toward host antigens presented on antigen-presenting cells (APCs). Apoptotic cells are capable of inducing tolerance by altering APC maturation. Apoptosis can be induced by extracorporeal photopheresis (ECP). We demonstrate that the use of ECP as a prophylaxis prior to conditioning significantly improves survival (P < .0001) after bone marrow transplantation (BMT) by inhibiting the initiation phase of acute GVHD in a murine BMT model. ECP-treated autologous splenocytes resulted in immune tolerance in the host, including reduced dendritic cell activation with decreased nuclear factor-κB engagement, increased regulatory T-cell (Treg) numbers with enhanced expression of cytolytic T lymphocyte-associated antigen 4, potentiating their suppressive function. The protective effect required host production of interleukin-10 and host Tregs. Conventional T cells that entered this tolerant environment experienced reduced proliferation, as well as a reduction of tissue homing and expression of activation markers. The induction of this tolerant state by ECP was obviated by cotreatment with lipopolysaccharide, suggesting that the inflammatory state of the recipient prior to treatment would play a role in potential clinical translation. The use of prophylactic ECP may provide an alternative and safe method for immunosuppression in the bone marrow transplant setting.

Total lymphoid irradiation (TLI) with antithymocyte globulin (ATG) is a unique regimen that prepares recipients for allogeneic hematopoietic cell transplantation by targeting lymph nodes, while sparing large areas of the bone marrow. TLI is reported to increase the frequency of CD4(+)CD25(+)FoxP3(+) T-regulatory cells (Treg) relative to conventional T cells. In this study, barriers to hematopoietic stem cell (HSC) engraftment following this nonmyeloablative conditioning were evaluated. TLI/ATG resulted in profound lymphoablation but endogenous host HSC remained. Initial donor HSC engraftment occurred only in radiation exposed marrow sites, but gradually distributed to bone marrow outside the radiation field. Sustained donor engraftment required host lymphoid cells insofar as lymphocyte deficient Rag2γc(-/-) recipients had unstable engraftment compared with wild-type. TLI/ATG treated wild-type recipients had increased proportions of Treg that were associated with increased HSC frequency and proliferation. In contrast, Rag2γc(-/-) recipients who lacked Treg did not. Adoptive transfer of Treg into Rag2γc(-/-) recipients resulted in increased cell cycling of endogenous HSC. Thus, we hypothesize that Treg influence donor engraftment post-TLI/ATG by increasing HSC cell cycling, thereby promoting the exit of host HSC from the marrow niche. Our study highlights the unique dynamics of donor hematopoiesis following TLI/ATG, and the effect of Treg on HSC activity.

Sequential analysis of chimerism after allogeneic blood stem cell transplantation (BSCT) has been shown to be predictive for graft failure and relapse. We have explored the impact of a novel approach for the quantitative determination of chimerism using a commercial PCR assay with multiplex amplification of nine STR-loci and fluorescence detection. The feasibility was studied in 121 patients transplanted from related or unrelated donors. Follow-up investigation was performed in 88 patients. Twenty-eight of these patients had received a transplantation after dose-reduced conditioning therapy. Results were compared to data obtained by FISH analysis in a subgroup of patients receiving grafts from sex-mismatched donors. The analysis was possible in all patients, the median number of informative alleles was 4 (range 1-8) compared to 7 (range 1-9) in the related and unrelated situation, respectively. A good correlation was seen in 84 samples from 14 patients analyzed in parallel with STR-PCR and FISH. Decreasing values of donor chimerism were detected prior to or concomitantly with the occurrence of graft failure and relapse of disease in all patients investigated prospectively. Using FACS-sorted material, eg peripheral blood CD34+ cells, the assay permitted the detection of residual recipient cells with high sensitivity (down to one CD34+ Kasumi cell in 40,000 normal WBC). Evaluation of the inter-laboratory reproducibility revealed that in 20 samples analyzed in three different centers, the median coefficient of variation was 2.1% (range 0.7-9.6%). Taken together, the results support the use of the test as a valuable tool in the follow-up of patients undergoing allogeneic BSCT. In cases lacking PCR-detectable disease-specific gene products, this assay may represent an alternative to recently established real-time PCR methods.

Steroid refractory gastrointestinal (GI) acute graft-versus-host disease (aGVHD) is a major cause of mortality in hematopoietic stem cell transplantation (HCT) without immune markers to establish a diagnosis or guide therapy. We found that T-cell receptor β (TCRβ) complementarity-determining region 3 repertoire sequencing reveals patterns that could eventually serve as a disease biomarker of T-cell alloreactivity in aGVHD. We identified T-cell clones in GI biopsies in a heterogeneous group of 15 allogeneic HCT patients with GI aGVHD symptoms. Seven steroid-refractory aGVHD patients showed a more conserved TCRβ clonal structure between different biopsy sites in the GI tract than 8 primary therapy–responsive patients. Tracking GI clones identified longitudinally at endoscopy in the blood also revealed an increased clonal expansion in patients with steroid-refractory disease. Immune repertoire sequencing-based methods could enable a novel personalized way to guide diagnosis and therapy in diseases where T-cell activity is a major determinant.

A major challenge following allogeneic hematopoietic stem cell transplantation (HCT) is to establish persistent engraftment of donor hematopoietic cells. Many strategies have been developed to permit engraftment involving high dose chemotherapy, serotherapy with anti-lymphocyte drugs or myeloablative irradiation resulting in highly toxic conditioning regimens. The introduction of less harmful therapies could result in less toxicity especially in the major mismatched setting and when reduced intensity conditioning is required. While recent studies have explored the mechanisms through which donor-type CD4+CD25+FoxP3+ regulatory T cells (Tregs) restrict the development of graft versus host and host versus graft reactions, less is known about the role of host-type Treg in the transplant setting. In syngeneic and minor mismatched HCT host Tregs comprise a major component of the Treg compartment in the first weeks after transplant. Moreover the transplant of in vitro primed host Tregs can improve donor engraftment in major mismatched models of HCT; therefore host Tregs could be one of the key controllers of the host versus graft reaction mediated by residual host CD4+ and CD8+ conventional T cells (Tcons), possibly influencing graft versus host disease (GvHD) onset and severity. In this study we investigated the role of host Treg after major mismatched HCT to understand their impact in graft facilitation and rejection and in GvHD induction and prevention. We investigated the mechanism through which this cell population works and we explored the feasibility and the effectiveness of host Treg adoptive transfer for cellular therapy in HCT animal models. Results CD4+CD25+FoxP3+ host Tregs persist for at least 28 days after total body irradiation (8 Gy) and transplantation of C57BL/6 (H-2b) T cell depleted bone marrow (TCD BM) into BALB/C (H-2d) mice. Host Treg could be found in spleen, lymph nodes and bone marrow with an increase in the Treg/CD4+ cell ratio. Moreover we observed that these residual host Tregs maintain their suppressive function in vitro if harvested 14 days after transplant and incubated with healthy mouse derived Tcons in a MLR. These results are even more relevant as transplanted mouse derived host Tcons lose their ability to proliferate confirming that host Tregs possess a numeric and functional advantage compared to residual host Tcons. Using FOXP3-DTR mice as hosts we observed that host Treg ablation results in reduced donor chimerism after major mismatched TCD BM transplant (p < 0.01, analysis performed 2 months after transplant). At the same time, the absence of host Tregs favors host CD4+ T cell persistence (p < 0.001) and delays B cell reconstitution (p < 0.001). Furthermore, we hypothesized that host Treg act as an immunological barrier for HSCs, providing a protective immunological niche. Confocal microscopic analysis of femurs performed at day 7 after TCD BM transplant confirmed that hypothesis showing host Tregs clustering in the epiphysis where donor hematopoietic stem cell (HSC) engraftment is mainly detectable. To strengthen these results and to provide a clinical translatable tool, we adoptively transferred 5x105/mouse highly purified unmanipulated host Tregs in a non myeloablative (TBI 5.5 Gy) major mismatched model of rejection. We found that the transferred host Tregs induce persistent full donor chimerism if injected together with a sublethal dose of donor Tcons (5x105/mouse, p=0.016) and transiently enhance donor chimerism in the first three weeks after transplant if injected with low dose interleukin-2 (IL-2, 50,000 IU bid for 7 days; p < 0.001) without impacting on GvHD incidence and lethality. The relatively low dose of injected Tregs, the possibility to stimulate and expand them in vivo with IL-2 and the safety of this model provide the first evidence of the feasibility of this clinical approach. Conclusion Our findings indicate that host Tregs facilitate bone marrow engraftment in major mismatched HCT models without impacting GvHD. Notably, our observations on the bone marrow environment after transplant strongly suggest that host Tregs can play a role in building the donor HSC cell niche. Finally host Treg adoptive transfer proved to be feasible and effective in animal models providing a new tool for cellular therapy and clinical translation. Disclosures: No relevant conflicts of interest to declare.

Impaired immunity is a fundamental obstacle to successful allogeneic hematopoietic cell transplantation. Mature graft T cells are thought to provide protection from infections early after transplantation, but can cause life-threatening graft-vs.-host disease. Human CMV is a major pathogen after transplantation. We studied reactivity against the mouse homologue, murine CMV (MCMV), in lethally irradiated mice given allogeneic purified hematopoietic stem cells (HSCs) or HSCs supplemented with T cells or T-cell subsets. Unexpectedly, recipients of purified HSCs mounted superior antiviral responses compared with recipients of HSC plus unselected bulk T cells. Furthermore, supplementation of purified HSC grafts with CD8 + memory or MCMV-specific T cells resulted in enhanced antiviral reactivity. Posttransplantation lymphopenia promoted massive expansion of MCMV-specific T cells when no competing donor T cells were present. In recipients of pure HSCs, naive and memory T cells and innate lymphoid cell populations developed. In contrast, the lymphoid pool in recipients of bulk T cells was dominated by effector memory cells. These studies show that pure HSC transplantations allow superior protective immunity against a viral pathogen compared with unselected mature T cells. This reductionist transplant model reveals the impact of graft composition on regeneration of host, newly generated, and mature transferred T cells, and underscores the deleterious effects of bulk donor T cells. Our findings lead us to conclude that grafts composed of purified HSCs provide an optimal platform for in vivo expansion of selected antigen-specific cells while allowing the reconstitution of a naive T-cell pool.

Backgrounds: CD4+FoxP3+ regulatory T cells (Treg) have been shown in pre-clinical murine models and human clinical trials to protect against acute graft-versus-host (GVHD) disease following hematopoietic cell transplantation (HCT). In murine models, Treg expression of CD62L is required for in vivo protection of GVHD (Strober 2005/Blazer 2004). Certain aspects of Treg isolation and administration could affect CD62L expression, such as the use of G-CSF mobilization or freeze-and-thaw. We hypothesized that freeze-thaw of Tregs could reduce CD62L expression and prevent Tregs from protecting against GVHD. Furthermore, we hypothesized that the loss of CD62L expression is mediated at least in part by metalloproteinases (MP). Methods and Results: We used established protocols of GVHD, in which lethal dose of fractionated total body irradiation was followed by murine bone marrow transplantation across major histocompatibility barriers. We sort-purified CD4+Foxp3+ Tregs based on CD25+(high) expression and measured baseline expression of CD62L. Cells were then gradually frozen in fetal calf serum/10%DMSO, and finally stored in liquid nitrogen for at least 7 days. After thawing Tregs showed an average of 58% of the baseline CD62L expression (CD62L expression fresh vs. frozen p=0.0009, n=5). Binding of frozen Tregs to plate bound L-Selectin ligand Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in vitrowas significantly reduced as compared to fresh Tregs (p=0.0003). To test the potential effect of freeze and thaw in vivo, we used an established murine model in which Tregs are given prior to conventional T cells (Tcon) to protect against GHVD. At the time of transplant conditioned BALB/c donors received 5x10^6 whole bone marrow and 5x10^5 CD4+CD25(high) Tregs (day 0), followed by 1x10^6 Tcon (day +2). Frozen/thawed Tregs and fresh Tregs respectively were given at the same time point at the same quantity of viable cells. Using luciferase-expressing cells (luc+) and BLI imaging we could show that homing of frozen luc+Tregs into pLN at day+5 was impaired as compared to fresh Tregs (p=0.0005). Relative weight loss as an indicator for GVHD was significantly higher and survival was significantly reduced in mice receiving frozen Treg in comparison to mice receiving fresh Treg. To confirm the importance of CD62L expression on Treg function we blocked CD62L in vivo by incubating fresh Tregs with monoclonal blocking antibody Mel14 (120ug/ml) for 1 hour prior to injection. A significantly higher proliferation of luc+ Tcon was observed in mice injected with Mel14-treated Tregs as compared to isotype control-treated Tregs. To test the potential role of metalloproteinases in cleaving CD62L following freeze and thaw, we added various metalloproteinase inhibitors (MPI) during the freezing process. We found that the addition of at least one MPI GM6001 (50nM) can diminish the loss of CD62L. Conclusion: Augmenting or manipulating Tregs of the donor graft is a promising area of cellular therapy that could be used to prevent GVHD in HCT and in potentially many other clinical contexts. We have found that the process of freeze and thaw reduces CD62L expression on Tregs and reduces protection against GVHD. Further, MPs may play role in the loss of CD62L expression and the use of MPIs for frozen products might preserve Treg function. Disclosures No relevant conflicts of interest to declare.

CD4+FoxP3+ regulatory T cells (Treg) are a rare cell population that is responsible for peripheral immune tolerance. Treg adoptive transfer has proved to be an effective treatment for graft versus host disease prevention in several preclinical and clinical studies. The impact of Treg on immune reconstitution and bone marrow engraftment after transplantation has been less well studied. We treated C57BL/6 FoxP3-DTR mice that carry the diphtheria toxin (DT) receptor in the promoter of the FoxP3 gene with DT resulting in a complete ablation of Treg (percentage of FoxP3+CD4+ cells over CD4+ cells < 0.2% in peripheral blood, spleen, lymph nodes, thymus and bone marrow). Transplantation of lethally irradiated (TBI 10 Gy) Treg depleted mice with allogeneic (BALB/C or FVB/N) T-cell depleted bone marrow (TCD BM) resulted in rejection or reduced donor chimerism (p < 0.01). Treg depletion favored host CD4+ (p < 0.001), CD8+ (p < 0.01) and GR1+ cell persistence (p < 0.01) and delayed B cell reconstitution (p < 0.001). Adoptive transfer of purified host type Treg in vitro activated with Interleukin-2 (IL-2) and anti-CD3/CD28 beads to DT treated mice rescued engraftment (p < 0.01) and boosted B cell reconstitution (DT treated mice that received Treg vs DT treated mice only p < 0.001; DT treated mice that received Treg vs untreated mice p < 0.01). Moreover Treg ablation resulted in rejection or reduced donor chimerism (p < 0.01) in mice transplanted with allogeneic (FVB/N) purified Lin-Sca1+cKit+ hematopoietic stem cells (HSCs) demonstrating that Treg promote donor HSC engraftment without interacting with donor derived facilitating cells. We explored the mechanism through which Treg impact donor engraftment and immune reconstitution and observed that Treg depleted mice transplanted with syngeneic (C57BL/6 CD45.1) TCD BM engrafted (p > 0.05) but had markedly delayed B cell reconstitution (p < 0.01) thus Treg promote donor B cell differentiation in manner not dependent upon alloreactivity. FACS analysis of bone marrow cells of syngeneic transplanted mice showed higher numbers of donor Lin-Sca1+cKit+ HSCs (p < 0.05) and donor Lin-Sca1+cKit+Flt3+ lymphoid progenitors (p < 0.05) while numbers of B220+IgM-CD19+cKit+ Pro-B cells (p < 0.05) and total CD19+ cells (p < 0.01) were reduced after Treg depletion demonstrating a block of maturation in the early phases of B cell differentiation. Confocal microscopic analysis of femurs after transplantation with TCD BM were used to determine the spatial relationship between Treg, B cells and HSCs. Treg localize near the endosteum (p < 0.05) and cluster in the epiphyseal areas where donor HSCs and B220+ B cells were mainly detectable suggesting that Treg act as an immunological barrier for HSCs and B cell progenitors, providing a protective immunological niche. Further, adoptive transfer of IL-2 and CD3/CD28 bead activated Treg induced B cell reconstitution in non irradiated immune deficient BALB/C rag2-/-γc-/- mice that received an infusion of allogeneic (C57BL/6) TCD BM. Bone marrow analysis of these mice revealed higher numbers of B220+IgM-CD19+cKit+ Pro-B cells (p < 0.05) and total CD19+ cells (p < 0.05) in mice that received Treg. Analysis of the sera after Treg adoptive transfer showed reduced production of inflammatory cytokines and chemokines such as Interferon-γ, Interleukin-12, Interleukin-13, Interleukin-1b, CXCR-9 and CXCR-10 and increased production of VEGF. In conclusion, our findings clearly indicate that Treg act as a key regulator of B cell differentiation promoting production of mature B cells. This effect is not dependent on alloantigen recognition and Treg localization after transplantation suggests that Treg play a role in building the donor HSC and B cell precursor niche. Finally, adoptive transfer of Treg enhances B cell immune reconstitution and induces tolerance to allogeneic bone marrow grafts even in the absence of conditioning providing a new tool for clinical translation in children with severe combined immune deficiencies or hemoglobinopathies and in patients undergoing organ transplantation. Disclosures No relevant conflicts of interest to declare.

Lag-3 has emerged as an important molecule in T cell biology. We investigated the role of Lag-3 in conventional T cell (Tcon) and regulatory T cell (Treg) function in murine GVHD with the hypothesis that Lag-3 engagement diminishes alloreactive T cell responses after bone marrow transplantation. We demonstrate that Lag-3 deficient Tcon (Lag-3(-/-) Tcon) induce significantly more severe GVHD than wild type (WT) Tcon and that the absence of Lag-3 on CD4 but not CD8 T cells is responsible for exacerbating GVHD. Lag-3(-/-) Tcon exhibited increased activation and proliferation as indicated by CFSE and bioluminescence imaging analyses and higher levels of activation markers such as CD69, CD107a, granzyme B, and Ki-67 as well as production of IL-10 and IFN-g early after transplantation. Lag-3(-/-) Tcon were less responsive to suppression by WT Treg as compared to WT Tcon. The absence of Lag-3, however, did not impair Treg function as both Lag-3(-/-) and WT Treg equally suppress the proliferation of Tcon in vitro and in vivo and protect against GVHD. Further, we demonstrate that allogeneic Treg acquire recipient MHC class II molecules through a process termed trogocytosis. As MHC class II is a ligand for Lag-3, we propose a novel suppression mechanism employed by Treg involving the acquisition of host MHC-II followed by the engagement of Lag-3 on T cells. These studies demonstrate for the first time the biologic function of Lag-3 expression on conventional and regulatory T cells in GVHD and identify Lag-3 as an important regulatory molecule involved in alloreactive T cell proliferation and activation after bone marrow transplantation.

Donor derived regulatory T cells (Tregs) effectively prevent graft versus host disease (GVHD) in mouse models and in early phase clinical trials. Interleukin 2 (IL-2) therapy in patients with chronic GVHD (cGVHD) can increase Treg number and the Treg/CD4+ T cell ratio resulting in organ damage reduction and symptom relief. Less is known regarding Treg-based treatment for acute GVHD (aGVHD). In this study we evaluated the role of donor Treg cellular therapy for aGVHD treatment in well established murine models. T cell depleted bone marrow (TCD BM) from C57BL/6 mice was transplanted into lethally irradiated (8 Gy) BALB/C recipients together with 7.5x105 to 1x106/animal donor derived luc+ Tcons. Naturally occurring CD4+CD25+FoxP3+ donor type Tregs (nTregs) were purified from C57BL/6 donor mice. 2.5x105/mouse nTregs were injected at day 6 or 7 after transplant in mice that showed clear clinical signs of aGVHD and Tcon proliferation assessed by bioluminescence imaging (BLI). Survival analysis showed a favorable trend for nTreg treated mice, but the impact of this treatment was modest and not statistically significant (p 0.08). aGVHD is a disease characterized by the activation and rapid proliferation of alloreactive donor conventional T cells (Tcons) directed against host antigens, so one of the major obstacles of this approach is to overcome the large number and effector function of activated Tcons. Several studies have utilized ex vivo expansion of Tregs to increase their number with the goal of maintaining suppressive function. We developed a different strategy with the intent to “educate” Tregs to specifically suppress the reactive Tcon population. We incubated 2.5x105 donor derived Tregs with irradiated (3000 cGy) blood of aGVHD affected mice for 20 hours without further stimulation and injected the entire pool of these cells, termed educated Treg (eTregs), at day 7 or 8 after transplant and Tcon injection. Interestingly eTregs significantly improved aGVHD affected mouse survival (p = 0.0025 vs Tcons alone). BLI showed no difference between the groups (p = 0.85) because the treatment intervened after Tcon proliferation and activation was initiated. To evaluate eTreg impact on graft versus tumor (GVT) effects, we transplanted BALB/C mice with C57BL/6 TCD BM and 1x104/mouse luc+ A20 tumor cells along with 1x106/mouse donor Tcons and 2.5x105 eTregs. Mice that received TCD BM and A20 tumor cells alone died from progressive tumor growth, while mice that received Tcons died from GVHD without tumor engraftment. Further animals that received both Tcon and eTreg treatment did not have tumor engraftment demonstrating that eTregs do not impact Tcon mediated GVT effects. Further studies are ongoing to characterize the eTreg population as compared to nTreg, with respect to expression of activation markers and in functional assays. Our observations indicate that Tregs can be ex vivo educated to suppress in vivo reactive and proliferating Tcons. Moreover our data demonstrate that eTreg adoptive transfer is clinically feasible and promising. These findings may be relevant for the development of clinical grade Treg based cellular therapy for the treatment of conditions caused by immune dysregulation such as aGVHD and autoimmune diseases and for transplant tolerance induction. Disclosures: No relevant conflicts of interest to declare.

Abstract 2366 The bone marrow (BM) is a complex microsystem that supports lifelong blood production and dynamically responds to injury, blood cell loss and senescence. At steady-state most hematopoietic stem cells (HSC) are quiescent. However, in situations of increased demand, their activation is triggered by an array of signals, such as cytokines released during infections. The susceptibility of BM to injury by T cells (TC) is long recognized, e.g. in aplastic anemia, and BM failure syndromes, and the inhibitory effect of IFNg on the proliferative activity of (HSC) is established. How donor TC given as part of an allogeneic hematopoietic cell transplantation (HCT) affect the recipient BM microenvironment and influence HSC activity has not been elucidated. Yet, graft TC are believed to be essential to overcome host barriers, especially following lower intensity (non-myeloablative) conditioning that spares host HSC and residual host immunity. We examined in an MHC-matched, minor-antigen-mismatched mouse model of non-myeloablative HCT the impact of graft TC subsets on HSC and hematopoietic reconstitution. BALB.K (H2k) and BALB.B (H2b) mice were prepared with sublethal radiation at a dose that allows donor cells (AKR/J [H2k] and C57BL/6 [H2b]) to engraft but also permits survival of host HSC and immune populations. Grafts consisted of purified HSC (KTLS; cKit+Thy1.1loLin−Sca-1+) given alone or supplemented with donor TC or TC subsets. Rather than improve engraftment, donor TC, specifically CD4+ cells, resulted in detainment of host HSC in their niches and consequent failure of donor HSC to engraft. These events appeared to be initiated specifically by conventional alloreactive donor CD4+CD25− cells. These CD4+CD25− cells interacted with immune-competent host cells, stimulating CD11c+ dendritic cells to upregulate the activation markers CD40 and MHC II. Following their activation dendritic cells produced excessive amounts of IL-12. IL-12, in turn, activated donor CD4+ cells to produce IFNg. This Th1-response was prominent in BM but absent in the spleen. In this Th1-cytokine environment, high levels of quiescent host HSC were observed, arrested at the short-term HSC stage, while more mature, multipotent progenitors were absent. Moreover, HSC arrest correlated with marrow aplasia, analogous to that seen in BM failure syndromes. This hypocellularity was only observed in recipients of CD4+ containing grafts, while recipients given HSC alone or HSC in combination with CD8+ TC had prompt blood regeneration. When resting HSC were FACS-purified from the BM of recipients of HSC+CD4+ and infused into secondary recipients they promptly expanded and gave rise to all blood lineages. Although recipients of HSC+CD4+ cells ultimately recovered blood formation, B lymphopoiesis was markedly delayed. No clinical signs of graft-versus-host disease were noted in these mice. Lymphocytes that recovered in these recipients of HSC+CD4+ cells were of host type only. In contrast, recipients of pure HSC demonstrated prompt donor cell engraftment, and mixed chimerism in all lymphoid lineages, including T cells. Our model corroborates the importance of the cytokine microenvironment in hematopoietic reconstitution and marrow failure post-transplant. Moreover, our findings underscore the importance of IFNg in BM aplasia and support the idea that IFNg can, under physiologic conditions, exert regulatory effects on hematopoiesis by controlling the exit of ST-HSC out of the marrow niche. Our studies shed new light on understanding the physiology of engraftment and marrow failure after HCT using nonmyeloablative conditioning, and indicate that the composition of the graft is of critical importance due to its effects on the marrow microenvironment We postulate that in some naturally occurring settings of BM inflammation (e.g., infections) detainment of resting HSC in their niche may provide protection of hematopoiesis and that comparable events are relevant for engraftment post-HCT. Disclosures: No relevant conflicts of interest to declare.

Abstract 228 Donor T cell alloreactivity in hematopoietic stem cell transplantation (HSCT) can result in acute graft-versus-host disease (GVHD) which is a major cause of morbidity and mortality that limits the clinical use of HSCT. Despite the potential large diversity in possible T cells (1015) it is thought that within each individual patient undergoing HSCT, a tractable and defined number of T cell clones mediate GVHD. We hypothesized that the identification of the most frequent T cell clones in the gastrointestinal (GI) tract of human patients with GVHD and subsequent tracking of these clones in the peripheral blood may provide a way to bracket and study disease related clones. To investigate T cell clonal dynamics in human GVHD, we collected endoscopic GI tract biopsy samples and matched blood specimens for twelve patients undergoing myeloblative HSCT with suspected GI GVHID prior to or within one day of corticosteroid therapy. We also collected peripheral blood thirty days after biopsy. Five of these patients were negative for GVHD or had mild grade I/II GI GVHD, whereas seven had severe and fatal grade III/IV GI GVHD. These patients had heterogenous indications, conditioning regimens and GVHD prophylaxis which included cyclosporine, tacrolimus and methotrexate. None of the patients had cytomegolovirus (CMV) colitis or other identifiable GI or significant systemic infectious complications, although four patients had low-level blood CMV positivity by PCR. As a control, we compared endoscopic GI biopsies from patients with inflammatory bowel disease as well as normal healthy screening subjects. We extracted genomic DNA and performed T cell receptor (TCR) β CDR3 repertoire sequencing at Stanford and with GigaGen GigaMune Rep-Seq™, both using the Illumina next generation sequencing MiSeq™ and HiSeq™ platforms. Results: For each tissue sample from each patient, between 1,000–4,000 unique T cell sequences were observed in endoscopic tissue samples (2–6 samples per patient). We sorted the most frequent clones in the GI tissue samples by rank order and fractionated the top 100 clones for analysis. Many of these top clones were found in the matched peripheral blood at the time of diagnosis with most of these occurring at frequencies well below those in the GI tissue. For the patients with negative or mild GVHD at the time of diagnosis, the GI-identified clones that were found in the peripheral blood had a mean frequency and standard deviation of 0.001369 +/− 0.0016. This is indistinguishable from peripheral blood samples from patients with severe GVHD which showed a mean frequency and standard deviation of 0.0013 +/− 0.0009 (P=0.36, 2 tailed t-test). We next tracked the GI-identified clones in the peripheral blood to the obtain fold change of the clones at thirty days after diagnosis to the time of diagnosis. The patients with negative or mild GVHD had a mean, median, and standard deviation in fold change of 1.9, 1.7, and 0.9 respectively. The patient with severe GVHD patients had a mean, median and standard deviation in fold change of 32.2, 30.7, and 17.3. A two tailed t-test comparison of the two groups showed they were statistically distinguishable with p-values of 0.0003 for examination of the mean fold change and 0.0005 for the median fold change. For patients with severe GVHD, the peripheral blood at 30 days after diagnosis showed a highly oligoclonal expansion of individually private GI-identified clones. Many of these clones were identified in their respective donor T cell pool. For those patients evaluated with upper and lower endoscopy, many clones were shared between the colon and duodenum or gastrum, however, the dominant clones between these sites appeared to be different. Conclusion: We found that a comparison of the fold change in the peripheral blood frequencies of the top one hundred clones identified in the GI tract biopsies can statistically distinguish five negative or mild GVHD patients from seven severe and fatal cases. These results support the use of T cell repertoire sequencing and associated approaches in human patients to both clarify the pathophysiology of the disease and eventually perhaps to provide an independent immune biomarker that could guide therapy. A major challenge will be to further phenotype and examine the T cell clones identified in the GI tissue and to investigate ways to determine which clones are highly associated or even causal to GVHD. Disclosures: Meyer: GigaGen Inc: Consultancy, Equity Ownership. Löhr:GigaGen Inc.: Employment, Equity Ownership. Hsu:GigaGen Inc.: Employment, Equity Ownership. Johnson:GigaGen Inc.: Employment, Equity Ownership.

Abstract 1900 GVHD is a major complication of bone marrow transplantation (BMT) and results from donor T cells becoming activated and reacting to host antigens. Recently, lymphocyte activation gene-3 (LAG-3) has emerged as an important molecule, negatively regulating T cell activation and has been proposed to play an important role in CD4+CD25+Foxp3+ regulatory T cell (Treg) function. We investigated the functional in vivo role of LAG-3 in Treg and conventional T cells in murine GVHD with the hypothesis that LAG-3 engagement diminishes alloreactive T cell responses after BMT. Using murine models of acute GVHD in which allogeneic bone marrow cells are transplanted into lethally irradiated hosts, we and others have shown previously that donor Treg are able to suppress GVHD induced by donor allogeneic conventional T cells (Tcon). The role of LAG-3 in Treg function was evaluated both in vitro and in vivo by directly comparing Treg isolated from LAG-3−/− donor mice to Treg isolated from wild type donors (WT Treg). In vitro, in a mixed lymphocyte reaction assay, LAG-3−/− Treg efficiently suppressed the proliferation of alloreactive T cells in a manner similar to WT Treg. In vivo, a bioluminescent imaging assay (BLI) was utilized that allows for quantitative assessment of Tcon proliferation in addition to traditional metrics of GVHD severity including weight loss, survival and GVHD score. Both LAG-3−/− Treg and WT Treg were equally potent at suppressing Tcon proliferation as illustrated by BLI of luc+ T cells and demonstrated a significant increase in median survival time (MST) as compared to mice receiving Tcon only (35 days for Tcon vs. 58 and 68 days for WT and LAG-3−/− Treg, respectively, P=0.03), but there was no significant difference in MST between the groups receiving WT and LAG-3−/− Treg. Interestingly, when LAG-3−/− Tcon were used to induce GVHD in the absence of Treg, GVHD lethality was accelerated. Thus, all mice receiving LAG-3−/− Tcon showed decreased survival and significantly lower body weights than mice receiving WT Tcon (P=0.017). GVHD scores of LAG-3−/− Tcon recipients were also significantly higher than WT Tcon recipients at Day 20 post BMT (6.0 vs. 2.2, P= Disclosures: No relevant conflicts of interest to declare.

Abstract 4037 Allogeneic hematopoietic cell transplantation (HCT) is an effective treatment for many hematologic malignancies such as leukemia, lymphoma and myelodysplastic syndromes. A potentially fatal complication of HCT is graft-versus-host disease (GVHD), where conventional T cells of alloreactive-specificities (CD4+ and CD8+ T cells; Tcon) infiltrate and destroy target organs such as the liver, gut and skin. While immunomodulation is necessary to control the adverse GVHD reactions, an effective immune response is required for successful tumor eradication, named graft-versus-tumor effect (GVT). Following allogeneic bone marrow transplantation, our and other laboratories have shown that the transfer of highly purified regulatory T cells (Treg), a key immunoregulatory cell population involved in the maintenance of immune tolerance, can prevent lethal GVHD induced by the donor-derived Tcon in multiple models across both major and minor histocompatibility barriers. Notably, when transferred into recipient mice with established leukemia or lymphoma, Treg cells effectively suppress Tcon cell proliferation and prevent lethal GVHD, while preserving GVT activity. We have recently adapted our pre-clinical model to an ongoing clinical trial, and are currently assessing the efficacy of highly purified human Treg cells in a clinical setting. However, recent evidence suggest that, under the influence of intense inflammatory signals, the Tregsuppressive phenotype can become unstable, a phenomenon that can culminate in Tregconversion into IL-17-producing pro-inflammatory cells. IL-17 is a proinflammatory cytokine that has been reported to promote GVHD in different models for human HCT. We hypothesized that under the influence of the intense proinflammatory signals, such as those released during an ongoing alloreaction, a fraction of the transferred Treg might be redirected to the Th17 cell fate, thereby losing immunosuppressive potential and acquiring proinflammatory capacity. Our preliminary in vitro studies demonstrated that Treg cells do indeed upregulate Il17 gene expression following activation by allogeneic antigen presenting cells (APCs). We, therefore, sought to evaluate the impact of Il17 gene ablation on Treg stability and immunosuppressive capacity in a major MHC mismatch model, in order to ultimately assess the possible therapeutic potential of IL-17 targeting for the enhancement of Treg-mediated GVHD suppressive ability following HCT. On the basis of our preliminary studies we anticipated that Treg cells purified from Il17 knockout (KO) donor mice would have enhanced immunomodulatory capacity compared to wild-type (WT) Treg, given their inability to produce IL-17. To monitor ongoing GVHD our laboratory has developed an imaging system that allows for the in vivo visualization of T cell homing and proliferation over time, without the necessity to sacrifice the experimental animals. Specifically, the allogeneic Tcon cells are isolated from donor mice where the gene coding for the enzyme luciferase (luc) is expressed by all hematopoietic cells. Importantly, the proliferation of the transferred luc+ Tcon can be directly assessed by the number of photons detected by the bioluminescent imaging system (BLI), which gives a measure of ongoing GVHD. Treg were purified from both WT and IL-17 KO animals and their GVHD-suppressive ability was directly compared in vivo following the transfer of luc+ Tcon. IL-17 KO Tregcells were as proficient as WT Treg in suppressing GVHD. Similarly, the GVHD-inhibitory capacity of Treg cells isolated from IL-1R KO mice, that were previously shown to be resistant to Th17 conversion, was comparable to that of WT Treg. Furthermore, upon ex vivo re-isolation of Tregthat were previously transferred into transplanted recipients, we could detect little or no IL-17 production by intracellular cytokine staining. Thus, we conclude that freshly isolated and highly purified Treg do not convert into IL-17 producing cells in our model, suggesting that IL-17 production is not a factor affecting Tregstability during ongoing GVHD, and excluding IL-17 targeting as a valuable strategy to improve Treg immunotherapy following bone marrow transplantation. Disclosures: No relevant conflicts of interest to declare.

Abstract 4030 Impaired immune function is a major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). Adoptively transferred donor T cells (TC) mediate graft-versus-host disease (GVHD), and make the use of pharmacological immunosuppression necessary, yet are believed to protect against infections post-HCT (pTX). In an MHC-matched, minor antigen mismatched mouse model, we studied the impact of allogeneic doTC on GVH-related damage of lymphoid organs, their recovery and immune function pTX. Lethally irradiated BALB.B mice received purified hematopoietic stem cells (HSC; cKit+Sca1+Thy1.1loLin−) +/− TC from C57BL/6 (B6) donors. Recipient lymphoid organs, marrow and livers were analyzed at multiple time points. Mice given HSC+TC promptly became full donor chimeras in all lineages and developed moderate GVHD. HSC recipients retained some residual host cells, and had no signs of GVHD. By tetramer analysis and ELISPOT assay, we showed that in pure HSC recipients early pTX residual host TC protected against murine cytomegalovirus infection. Once a nascent donor HSC-derived TC pool was established, these TC reacted robustly against the virus. In contrast, transferred mature allogeneic TC proliferated and expanded in HSC+TC recipients, but did not respond or protect against the virus. To better understand why MHC-matched mature donor TC did not react against this pathogen, even when derived from pre-immunized donors, we studied the lymphatic microenvironment, hypothesizing that only highly organized interactions between cellular and humoral immunity, antigen-presenting and innate immune cells provide full immune function. Comparing tissues of mice given pure HSC +/− TC we found that: (i) lethal radiation caused marked reduction in lymphoid organ cellularity at 2 wks pTX in all transplanted groups. While the cellularity increased rapidly in HSC recipients, severe hypocellularity persisted in HSC+TC recipients, was associated with significantly decreased organ size (lymph nodes [LN] and thymuses) and marked disruption of the histological architecture; (ii) TC phenotypes present in reconstituted mice comprised a mixture of naïve (CD62L+CD44−), central memory (CM; CD62L+CD44+) and effector memory (EM; CD62L−CD44+) cells in HSC recipients, but were largely EM and rarely naïve TC in HSC+TC recipients; (iii) at ∼3 wks pTX donor B cells (BC) dominated lymphoid tissues in HSC recipients, but were absent in HSC+ToTC recipients; (iv) NK cells regenerated promptly and steadily in HSC recipients, constituting up to 20% of all lymphoid cells in the liver at 3 wks pTX, while in HSC+TC recipients they incessantly decreased to near-complete absence; (v) in mesenteric (m) LN of HSC-, but not HSC+TC- recipients, CD4+CD8+CD3− TC were observed, derived from donor HSC. This latter population resembled immature thymic TC, suggesting that peripheral lymphoid organs participate in lymphoid reconstitution in adult recipients; (vi) at 2 wks pTX we noted only in HSC, but not HSC+ToTC recipients persisting and regenerating innate lymphoid cells (ILC). No ‘classical' lymphoid tissue inducer cells, as described in embryogenesis of lymphoid organs, but cells with features of ILC were identified. Donor HSC-derived NKp46+RORgt+CD3− NK-like cells and IL-17A-secreting residual host CD4 TC were present, especially in livers and mLN. Cells that secreted IL-17A only and IL-17A+IFNγ were identified. IL-17A was not detected in recipients of HSC+ToTC, rather their donor TC were characterized by high IFNγ levels. Our studies show that, paradoxically, pure HSC grafts have advantages over TC-replete grafts with regard to immune function pTX. We hypothesize this superiority may be due to the presence of ILC that participate in the repair of lymphoid organs, and the whole spectrum of immune cells, including TC, BC, innate and immature cells emerging in HSC recipients. An intact microenvironment appears to be critical for optimal activation of TC to respond to pathogens. Moreover, our studies imply that donor TC cannot exert their protective immune function, when rapidly expanding alloreactive TC prevent the development of other lymphoid and innate immune cell, and disrupt the lymphoid tissue architecture. Controlling the negative effects of mature donor TC remains a challenge, and requires better understanding of the complexity of functional immune reconstitution pTX. Disclosures: No relevant conflicts of interest to declare.

Abstract 4025 Graft versus host disease (Gvhd) remains the major cause of morbidity and mortality after bone marrow transplantation (BMT). To prevent Gvhd the graft has to be rendered tolerant to avoid alloreactivity towards the host. One promising approach to induce tolerance in the graft is to administer apoptotic cells to the host. Apoptosis can be induced by extracorporeal photopheresis (ECP), a therapy based on exposure of cells to photoactivable 8-methoxypsoralen (8MPO) and UVA irradiation. The effect of ECP treatment is at least in part, due to alteration of dendritic cell (DC) maturation, leading to reduced antigen presentation and Tcell activation. Moreover, ECP treatment increases the number of regulatory Tcells (Treg), further reducing Tcell activation. In contrast to clinical practice where ECP is only applied to established Gvhd, we have developed a pre-emptive murine model with the aim of inhibiting the initiation phase of acute Gvhd and improving survival. Here we describe our model and characterization of the mechanisms of action. Apoptotic host-type splenocytes were infused into MHC matched or mismatched recipients 5 and 2 days prior to lethal irradiation and BMT. Apoptotic cells were generated by incubation with 8-MPO and UVA light (UVAR light set, Therakos). Mice were followed for Gvhd score and survival. ECP-treated mice survived longer both across major (median survival 51 versus 26 days, p To address the mechanisms of this beneficial outcome we investigated Tcell proliferation in vivo (i), ex vivo (ii) and in vitro (iii). (i) Luciferase-expressing Tcon were used to quantify Tcell proliferation in vivo by bioluminescent imaging. ECP-treated mice showed reduced CD4+Tcell proliferation during the initiation phase of Gvhd (day+4) compared to mice receiving Tcon only. (ii) Re-isolation of CFSE labeled Tcon on day+4 after BMT showed a reduction in CD4+Tcell proliferation. (iii) FACS-sorted DC incubated with apoptotic cells for 2 days in vitro showed a diminished capability to induce Tcell proliferation. To evaluate the impact of ECP treatment on the expression of homing (P-Selectin, α4β7) and activation (CD44, CD69) markers on donor Tcells we performed re-isolation experiments on day+3 and day+7. At day +3 ECP-treated groups showed a reduced expression of these markers in comparison to control groups. These studies demonstrate slower trafficking to Gvhd target organs. Furthermore, at day+7 ECP-treated groups showed increased FoxP3-expressing host Tregs in comparison to control groups whereas donor Treg were similar in both groups. Ongoing experiments will utilize a mouse model in which it is possible to selectively deplete either donor or host Tregs to examine the role of these two subpopulations of Treg in improving Gvhd outcome. In summary, ECP treatment prior to transplantation led to (i) a significant survival benefit with no impact on graft versus tumor effect; (ii) reduced CD4+ Tcell proliferation; (iii) delayed expression of homing and activation markers during the initiation phase of Gvhd and (iv) an increase in host Treg. Overall our work suggests that pre-emptive ECP of host cells prior to BMT may be a clinically relevant strategy to prevent acute Gvhd. Disclosures: No relevant conflicts of interest to declare.

Abstract 1000 Natural killer (NK) cells exhibit in vitro cytotoxicity against many tumor cell types and have an important role in controlling tumor growth, as depletion of NK cells from tumor-bearing mice hastens tumor growth and impairs survival. These data, in combination with results from clinical trials of haploidentical killer immunoglobulin-like receptor (KIR)-ligand mismatched bone marrow transplantation, led to interest in the use of adoptive NK immunotherapy for the treatment of malignancy. Recent clinical results have shown that allogeneic NK cells can be safely administered after chemotherapy and/or irradiation but have also demonstrated limited persistence of the infused NK cells without clear evidence of efficacy. We traced the fate of adoptively infused NK cells in order to delineate the barriers to successful NK immunotherapy using several NK-sensitive murine tumor models. Mice bearing established lymphoma or leukemia received an intravenous infusion of 0.5–1.0×106 (approximately 2.5–5×107/kg body weight) NK cells from allogeneic or syngeneic donors after or concurrently with total body irradiation and bone marrow rescue. Using luciferase +ve NK cells, we first showed that in animals bearing subcutaneous tumors, NK cells homed to lymphoid organs in the first week, followed by progressive localization to and accumulation within the tumor site (Figure 1). In contrast, in non-tumor bearing animals NK cells homed to lymph nodes, spleen and liver with maximal proliferation at the end of the second week. These observations indicated that NK cells fail to eradicate the tumor despite prior demonstration of in vitro sensitivity, successful homing and local accumulation. As expected from these findings, survival was not prolonged by the NK cell infusion. Reisolation of donor NK cells within 18 hours of transfer showed enhanced cytotoxicity (14% vs 2%, p = 0.004) and IFNγ production (48% vs 22%, p = 0.04) compared with naive resting NK cells. In contrast, NK cells isolated from tumor-bearing mice at later time-points beginning d+5 showed loss of IFNγ production (48% early vs 3% late, p = 0.01), decreased expression of the activating receptor NKG2D, and impaired cytotoxicity in chromium release assays. These observations did not relate to over-stimulation through NKG2D, as NK cells from NKG2D−/− animals were also susceptible to acquired dysfunction (although their baseline cytotoxicity was lower than WT NK toward A20 lymphoma).Fig. 1Adoptively transferred luciferase-transgenic NK cells accumulate within the tumor over timeFig. 1. Adoptively transferred luciferase-transgenic NK cells accumulate within the tumor over time Eomesodermin and T-bet are transcription factors with important roles in effector functions of CD8+ T cells and NK cells. In T cells T-bet downregulation has been shown to correlate with exhaustion (Kao et al, Nat Imm 2011). Flow cytometry of reisolated NK cells revealed downregulation of Eomesodermin (naive splenic control, d+1 reisolated and d+10 reisolated cells showing 85%, 96%, and 29% expression, respectively) and T-bet (naive splenic control, d+1 reisolated and d+10 reisolated cells showing 82%, 99%, and 59% expression, respectively), correlating with loss of IFNγ production. The phenotype described herein was most dramatic within the tumor and within mice carrying high tumor burdens, but was also present in NK cells reisolated from non-tumor bearing animals that received NK cells, suggesting that homeostatic proliferation after transfer of mature NK cells could also contribute to exhaustion. CFSElo proliferated NK cells showed the most dramatic loss of effector function (chromium release = 42% in unproliferated vs 18% in proliferated cells, p = 0.03) and transcription factor expression (Eomesodermin positive 83% in unproliferated cells vs 18% in proliferated cells, p = 0.002). Collectively, our results suggest that the success of NK cell immunotherapy is limited by an acquired dysfunction that occurs within days after homeostatic proliferation and target encounter and that may be related to the downregulation of transcription factors required for NK effector function. These findings illuminate a previously unappreciated phenomenon and explain why short-term in vitro killing assays have limited utility in predicting the in vivo behaviour of transferred NK cells. Hence, these findings suggest that transferred NK cells become dysfunctional in vivo and that novel approaches may be required in order to circumvent the described dysfunction phenotype. Disclosures: No relevant conflicts of interest to declare.

Abstract 243 Mast cells are important effector cells in many innate and adaptive immune responses, and can influence the function of several immune cells including granulocytes, monocytes/macrophages, dendritic cells, T cells, B cells, NK cells, and NKT cells. In contrast to their well-know pro-inflammatory roles, in certain models, mast cells have been shown to decrease inflammation and tissue injury. We explored the biological activity of mast cells in a well-established model of mouse graft-versus-host disease (GVHD) by comparing C57BL/6-KitW-sh/W-sh mice (which lack mast cells) to wild-type (WT) C57BL/6 (H-2b) recipients in a major-MHC mismatched model of allogeneic transplantation. After myeloablative irradiation, recipients received 5×106 T-cell depleted bone marrow cells from FVB/N donors (H-2q) on day 0 followed by transfer of 2×106 FVB/N CD4 and CD8 conventional T cells (Tcon) at a 2:1 ratio on day 4. A dramatic decrease in survival was observed in the animals lacking mast cells where 100% of KitW-sh/W-sh recipients died by day 15 following transplantation while over 50% of WT recipients were alive at day 60 (p < 0.0001). The exacerbated GVHD in KitW-sh/W-sh mice correlated with an increase in Tcon proliferation as indicated by bioluminescence imaging (BLI), particularly in lymph node, liver, and gastrointestinal tract tissue sites (p < 0.001). The percentage of Foxp3 positive Treg cells was similar before and after transplantation in KitW-sh/W-sh and WT recipients, and CD4+CD25hi Treg from KitW-sh/W-sh mice were capable of suppressing T cell proliferation in a mixed leukocyte reaction. When KitW-sh/W-sh mice were given 5×106 bone marrow-derived cultured mast cells (BMCMCs) i.p., mast cell re-population of the gut and peritoneal cavity was indicated by both BLI and traditional staining methods. Preliminary experiments with KitW-sh/W-sh recipients engrafted with BMCMCs i.p. 6 weeks before allogenic transplantation demonstrated improved survival in the above GVHD model, but not if the BMCMCs were derived from IL-10-/- C57BL/6 mice. Together, these results support the hypothesis that mast cells can decrease Tcon proliferation and reduce GVHD severity by a mechanism that involves production of IL-10 by mast cells but which is independent of CD4+CD25+ Treg. Disclosures: No relevant conflicts of interest to declare.

Abstract 75 While non-myeloablative conditioning significantly reduces morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT), the risk of graft rejection increases, because persistent host cells mediate host-vs-graft reactivity. Here we studied the dynamics of engraftment and hematopoietic reconstitution after non-myeloablative radiation (XRT) in a minor antigen-mismatched model with high barriers to engraftment. BALB.K mice received purified hematopoietic stem cells (HSC: cKit+Thy1.1loSca1+Lin−) +/− TC from AKR/J donors. Recipients of pure HSC or HSC+CD8 TC regularly achieved stable mixed chimerism; however, if grafts contained CD4+CD25− TC (CD4con), recipients failed to engraft. This lack of engraftment was associated with BM hypocellularity (median 3.8 vs 13.3 x10^6 cells/2 legs in HSC+CD4con vs HSC recipients, respectively; p=0.0003), and lymphopenia (40%) at 2 weeks (w) post-HCT. In addition, 2w post-HCT the BM of mice given HSC+CD4con contained increased proportions of CD4 TC which expressed high levels of IFNγ (median 45% of donor vs. 13% of host CD4 TC; pLT) HSC was enlarged. These HSC were not apoptotic (as assessed by annexin V staining), but were cell cycle arrested (assessed by DNA content analysis). To study the functionality of these HSC outside the inflammatory environment, LT+ST HSC from HSC+CD4con recipients were FACS purified at 2w post-HCT and infused into secondary Rag2γc-/- recipients. These adoptively transferred HSC and promptly sustained multilineage hematopoiesis derived from the primary BALB.k host. Our results suggest that interactions between naïve donor CD4 TC and immunocompetent residual host cells, such as DC, create a pro-inflammatory environment involving the IL-12/IFNγ axis. We evaluated several other MHC-identical strain combinations which demonstrated lower levels of cytokine production with improved hematopoiesis compared to the AKR into BALK.K combination. These differences imply that impaired hematopoiesis is due to activation of donor CD4 TC in response to disparate minor alloantigens, and not due to non-specific inflammation. Such immune reactions appear to prevent engraftment of donor HSC and also inhibit the HSC expansion required to restore hematopoiesis early post-HCT. We conclude that HCT with pure HSC can result in superior immune reconstitution and donor chimerism after non-myeloablative conditioning. Pure HSC are immunologically anergic, and do not trigger inflammation in the BM microenvironment. As it is generally believed that TC augment donor HSC engraftment, our results are of broad significance because they reveal critical interactions between donor and host populations after non-myeloablative conditioning. Disclosures: No relevant conflicts of interest to declare.

Abstract 3528 Poster Board III-465 In the last decade the field of allogeneic hematopoietic cell transplantation has made significant strides in the reduction of morbidity and mortality using non-myeloablative conditioning. However, with reduced conditioning intensity the risk of allograft rejection has increased due to the persistence of host cells that mediate host-vs-graft alloreactivity or that occupy niches in the host hematopoietic microenvironment. Here we studied hematopoietic reconstitution and chimerism after non-myeloablative total body irradiation (TBI: 4 Gy [sublethal]; 7 Gy [lethal]) or total lymphoid irradiation (TLI: 17×240 cGy) in a MHC-matched mouse model with known high resistance to engraftment. BALB.K (H-2K) hosts received FACS-purified hematopoietic stem cells (HSC: cKIT+Thy1.1loSca1+Lin-) alone or supplemented with T cells (TC) from AKR/J (H-2K) donors. Peripheral blood (PB), bone marrow (BM), and spleen (SP) were analyzed 2 and 4 weeks (w) post transplant (pTX), and PB chimerism was followed beyond day (d) 100. Mice conditioned with lethal TBI survived, when grafts contained HSC only, and became mixed chimeric for TC, but converted to primarily donor type for B cells and the myeloid lineage. When grafts were supplemented with TC, chimerism converted to full donor type, but hosts developed fulminant acute GVHD. In contrast, sublethal TBI resulted in only mild symptoms of GVHD and mice recovered rapidly. Recipients of HSC alone or HSC+CD8 TC became mixed chimeras in all lineages, even long-term (>100d). However, mice given grafts of either HSC with total TC, or HSC+CD4 TC remained host type for T and B cells, and achieved only low levels of donor engraftment in the myeloid lineage. These findings suggested that the addition of peripheral donor CD4 TC resulted in powerful lymphoid suppression as well as retardation of myeloid engraftment. Indeed at 2w pTX recipients of HSC+CD4 TC displayed severe lymphopenia (40% in recipients of HSC alone]) and B cell reconstitution was the most severely affected (6% vs 75% of lymphocytes). Graft suppression was also evidenced by absolute cytopenia as mice given HSC+CD4 TC vs HSC alone, had reduced cellularity in BM (median 3.8×10∧6 [n=7] vs 13.3 ×10∧6 cells [n=4]; p=0.0003) and spleen (median 4.4×10∧6 vs 14×10∧6 cells; p=0.03). Suppression of hematopoiesis by CD4 TC grafts was accompanied by an expansion of donor and host CD4 TC, each comprising ∼6% of BM cells as compared with Disclosures: No relevant conflicts of interest to declare.

We investigated a novel graft-versus-host disease (GVHD) prophylactic regimen, sirolimus and mycophenolate mofetil (MMF), in patients with advanced hematologic malignancies receiving myeloablative hematopoietic cell transplantation (HCT) from matched related donors (MRD). The sirolimus and MMF combination was chosen based on intriguing evidence of sirolimus, and less dramatically MMF, conserving the inhibition of T cell proliferation compared to cyclosporine (CSP) in an AGVHD murine model as well as multiple reports supporting sirolimus’ preservative or augmentative effects on the T regulatory cell population (CD4+CD25+, FoxP3+) in animals and humans in comparison to cyclosporine (CSP) and a theoretical reduction in the incidence and severity of AGVHD. In addition, this regimen may cause less severe toxicities compared with standard methotrexate and/or calcineurin inhibitor (CI)-containing GVHD prophylactic regimens. Sirolimus was begun as an oral medication on day -3 with a 12 mg loading dose followed by 4 mgs daily, dose adjusted to maintain serum trough levels of 3–12 ng/ml. MMF was begun on D0 at 15 mg/kg IV every 12 hours. Mycophenolic acid (MPA) pharmacokinetics were conducted on days 2 and 21 post-HCT. Sirolimus and MMF were scheduled to be tapered simultaneously beginning 100 days post-HCT. FoxP3+ cell populations in the peripheral blood (PB) were analyzed before and after HCT in the patients receiving sirolimus and MMF GVHD prophylaxis as well as in 15 control patients with similar disease categories and preparative regimens receiving CI-containing GVHD prophylaxis. Based on pre-study stopping rules of expected grade II–IV AGVHD incidence, the trial was closed to accrual after enrollment of 11 patients. The median age of the 11 patients was 51 years (26–59). The diagnoses of the study patients included myelodysplasia (5), acute myelogenous leukemia (3) and nonhodgkin lymphoma (3). Seven patients received busulfan, etoposide and cyclophosphamide (CY); three patients received carmustine, etoposide and CY and one patient received total body irradiation, etoposide and CY as the preparative regimens. Sirolimus was discontinued in 4 patients due to concern for toxicity-related events including severe sinusoidal obstructive syndrome (1), altered mental status (1), portal vein thrombosis (1) and risk of poor wound healing after abdominal exploratory surgery (1) 9 days, 9 days, 61 days and 39 days post-HCT, respectively. Six of 11 patients developed grade II-IV AGVHD a median of 15.5 days post-HCT (range 11–25 days), 3 of the 6 with grade IV AGVHD. Two of the 3 patients with grade IV AGVHD required discontinuation of sirolimus 9 days post-HCT due to toxicity. Five of the 6 AGVHD patients had skin-only and one had liver, gut and skin involvement. All patients responded to AGVHD therapy and there were no AGVHD-related deaths. One patient died 54 days post-HCT due to complications of colonic ulceration believed related to oral MMF therapy. Two patients died of relapsed disease at 103 and 304 days post-HCT. At a median follow-up of 416 days (328–652 days), 8 of 11 patients were alive without disease, 5 with active chronic GVHD. The median MPA area under the curve (AUC) on day 2 was 12.8 mcg*hr/mL (6.4–17.4) and day 21 was 17.9 mcg*hr/mL (8–26.6). We measured a statistically significant increase in the percentage of CD4+FoxP3+ cells per total CD4+ cells in the sirolimus/MMF study group compared to the CI historical control group with a mean of the individual patient means over the first 100 days post-HCT of 16% versus 6%, respectively (p.0005). Despite the increased percentage of CD4+FoxP3+ cells per total CD4+ cells identified in the PB of the sirolimus/MMF cohort, the incidence of grade II-IV AGVHD was similar to the CI cohort (6 of 11 versus 7 of 15, respectively). However, all but one patient had skin-only AGVHD and there were no GVHD-related deaths in the sirolimus/MMF group. All patients requiring early withdrawal of sirolimus had received the busulfan-containing preparative regimen with two of these patients developing severe AGVHD. In a subsequent GVHD prophylaxis trial including sirolimus and MMF, the busulfan-containing preparative regimen will be eliminated to optimize sirolimus and MMF administration with the continued goal of reducing the incidence and severity of AGVHD.

Regulatory T cell (Treg) immunotherapy is a promising strategy for the treatment of graft rejection responses and autoimmune disorders. Our and other laboratories have shown that the transfer of highly purified CD4+CD25+Foxp3+ natural Treg can prevent lethal graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation across both major and minor histocompatibility barriers. However, recent evidence suggests that the Treg suppressive phenotype can become unstable, a phenomenon that can culminate in Treg conversion into IL-17–producing cells. We hypothesized that the intense proinflammatory signals released during an ongoing alloreaction might redirect a fraction of the transferred Treg to the Th17 cell fate, thereby losing immunosuppressive potential. We therefore sought to evaluate the impact of Il17 gene ablation on Treg stability and immunosuppressive capacity in a major MHC mismatch model. We show that although Il17 gene ablation results in a mildly enhanced Treg immunosuppressive ability in vitro, such improvement is not observed when IL-17–deficient Treg are used for GVHD suppression in vivo. Similarly, when we selectively blocked IL-1 signaling in Treg, that was shown to be necessary for Th17 conversion, we did not detect any improvement on Treg-mediated GVHD suppressive ability in vivo. Furthermore, upon ex vivo reisolation of transferred wild-type Treg, we detected little or no Treg-mediated IL-17 production upon GVHD induction. Our results indicate that blocking Th17 conversion does not affect the GVHD suppressive ability of highly purified natural Treg in vivo, suggesting that IL-17 targeting is not a valuable strategy to improve Treg immunotherapy after hematopoietic cell transplantation.