Graft-versus-host Response Research Articles

Application of Immune Checkpoint Inhibitors in Solid Organ Transplantation Recipients: A Systematic Review.

Solid organ transplantation (SOT) is a treatment method for end-stage organ diseases and improve their life quality, while using long-term immunosuppressant drugs (ISD) is needed to suppress the function of the immune system. Immune checkpoint inhibitors (ICIs) are a class of anti-tumor drugs that kill tumors by activating the autoimmune system. The primary objective of our systematic review is to investigate the risk factors for organ rejection and the efficacy of ICIs in solid organ transplantation recipients (SOTRs). We searched four databases to find relevant articles up to January 2021. A total of 61 articles involving 106 SOTRs met the screening criteria and were included in our systematic review. The collected data were statistical described, and the risk factors were analyzed by logistic regression. Forty-four patients (41.5%) developed host-versus-graft response (HVGR) after ICIs. mTOR inhibitors (pre-ICIs) (p = 0.069, OR = 0.377, 95% CI 0.132-1.078) and calcineurin inhibitors (post-ICIs) (p = 0.056, OR = 0.375, 95%CI 0.137-1.025) may help reduce the incidence of HVGR. Hormones (pre-ICIs) (p = 0.026, OR = 3.150, 95%CI 1.150-8.628) and anti-metabolites (pre-ICIs) (p = 0.022, OR = 3.214, 95%CI 1.185-8.720) may adversely affect the efficacy of ICIs. Only 35.6% of patients both responded well to ICIs treatment and did not develop HVGR. Our systematic review summarizes the use of ICIs in SOTRs and evaluates the efficacy of ICIs and the risk factors that induce HVGR. Through risk factor analysis, we found that mTOR inhibitors and calcineurin inhibitors may help to reduce the occurrence of HVGR;hormones and anti-metabolic drugs may have adverse effects on the efficacy of ICIs. In addition, there is a contradictory relationship between the occurrence of HVGR and the efficacy of ICIs.

Abstract 3245: FT819 path to IND: First-of-kind off-the-shelf CAR19 T-cell for B cell malignancies

Abstract Genetic engineering of T cells using a chimeric antigen receptor targeting CD19 antigen (CAR19) is now a well-established treatment of B cell malignancies. While cellular immunotherapies are entering front line treatment, substantial limitations currently hamper the broad application of adoptive T cell therapies in diverse patient population including dysfunctional starting material, lack of product consistency and purity post genetic engineering and inefficient quantity produced for true on-demand availability. FT819 is a first-of-kind off-the-shelf CAR19-T cell product generated from a renewable pluripotent stem cells for large-scale clinical manufacturing. We previously reported the engineering and characterization of the FT819 clonal master cell bank (MCB) derived from a single cell comprising targeted integration of a novel CD19 1XX CAR into the T-cell receptor (TCR) α constant locus to provide optimally regulated CAR expression and elimination of graft versus host (GvH) response. Here we preview the nonclinical study for the original investigational new drug application of FT819. Derived in a manufacturing process analogous to pharmaceutical drug product development, pilot runs from the MCB demonstrated FT819 can be consistently and uniformly manufactured in cGMP compliance, cryopreserved at clinical scale to support off-the-shelf clinical application with greater than 1e5 fold increase in cellular yield from the starting MCB and can be thawed and directly used for facilitated treatment. Repeatedly, FT819 displayed a uniform product profile of ≥95% CAR+, TCR-, CD45+, CD7+ and CD3+ [intracellular] with majority of CD8 T cells expressing CD8β. FT819 global gene expression profile displayed high similarity to primary CAR19-T cells confirming its identity as a T lymphocyte. Functional assessment demonstrated that FT819 possesses potent antigen specific cytolytic activity against leukemia and lymphoma cell lines (p=0.0004). Additional specificity studies demonstrated on-target, off-tumor cytolysis of CD19+ B cells in mixed lymphocyte reaction assay (85% lysis of CD19+ B cells versus < 2% lysis of T cells). Inability of FT819 to produce a GvH response was confirmed in a co-culture assay with anti-TCR crosslinking antibodies. Disseminated leukemia xenograft mouse studies demonstrated the ability of directly thawed and infused FT819 to control tumor growth (p=0.0003 at day 21). In a systemic administered leukemia model FT819 also showed sustained localization in the bone marrow up to 45 days post injection. Ongoing in vivo studies will assess long-term survival and avoidance of GvH disease. Collectively, these studies demonstrate that FT819 is a potent, consistent and uniform CAR19 T cell product and can be effectively and safely used off-the-shelf in the treatment of B cell malignancies with an original Phase 1 clinical trial planned in 2020. Citation Format: Mili Mandal, Raedun Clarke, Sjoukje van der Stegen, Chia-Wei Chang, Yi-Shin Lai, Alec Witty, Mushtaq Husain, Cheng-Jang Wu, Bi-Huei Yang, Chad Dufaud, Gloria Hsia, Helena Shaked, Laurel Stokely, Helen Chu, Mochtar Pribadi, Gilberto Hernandez, Jason ORourke, Alma Gutierrez, Ramzey Abujarour, Tom Lee, Jolanta Stefanski, Juan Zhen, Meilan Wu, Isabelle Riviere, Michel Sadelain, Bahram Valamehr. FT819 path to IND: First-of-kind off-the-shelf CAR19 T-cell for B cell malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3245.

Maintenance of long remission in adult T-cell leukemia by Tax-targeted vaccine: A hope for disease-preventive therapy.

Adult T‐cell leukemia/lymphoma (ATL) is an aggressive lymphoproliferative disease caused by human T‐cell leukemia virus type 1 (HTLV‐1). Multi‐agent chemotherapy can reduce ATL cells but frequently allows relapses within a short period of time. Allogeneic hematopoietic stem cell transplantation (allo‐HSCT) following chemotherapy is now a standard therapy for ATL in Japan as it can achieve long‐term remission in approximately one‐third of recipient ATL patients; however, it also has a risk of treatment‐related mortality. Allo‐HSCT often induces HTLV‐1 Tax‐specific cytotoxic T cells (CTL) as well as graft‐versus‐host (GVH) response in ATL patients. This observation led to development of a new therapeutic vaccine to activate Tax‐specific CTL, anticipating anti‐ATL effects without GVH response. The newly developed Tax‐DC vaccine consists of autologous dendritic cells pulsed with Tax peptides corresponding to CTL epitopes that have been identified in post‐allo‐HSCT ATL patients. In a pilot study of Tax‐DC therapy in three ATL patients after various initial therapies, two patients survived for more than 4 years after vaccination without severe adverse effects (UMIN000011423). The Tax‐DC vaccine is currently under phase I trial, showing a promising clinical outcome so far. These findings indicate the importance of patients’ own HTLV‐1‐specific T‐cell responses in maintaining remission and provide a new approach to anti‐ATL immunotherapy targeting Tax. Although Tax‐targeted vaccination is ineffective against Tax‐negative ATL cells, it can be a safe alternative maintenance therapy for Tax‐positive ATL and may be further applicable for treatment of indolent ATL or even prophylaxis of ATL development among HTLV‐1‐carriers.

Role of Graft-derived Graft-versus-Host T cells in Facilitating Multilineage Blood Chimerism after Human Intestinal Transplantation

Long-term multilineage chimerism often appears in blood following human intestinal transplantation (ITx), especially in multivisceral transplant (MVTx) recipients. Patients with donor T cell macrochimerism (>4%) in blood, frequently without graft-versus-host disease (GVHD), displayed less graft rejection and donor-specific antibody (DSA) production and slower graft T cell replacement by the recipient. To address the hypothesis that the balance of graft-versus-host (GvH) and host-versus-graft (HvG) alloreactivity might underly these relationships, we analyzed the phenotype, repertoire and alloreactivity of donor- and recipient-derived T cells in the intestinal allograft, peripheral blood and bone marrow (BM) of ITx recipients. We identified GvH and HvG clones on the basis of high throughput TCRβ CDR3 sequencing from pre-Tx donor and recipient mixed lymphocyte reactions as we have described1. Enrichment of GvH compared to HvG clones in early intestinal biopsies and absence of Class I DSA were associated with donor T cell macrochimerism. Local expansion of GvH clones in the graft was likely driven by uniformly rapid replacement of recipient antigen presenting cells (APCs) within allografts early post-Tx. Cumulative frequency of GvH clones in blood early post-Tx was positively correlated with the peak level of donor T cell chimerism (Fig.1).Unlike donor ileal lymphocytes, which display a tissue-resident memory phenotype, long-term circulating donor T cells were markedly enriched for the recent thymic emigrant phenotype compared with recipient cells, and GvH clones were undetectable among these late circulating naïve donor-derived T cells. Enrichment of donor circulating naïve B cells and T cells and recurrent myeloid chimerism late post-Tx are consistent with our observation that donor-derived hematopoietic stem and progenitor cells are present in intestinal allografts and are detectable in the circulation of patients with macrochimerism. Multilineage chimerism might reflect a lymphohematopoietic GvH response (LGVHR) migrating from recipient circulation to the BM, making “space” for engraftment of progenitor cells from the graft. Indeed, 0.0356% of CD45+ cells in the BM of an MVTx patient on day 105 post-Tx were donor-derived, among which 72.9% were T cells and 1.37% were CD34+ progenitors. TCR sequencing of sorted donor cells in recipient BM identified donor-mappable clones, among CD4s of which 60% by frequency were GvH-reactive. GvH CD4 cells did not show marked enrichment in donor-mappable PBMCs on the same day. (Fig.2)Together, these data suggest that donor GvH-reactive T cells expanding within the graft in response to recipient APCs entering the graft migrate into the recipient circulation and BM, playing a key role in promoting and maintaining mixed chimerism. LGVHR without GVHD in ITx recipients suggests a novel approach to achieving intestinal allograft tolerance. 1. Zuber et al., 2016 Science Immunology. The study was funded by the National Institute of Allergy and Infectious Diseases grant P01 AI106697. Research reported in this study was performed in the CCTI Flow Cytometry Core, supported in part by the Office of the Director, National Institutes of Health under awards S10RR027050 and S10OD020056.

Mixed Chimerism in Peripheral Blood and Allograft after Human Intestinal Transplantation and its Relationship with Clinical Outcomes

Donor T cell macrochimerism (>4%) often appears in blood without graft-versus-host disease (GVHD) following intestinal transplantation (ITx), and is associated with less graft rejection and donor-specific antibody (DSA) production.[1][2] Blood chimerism is also observed in other lymphoid and myeloid lineages.[1] We investigated the mechanisms underlying multilineage chimerism in blood. We analyzed the repertoire, phenotype and origin of graft-resident and circulating donor T cells in ITx recipients using flow cytometry, mixed lymphocyte reaction and high-throughput TCR sequencing to identify and track alloreactive T cells. Expanded graft-versus-host (GvH) clones triggered by rapidly replaced recipient antigen-presenting cells were detected early in intestinal biopsies (Figure 1A). Enrichment of graft GvH clones and absence of Class I DSA in the circulation were associated with donor T cell macrochimerism. The cumulative frequency of GvH clones in blood early post-Tx was correlated with peak donor T cell chimerism levels (Figure 1B). Frequencies of host-versus-graft (HvG) clones subsequently declined in the blood of a patient with a high level of T cell chimerism (>38%), suggesting that a lymphohematopoietic GvH response (without GVHD) may attenuate HvG responses (Figure 1C). While donor ileum lymphocytes display a tissue-resident memory (Trm) phenotype, circulating donor T cells include naïve and other non-Trm phenotypes (Figure 2A-B). Long-term circulating donor T cells were markedly enriched for the recent thymic emigrant phenotype and for T-cell receptor excision circles (TRECs) compared with recipient cells, suggesting they were recent thymic emigrants (Figure 2C-D). Donor circulating B cells and γδ T cells were also enriched for naïve phenotype compared to recipient cells. Myeloid lineage chimerism recurred at low levels late post-Tx in patients with macrochimerism. Consistent with the possibility that blood chimerism originated from progenitors in the graft, we detected donor-derived hematopoietic stem cells (HSCs) and/or hematopoietic progenitors (HPs) in human intestinal mucosa. Our findings suggest that locally expanded GvH-reactive donor T cells in the graft enter the recipient circulation and attack host hematopoietic cells, which may allow the engraftment of graft-derived HSCs/HPs, thereby promoting sustained mixed chimerism. This pathway may help to reduce graft rejection and achieve tolerance after ITx.FIGURE 1: (A) Enrichment of GvH clones in intestinal biopsies and absence of Class I DSA (POD <90) in the circulation were associated with donor T cell macrochimerism in blood. (B) Correlation between the cumulative frequency of GvH clones in blood early post-Tx and the peak level of donor T cell chimerism. (C) Cumulative frequency of HvG clones in blood post-Tx.FIGURE 2: (A-B) Differential phenotypes of circulating versus gut-resident donor T cells. (C-D) Long-term circulating donor T cells were markedly enriched for the recent thymic emigrants (RTEs) and T-cell receptor excision circles (TRECs) compared with recipient cells.Research reported in this publication was performed in the Columbia Center for Translational Immunology (CCTI) Flow Cytometry Core, supported in part by the Office of the Director, National Institutes of Health under awards S10RR027050 and S10OD020056. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Differing Mechanisms for Early Versus Persistent Donor T cell Chimerism in Peripheral Blood of Human Intestinal Transplant Recipients

Donor T cell macrochimerism (>4%) often appears in blood following intestinal transplantation (ITx), usually without graft-versus-host disease[1]. Patients with donor T cell macrochimerism displayed less graft rejection, slower recipient T cell replacement within the graft and less donor-specific antibody (DSA) production[2]. However, the reasons for the development and persistence of donor T cell chimerism are unknown. We analyzed the repertoire, phenotype and origin of circulating donor T cells in ITx recipients using flow cytometry, mixed lymphocyte reaction (MLR) and high-throughput TCR sequencing. Expanded graft-versus-host (GvH) clones triggered by rapidly replaced recipient antigen-presenting cells were detected early in ileum biopsies (Figure 1A). Enrichment of GvH clones in the graft and absence of Class I DSA in the circulation were associated with donor T cell macrochimerism. Cumulative frequencies of GvH clones in blood early post-Tx correlated with the peak level of donor T cell chimerism (Figure 1B). Frequencies of host-versus-graft (HvG) clones subsequently declined in the blood of a patient with a high level of T cell chimerism (>38%), suggesting that lymphohematopoietic GvH responses may attenuate HvG responses (Figure 1C). Long-term donor T cells were markedly enriched for the naïve recent thymic emigrant phenotype compared with recipient cells regardless of donor age (Figure 2A). GvH clones were absent among long-term circulating naïve donor-derived T cells, which were enriched for CD31 expression and T-cell receptor excision circles (TRECs), suggesting they developed de novo in the recipient thymus. Long-lasting (POD > 200) circulating donor cells lacked GvH reactivity yet were immunocompetent in MLR, partially due to the presence of recipient-specific Tregs (Figure 2B). We also detected donor-derived hematopoietic stem cells (HSCs) and/or progenitors (HPs) in blood and stoma specimens post-Tx and in donor liver, ileum and perfusates pre-Tx. HSCs/HPs from ileum and bone marrow of each organ donor showed similar properties on CyTOF (mass cytometry) analysis, suggesting they may have similar functional phenotypes. Together, donor GvH-reactive T cells and HSCs/HPs carried within the graft promote and maintain donor T cell chimerism in blood, which correlates with better clinical outcomes and suggests an approach to reduce graft rejection and achieve tolerance post-ITx.FIGURE 1: (A) Enrichment of GvH clones in intestinal biopsies and absence of Class I DSA (POD <90) in the circulation were associated with donor T cell macrochimerismin blood. (B) Correlation between the cumulative frequency of GvH clones in blood early post-Tx and the peak level of donor T cell chimerism. (C) Cumulative frequency of HvG clones in blood post-Rx.FIGURE 2: (A) Circulating donor-derived naïve T cells display a recent thymic emigrant (RTE) phenotype. (B) Long-lasting (POD>200) circulating donor cells lacked GvH reactivity yet were immunocompetent in MLR, partially due to the presence of recipient-specific Tregs.Research reported in this publication was performed in the Columbia Center for Translational Immunology (CCTI) Flow Cytometry Core, supported in part by the Office of the Director, National Institutes of Health under awards S10RR027050 and S10OD020056. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

CD8 Tregs Promote Gvhd Prevention and Restore Impaired GVL Effect Mediated By CD4 Tregs in Mice

Adoptive regulatory T-cell (Treg) therapy has enhanced the outcome of patients suffering from graft-versus-host (GVH) disease following allogeneic hematopoietic stem cell transplantation (allo-HCT); however, fear of broad immune suppression and subsequent dampening of the beneficial graft-versus-leukemic (GVL) responses remains a challenge. In order to subvert broad immune suppression, we generated alloantigen-specific induced Tregs (iTregs) from resting CD4 or CD8 T cells and tested the ability of iTregs to suppress GVH and maintain GVL responses. We utilized a clinically relevant murine model of haploidentical-HCT with the addition of host-original leukemia cell line to evaluate the effects of CD4 and CD8 iTregs in GVH and GVL responses. While alloantigen-specific CD4 iTregs were effective in preventing GVHD (Fig. 1 A and C), they completely abrogated the GVL effect against aggressive leukemia resulting in 100% tumor mortality (Fig. 1 B and D). Mechanistically, these CD4 iTregs were found to potently suppress the expansion of effector T cells (Teffs) and their ability to secrete IFNγ and granzyme B in the recipient spleen and liver, which may contribute to the impaired GVL activity. Using similar approach, we generated alloantigen-specific CD8 iTregs and found they express higher levels of granzyme B and CTLA-4 compared to nTreg and CD4 iTregs. In vivo studies showed these CD8 iTregs moderately attenuated GVHD (Fig. 1 A and C)while completely sparing the GVL effect (Fig. 1 B and D). We thus further reasoned that the combination of CD4 and CD8 iTregs could achieve the optimal goal of allo-HCT: GVHD suppression with GVL preservation. Indeed, the combination therapy potently suppressed GVHD resulting in increased survival and decreased pathological injury to target organs than either CD4 or CD8 iTreg singular therapy (Fig. 1 A and C). More importantly, the combination therapy maintained potent GVL responses reflected by significantly decreased tumor mortality and load (Fig. 1 B and D).Mechanistically, we observed addition of CD8 iTregs maintained the suppression of Teff expansion but restored the ability of Teffs in producing inflammatory cytokines (e.g. IFNγ and TFNα) and cytolytic effector molecules (e.g. granzyme B and TRAIL). To our knowledge the current findings are the first to support the use of combinational iTreg therapy to achieve optimal suppression of GVHD while maintaining GVL responses.This work was supported by NIH grants: R01 CA118116 and R01 CA169116 [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Benzimidazoisoquinolines: A New Class of Rapidly Metabolized Aryl Hydrocarbon Receptor (AhR) Ligands that Induce AhR-Dependent Tregs and Prevent Murine Graft-Versus-Host Disease

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that plays multiple roles in regulation of immune and inflammatory responses. The ability of certain AhR ligands to induce regulatory T cells (Tregs) has generated interest in developing AhR ligands for therapeutic treatment of immune-mediated diseases. To this end, we designed a screen for novel Treg-inducing compounds based on our understanding of the mechanisms of Treg induction by the well-characterized immunosuppressive AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We screened a ChemBridge small molecule library and identified 10-chloro-7H-benzimidazo[2,1-a]benzo[de]Iso-quinolin-7-one (10-Cl-BBQ) as a potent AhR ligand that was rapidly metabolized and not cytotoxic to proliferating T cells. Like TCDD,10-Cl-BBQ altered donor CD4+ T cell differentiation during the early stages of a graft versus host (GVH) response resulting in expression of high levels of CD25, CTLA-4 and ICOS, as well as several genes associated with Treg function. The Treg phenotype required AhR expression in the donor CD4+ T cells. Foxp3 was not expressed in the AhR-induced Tregs implicating AhR as an independent transcription factor for Treg induction. Structure-activity studies showed that unsubstituted BBQ as well as 4, 11-dichloro-BBQ were capable of inducing AhR-Tregs. Other substitutions reduced activation of AhR. Daily treatment with 10-Cl-BBQ during the GVH response prevented development of GVH disease in an AhR-dependent manner with no overt toxicity. Together, our data provide strong support for development of select BBQs that activate the AhR to induce Tregs for treatment of immune-mediated diseases.

102-P: LOSS OF DONOR CHIMERISM IN PATIENT UNDERGOING ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANT FOR MDS

Aim The aim of this study was to address unexpected chimerism results that were not consistent with the clinical symptoms of a 49 year old female patient with MDS who had received nonmyeloablative hematopoietic cell transplantation (HCT) from her haploidentical sibling. Methods Donor chimerism for peripheral blood, CD3 and CD33-selected cells was determined using a quantitative PCR-based amplification of Short Tandem Repeat (STR) sequences. Results The first chimerism test at day 30 post BMT resulted in > 99% recipient. This was puzzling as the recipient had normalized blood counts and exhibited skin rash primarily on her face which had responded well to steroids, suggesting the presence of GVHD. Results of the second samples of donor blood and host buccal swabs confirmed the initial findings. Peripheral blood and CD3 and CD33-selected cells on day 60 was >99% recipient. Bone marrow biopsy was normal. Total WBC stabilized at count of 6.6. Repeat HLA typing confirmed the patient’s pre-transplant HLA typing. Conclusions This patient has clearly undergone immune mediated rejection of the donor graft. The skin rash that was suspected to be GVHD is likely triggered by her rosacea flare-up. The surprising observation that she has maintained tumor remission despite loss of donor chimerism suggests the presence of a possible antitumor response. It is plausible that host-versus-graft (HVG) alloresponse may have induced tumor-specific response where the recipient’s antigen-presenting cells process and present donor alloantigens and tumor antigens as peptides in the context of host MHC. Alternatively, HVG response may involve release of cytokines that upregulate the expression of MHC class I on tumor cells, rendering them more susceptible to CTL killing. While the complete donor chimerism is a desirable outcome of allogeneic transplant for hematologic malignancies, discovering the mechanisms involved in this case may provide future strategies that will help in achieving antitumor effects following HCT.

Allogeneic T cell responses are regulated by a specific miRNA-mRNA network

Donor T cells that respond to host alloantigens following allogeneic bone marrow transplantation (BMT) induce graft-versus-host (GVH) responses, but their molecular landscape is not well understood. MicroRNAs (miRNAs) regulate gene (mRNA) expression and fine-tune the molecular responses of T cells. We stimulated naive T cells with either allogeneic or nonspecific stimuli and used argonaute cross-linked immunoprecipitation (CLIP) with subsequent ChIP microarray analyses to profile miR responses and their direct mRNA targets. We identified a unique expression pattern of miRs and mRNAs following the allostimulation of T cells and a high correlation between the expression of the identified miRs and a reduction of their mRNA targets. miRs and mRNAs that were predicted to be differentially regulated in allogeneic T cells compared with nonspecifically stimulated T cells were validated in vitro. These analyses identified wings apart-like homolog (Wapal) and synaptojanin 1 (Synj1) as potential regulators of allogeneic T cell responses. The expression of these molecular targets in vivo was confirmed in MHC-mismatched experimental BMT. Targeted silencing of either Wapal or Synj1 prevented the development of GVH response, confirming a role for these regulators in allogeneic T cell responses. Thus, this genome-wide analysis of miRNA-mRNA interactions identifies previously unrecognized molecular regulators of T cell responses.

Suppression of acute graft-versus-host response by TCDD is independent of the CTLA-4-IFN-γ-IDO pathway.

Activation of the aryl hydrocarbon receptor (AhR) by its prototypic ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), induces potent suppression of an acute graft-versus-host (GVH) response and prevents GVH disease (GVHD). Suppression is associated with development of a regulatory population of donor CD4(+) CD25(+)T-cells that express high levels of cytotoxic T-lymphocyte antigen 4 (CTLA-4). However, a direct link between these AhR-induced Tregs (AhR-Tregs) and suppression of GVHD remains to be shown. CTLA-4 is a negative regulator of T-cell responses and is associated with the induction of tolerogenic dendritic cells (DCs) that produce indoleamine 2,3-dioxygenase (IDO). We hypothesized that AhR-Tregs mediate suppression via their enhanced expression of CTLA-4, which, in turn, induces IFN-γ and IDO in host DCs. Subsequent depletion of tryptophan by IDO leads to termination of the donor T-cell response prior to development of effector CTL. Here, we show that despite increased expression of Ifng, Irf3, Irf7, Ido1, and Ido2 in the lymph nodes of TCDD-treated host mice, inhibition of IDO enzyme activity by 1-methyl-tryptophan was unable to relieve TCDD-mediated suppression of the GVH response. Furthermore, treatment with an anti-CTLA-4 antibody that blocks CTLA-4 signaling was also unable to alleviate TCDD-mediated suppression. Alternatively, we investigated the possibility that donor-derived AhR-Tregs produce IFN-γ to suppress effector CTL development. However, suppression of GVHD by TCDD was not affected by the use of Ifng-deficient donor cells. Together, these results indicate that neither overexpression of CTLA-4 nor production of IFN-γ by AhR-Tregs plays a major role in the manifestation of their immunosuppressive function in vivo.

Biology of Graft-versus-Host Responses: Recent Insights

Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for many malignant and non-malignant hematological diseases. The curative effect of allo-HSCT results from both the chemotherapy and/or radiation in the conditioning regimen and the donor T mediated graft-versus-host (GVH) response against the malignancy known as a graft-versus-leukemia (GVL) effect. In most cases, the GVH response is not limited against the malignant cells, but also mediated against host epithelial cells causing graft-versus-host disease (GVHD). The safety and effectiveness of allogeneic transplants has increased substantially over the last several years. Despite the progress, GVHD and relapse remain the biggest hurdles for a more widespread and effective use of this potent therapy. Experimental data from recent years have provided deeper and better insights into the process of GVH responses. Emerging data have refined our understanding of the role of antigen presenting cells (APC) in GVHD. Novel molecular targets have been identified in donor T cell subsets and host non-hematopoietic cells that can potentially be exploited for mitigating GVHD and to better harness GVL. During recent years the role of various cytokines in GVHD continues to be explored. This work has led to improved understanding of potential causes for tissue specificity of GVHD. The role of host and donor microbiome in regulating GVH responses has been a subject of intense focus as well. Notable advances have been made in the defining various other aspects of GVH responses , but these go beyond the scope of this brief review. Herein we will focus on three specific aspects of GVH responses, namely 1) the evolving role of hematopoietic APCs as the regulators of GVHD, 2) novel strategies for enhancing GVL responses and 3) the recently identified molecular targets for mitigating GVH responses.

Immunosuppressive properties of a series of novel inhibitors of the monocarboxylate transporter MCT-1

We have recently described the immunosuppressive properties of AR-C117977 and AR-C122982, representatives of a group of compounds identified as inhibitors of lactate transporters (monocarboxylate transporters; MCTs). These compounds demonstrate the potential therapeutic usefulness of inhibiting MCT-1, but their physical and metabolic properties made them unsuitable for further development. We have therefore tried to find analogues with similar immunosuppressive efficacy and a more suitable profile for oral administration. Five analogues of AR-C117977 were synthesised and screened for binding to the transporter, for inhibition of proliferation of both human and rat lymphocytes, for in vivo activity in a model of graft-versus-host (GvH) response in the rat, and in high- and low-responder cardiac transplant models in the rat. There was a good correlation between levels of binding of the five analogues to MCT and their inhibition of lymphocyte proliferation in human and rat cells. Furthermore, activity in both the GvH response and the cardiac transplant models correlated well with the determined concentrations of test compound in plasma. These findings on new analogues of MCT-1 inhibitors have taken us further towards defining the pharmacokinetic properties that may help to identify future drug candidates among inhibitors of MCT-1.

Relevance of HLA compatibility in living donor liver transplantation: the double‐edged sword associated with the patient outcome

HLA compatibility in living donor liver transplantation (LDLT) seems relevant to the acceptability of graft livers because LDLT recipients often share most or some part of HLAs with the respective donors. This study retrospectively investigated whether HLA compatibility affected the outcome of LDLT. Three hundred ninety LDLTs were performed in this hospital, and 346 pairs of HLAs (HLA-A, B, DR) were retrieved from the medical record between October 1996 and March 2011. The dates of the deaths were censored when a recipient apparently died of or was retransplanted by other causes than graft failure because of host-versus-graft (HVG) response to purely analyze the outcomes of LDLT in view of HVG response. The relationship between HLA compatibility and graft-versus-host disease (GVHD) was also analyzed. No recipients with recipient-against-donor HLA mismatch (R→D MM) 0 experienced graft failure by HVG response. On the other hand, three of five recipients with "R→D MM 0" together with "donor-against-recipient MM 3" died of fatal GVHD. HLA compatibility in LDLT not only affected the long-term acceptance of graft livers but also the risk of fatal GVHD.

Blockade of IL-23 Signaling Results in Targeted Protection of the Colon and Allows for Separation of Graft Versus Host and Graft Versus Leukemia Effects.

Abstract 231Separation of graft versus leukemia (GVL) and graft versus host (GVH) reactivity has been a longstanding but elusive goal in allogeneic bone marrow transplantation (BMT). The difficulty in dissociating these two responses stems in part from the fact that alloreactive donor T cells are equally capable of trafficking to sites of disease as well as GVHD target organs such as the colon, liver and skin. Recent studies from our laboratory have shown that donor APC-derived IL-23 secretion has a critical and selective role in mediating pathological damage in the colon during GVHD (Das et al, Blood, 2009). These results demonstrate the feasibility of regional GVHD protection and raise the question as to whether such localized protection might be a strategy to separate GVH and GVL responses. To directly address this question, we performed studies using a Balb/c-derived luciferase-transfected A20 leukemia cell line (A20-luc) in GVL experiments. Lethally irradiated Balb/c mice were administered 1 × 105 A20 cells and then transplanted with C57BL/6 (B6) BM alone or BM and spleen cells from B6 or IL-23−/− donors. Transplantation with BM and spleen cells from IL-23−/− donor mice resulted in prolonged survival when compared to mice reconstituted with similarly composed marrow grafts from B6 animals (63% versus 26% at day 70, p=0.03). A five-fold escalation of the A20 cell dose produced similar protective results in recipients of IL-23−/− marrow grafts indicating that the effects were not leukemia dose dependent. Studies were then conducted using an IL-23 specific (p19) antibody to block IL-23 signaling as a more clinically relevant strategy. To confirm that antibody blockade would also attenuate GVHD, lethally irradiated Balb/c mice were transplanted with B6 BM and spleen cells to induce GVHD. Mice that were administered p19 antibody (once/week for four weeks) had selective protection from colonic GVHD and a significant reduction in colonic proinflammatory cytokine production when compared to isotype control-treated mice, confirming results observed with use of transgenic IL-23−/− donors. Administration of p19 antibody on a similar schedule to A20-luc-bearing animals resulted in increased survival relative to isotype control antibody-treated mice demonstrating that antibody blockade also resulted in preservation of the GVL response. Serial bioluminescence imaging (BLI) studies and pathological examination confirmed that animals treated with p19 antibody had no evidence of leukemia recurrence. Since the colon is preferentially protected after blockade of IL-23 signaling, we considered that this site might serve as a sanctuary site for residual leukemia cells. BLI studies revealed that A20-luc cells migrated to the liver, lung and nodal sites but were not detectable in the colon. For that reason, we examined this question using a novel, clinically relevant murine model of chronic myelogenous leukemia (CML). In this model, FVB animals that have the bcr/abl oncogene under the control of a tetracycline-inducible repressor are used as donor animals (i.e. CML mice). Lethally irradiated normal FVB mice were transplanted with equivalent numbers of T cell depleted BM from B6 and FVB CML animals. Withdrawal of tetracycline from the drinking water induces expression of bcr/abl oncogene, the development of neutrophilia, and leukemic infiltration in the colon, liver and spleen. Real time PCR studies also demonstrated a 600-fold increase in bcr/abl mRNA levels in the colon of these mice, relative to controls, indicative of CML in this organ. To determine whether the colon would serve as a sanctuary site and limit the effectiveness of the GVL response, lethally irradiated FVB mice were transplanted with the same BM inoculum plus 3.5 × 106 T cells from wild type or IL-23−/− donors. Whereas mice that received adjunctive B6 T cells succumbed from GVHD, animals transplanted with T cells from IL-23−/− mice had significantly prolonged survival and no evidence of leukemia by either blood counts or pathological examination. Real time PCR studies also demonstrated absence of the bcr/abl oncogene in the colon of mice indicating that this organ did not serve as a reservoir for subsequent leukemic relapse. In conclusion, these studies demonstrate that targeting of IL-23 is able to reduce GVHD without loss of the GVL effect and suggest that this approach may be a viable clinical strategy for the dissociation of GVH and GVL responses. Disclosures:No relevant conflicts of interest to declare.

Ikaros Deficiency Enhances Notch Signaling in Host Antigen Presenting Cells and Differentially Regulates Gvhd and GVL Responses After Experimental BMT.

Abstract 690Host antigen presenting cells (APCs) are necessary for induction of graft-versus-host (GVH) responses. But the APC autonomous molecular mechanisms that are critical for modulation of GVH are not known. Ikaros (Ik) is a zinc-finger DNA binding protein that is essential for the development of lymphoid cells and hematological malignancies. Because Ik is known to negatively regulate certain dendritic cell (DC), the most potent APCs, functions (Wu et al, Immunity, 1997) we hypothesized that its deficiency in host APCs will reduce GVHD. We generated [B6→B6] and [Ik-/-B6→B6] chimeras and utilized them as recipients in well characterized MHC matched, minor mismatched C3H.SW B6 model of acute GVHD. [B6→B6] and [Ik/-B6 → B6] animals received 9 Gy and were transplanted with 3 × 106 T cells and 5 × 106 BM from either syngeneic B6 or allogeneic C3H.SW donors. Contrary to our hypothesis, [Ik-/-B6 < 0.001). The increase in mortality was associated with a significant increase in GVHD specific clinical severity, histopathological damage of the liver and GI tract, donor T cell expansion, serum levels TNF-αa, IFNγ and IL-17 on days +7 and +14 after BMT. Similar results were obtained in a second, BALB/c→B6 model of GVHD and also in recipient chimeras generated from dominant negative Ikaros (IkDN) mice, demonstrating a role for Ik in a strain independent manner. We next performed systematic analyses of DCs to uncover the mechanisms underpinning our surprising results. Phenotypic analysis of spleen and BM derived DCs demonstrated similar numbers of CD8+ and CD8-CD11c+DCs but a reduced number of pDCs were present in Ik-/- animals when compared with WT animals. Ik-/-DCs demonstrated an immature phenotype as determined by the expression of CD40/80. By contrast, functional analyses of Ik-/- DCs, when stimulated with LPS showed significantly greater secretion of proinflammatory cytokines TNF-αa and IL-6 and greater stimulation of allo-T cells in MLRs when compared with WT B6 DCs. CFSE, annexin and CD107a labeling studies demonstrated that Ik-/-DCs caused greater proliferation without altering the rate of apoptosis or the cytotoxic function of the allo-T cells. To characterize the molecular mechanisms we evaluated the role of putative molecular targets of Ikaros, the Notch signaling pathway because Ikaros and Notch antagonistically regulate target genes (Chari et al. J Immunol 2008 and Bugeon, et al. J Immunol 2008). Both Ik-/- and IkDN DCs, at steady state, showed an increase in the expression of several Notch target genes such as Hes-1, Hex-1, Deltex1, Notch3, Jagged-1 and Jagged-2. Blockade of Notch signaling with γ-secretase inhibitor (DAPT) in MLR mitigated the enhanced allo-stimulatory capacity of the Ik-/- DCs, thus demonstrating a functional relevance for enhanced notch signaling in the absence of Ikaros. We next hypothesized that given the enhanced GVHD response that was associated with increased proliferation and preserved cytotoxicity of allo-T cells, the GVL response will also be enhanced in the recipients with Ik-/- APCs. Unexpectedly, the [B6→ B6] and [Ik-/-B6 → B6] chimeras when transplanted with allogeneic T cells from C3H.SW cells along with two different doses of syngeneic 0.5 or 1×104 MBL-2 tumor cells demonstrated equivalent GVL responses despite greater severity of GVHD. Together our data demonstrate (a) for the first time, to our knowledge, suggest differential regulation of GVHD and GVL at the level of host APCs and (b) show a role for a novel molecular pathway, the Ik-Notch axis, in the host APCs as an important modulator of GVH responses. Disclosures:Maillard:University of Michigan Comprehensive Cancer Center: Research Funding; Damon Runyon Cancer Foundation: Research Funding; ASH Scholar Awards : Research Funding.

Regulatory T cells and liver pathology in a murine graft versus host response model

We have previously reported in mice the hepatic inflammatory in graft versus host response (GVHR) model due to the disparity of major histocompatibility complex class-II. The regulatory T (Treg) cells have been reported to control excessive immune response and prevent immune-related diseases. This study aimed to investigate the pathogenesis profiles of chronic GVHR progression, focusing on the Treg cells. GVHR mice induced by parental spleen CD4(+) T cell injection were sacrificed after 0, 2, 4, and 8 weeks (G0, G2, G4, G8). Further, one GVHR group received anti-IL-10 antibody in advance and were maintained for 2 weeks. Pathologic profiles of hepatic infiltrating inflammatory cells were evaluated by haematoxylin and eosin and immunohistochemistry staining with surface markers including Treg cell markers. Remarkable hepatic inflammatory in G2 significantly and gradually improved over time up to G8. In immunohistochemical staining, the increased IL-10 receptor beta(+) Tr1 cells in G2 were maintained through to G8; although other inflammatory cells decreased from G2 to G8. By contrast, in the anti-IL-10 antibody received-GVHR mice, the Tr1 cells were not detectable with significant inflammatory aggravation, while FoxP3(+) Treg cells significantly enhanced. These findings in the GVHR mice suggest that the expression and activity of Treg cells, especially the Tr1 cells, might be key factors for pathologic alteration in immune-related liver disease.

Targeted Protection of the Colon in the Absence of Donor Antigen-Presenting Cell -Derived Interleukin 23 Allows for Separation of Graft Versus Host and Graft Versus Leukemia Effects

Separation of graft versus leukemia (GVL) and graft versus host (GVH) reactivity has been a longstanding but elusive goal in allogeneic bone marrow transplantation (BMT). Recent studies from our laboratory have shown that donor APC-derived IL-23 secretion has a critical role in mediating pathological damage in the colon during GVHD, but has no protective effect in other GVHD target organs such as the lung and liver. These results demonstrate the feasibility of regional GVHD protection and raise the question as to whether such localized protection might be a strategy to separate GVH and GVL responses.

STAT3 Signaling in Donor-Derived CD4+ T-Cells Plays a Critical Role in the Induction of Acute and Chronic GVHD in Murine Models of alloBMT.

Growing evidence from several murine models suggests that STAT3 signaling in CD4+ T-cells plays a pivotal role in the pathogenesis of autoimmunity. We herein tested the hypothesis that the same pathway plays a critical role in the induction of GVHD in murine models of allogeneic BMT. To test our hypothesis and to compare the differential ability of STAT3 signaling in CD4+ T-cells to mediate GVHD in MHC-mismatched and-matched murine models of alloBMT, CD4-Cre × STAT3flox/flox (STAT3KOCD4+), or corresponding wild-type control mice were used as donors of CD4+ T-cells. In an MHC-mismatched model, groups of lethally irradiated (775 cGy) BALB/c (H-2d) mice received T-cell depleted (TCD) bone marrow (BM) from wild-type C57BL/6 (H-2b) mice alone, TCD B6 BM plus 4 × 105wild-type, or STAT3KOCD4+ T-cells, all on a C57BL/6 background. In repeated experiments we observed significantly decreased clinical signs of GVHD in animals receiving STAT3KOCD4+ in comparison to those receiving wild-type CD4+ T-cells (median GVHD score of 2.25 vs. 5.29; P <.001) but no difference in lethality in chimeras monitored up to day 60 posttransplant. In an MHC-matched, minor histocompatibility mismatched setting, we conducted experiments using the B10.D2 (H-2d) BALB/c (H-2d) model in which GVHD is CD4+ T-cell-mediated and has clinicopathologic consistent with human chronic GVHD. Since the dominant clinical manifestation in this model is cutaneous sclerodermatous GVHD that is associated with in situ dermal mononuclear infiltrates, the use of BALB/c-CD45.1 (CD45.1+) mice as recipients enabled us to examine the fate of skin CD11c+ dendritic cells. After conditioning (775 cGy) recipient mice received B10.D2 (CD45.2+) TCD BM and 9.3 ×106 TCD splenocytes, repleted with 106 wild-type CD8+ and 1.9 ×106 wild-type, or STAT3KOCD4+ T-cells. We reproducibly induced all signs of chronic GVHD in chimeras receiving wild-type CD4+ T-cells, but not in chimeras injected with STAT3KOCD4+ T-cells (median GVHD score of 5.2 vs. 0.0; P <.001). We found that development of cutaneous GVHD was accompanied by prominent dermal infiltration of donor-derived inflammatory monocytes and complete turnover to donor CD11c+ epidermal DC chimerism (88.4±4.3% vs. 0.9±0.6%; P <.001). Interestingly, splenic CD11c+ DC (99.9±0.1% vs. 99.8±0.1%), CD4+ (99.9±0.2% vs. 99.6±0.7%) and CD8+ (99.9±0.2% vs. 99.9±0.2%) T-cell chimerism was nearly completely donor-derived and did not differ between the two sets of described chimeras. In summary, our data suggest that intact STAT3 signaling in CD4+ T-cells is required for clinical manifestations of delayed acute and/or chronic GVHD, but it does not interfere with lymphohematopoietic graft-versus host response and achievement of full donor chimerism. Further exploration of the STAT3 signaling pathway in pathophysiology of delayed acute and/or chronic GVHD is warranted.

The Specific Monocarboxylate Transporter-1 (MCT-1) Inhibitor, AR-C117977, Induces Donor-Specific Suppression, Reducing Acute and Chronic Allograft Rejection in the Rat

In a search for immunosuppressive drugs having novel mechanisms, monocarboxylate transporter (MCT-1) inhibitors were identified that markedly inhibited immune responses. Here, we report the effects of AR-C117977, a potent MCT-1 inhibitor, on alloimmune responses in the rat. In vitro activity was determined in a rat mixed lymphocyte response (MLR). In vivo activity was tested in a graft versus host response (GVHR) and in both high (DA to PVG) and low (PVG to DA) responder cardiac allograft models. To assess induction of donor-specific suppression recipients of allogeneic hearts surviving longer than 100 days received a second transplant either of the same donor strain or a third-party donor strain. Effects on chronic graft rejection were assessed histologically by evaluating vasculopathy in long-term surviving grafts and in an obliterative bronchiolitis (OB) model. AR-C117977 inhibited the rat MLR and was more potent than cyclosporin A (CsA). In the rat GVHR model, AR-C117977 gave a dose-related inhibition. In the high responder cardiac allograft model, graft survival in excess of 100 days was achieved with AR-C117977 compared with 20 days with CsA and all the long-term survivors exhibited donor-specific suppression on retransplantation. In the low responder model, both AR-C117977 and CsA induced survival in excess of 100 days. Histology of the long-term surviving grafts suggested reduced vasculopathy associated with chronic rejection. Furthermore, AR-C117977 inhibited the occlusion of transplanted trachea in a OB model. This report describes a MCT-1 specific inhibitor having immunosuppressive activity on alloimmune responses and inducing donor-specific suppression.