Graft-versus-host Disease Target Organs Research Articles

Graft-Versus-Host Disease Prevention by In Vitro-Generated Myeloid-Derived Suppressor Cells Is Exclusively Mediated by the CD11b+CD11c+ MDSC Subpopulation.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid progenitor cells that dampen overwhelming adaptive immune responses through multiple mechanisms and are recognized as an attractive novel immune intervention therapy for counteracting the destructive effects of graft- versus -host disease (GVHD) developing after allogeneic bone marrow transplantation (BMT). MDSCs can be produced in great numbers for cellular therapy, but they present a mixture of subsets whose functions in GVHD prevention are undefined. Here, we generated MDSCs in vitro from murine BM cells in the presence of GM-CSF and defined the integrin CD11c as a marker to subdivide MDSCs into two functional subgroups: CD11b+CD11c+ and CD11b+CD11c− MDSCs. Isolated CD11b+CD11c+ and CD11b+CD11c− MDSCs both inhibited alloantigen-stimulated T-cell proliferation in vitro, although CD11b+CD11c+ MDSCs were more efficient and expressed higher levels of different immunosuppressive molecules. Likewise, expression of surface markers such as MHC class II, CD80, CD86, or PD-L1 further delineated both subsets. Most importantly, only the adoptive transfer of CD11b+CD11c+ MDSCs into a single MHC class I-disparate allogeneic BMT model prevented GVHD development and strongly decreased disease-induced mortality, while CD11b+CD11c− MDSCs were totally ineffective. Surprisingly, allogeneic T-cell homing and expansion in lymphatic and GVHD target organs were not affected by cotransplanted CD11b+CD11c+ MDSCs indicating a clear contradiction between in vitro and in vivo functions of MDSCs. However, CD11b+CD11c+ MDSCs shifted immune responses towards type 2 immunity reflected by increased Th2-specific cytokine expression of allogeneic T cells. Induction of type 2 immunity was mandatory for GVHD prevention, since CD11b+CD11c+ MDSCs were ineffective if recipients were reconstituted with STAT6-deficient T cells unable to differentiate into Th2 cells. Most importantly, the beneficial graft- versus -tumor (GVT) effect was maintained in the presence of CD11b+CD11c+ MDSCs since syngeneic tumor cells were efficiently eradicated. Strong differences in the transcriptomic landscape of both subpopulations underlined their functional differences. Defining CD11b+CD11c+ MDSCs as the subset of in vitro-generated MDSCs able to inhibit GVHD development might help to increase efficiency of MDSC therapy and to further delineate relevant target molecules and signaling pathways responsible for GVHD prevention.

Post-Transplantation Cyclophosphamide Induces Alloreactive T-Cell Functional Impairment and Depends on Foxp3+ Regulatory T Cells Not Selective Elimination or Intrathymic Clonal Deletion of Alloreactive T Cells

Our understanding of how post-transplantation cyclophosphamide (PTCy) prevents graft-versus-host disease (GVHD) in hematopoietic cell transplantation (HCT) is largely extrapolated from major histocompatibility complex (MHC)-matched murine skin allografting models. This current paradigm of understanding posits that PTCy preferentially eliminates alloreactive T cells and is also dependent on intrathymic clonal deletion of alloreactive T cells. Skin allografting data used to generate this model leveraged strain-specific differential expression of certain T-cell receptor (TCR) Vβs as markers of alloreactive cells.To test the hypothesis that PTCy works via selective elimination of alloreactive T cells and investigate the biology of PTCy, we developed a murine MHC-haploidentical HCT model that directly parallels human sibling haploidentical HCT. In this model (B6C3F1→B6D2F1), the administration of 40 x 106 splenocytes and 10 x 106 T-cell-depleted bone marrow cells following 10.5 Gy irradiation induced universally fatal GVHD, with the Vβ6 TCR serving as a marker of alloreactive T cells. Administration of PTCy on days +3 and +4 at doses ranging from 10 to 50 mg/kg effectively prevented lethal GVHD.In assessments of the blood, lymph nodes, spleen, and liver, PTCy 25 mg/kg did reduce proliferation of CD4+ T cells but not CD8+ T cells at day +7 post-transplant. Vβ6+ T cells in both the CD4+ and CD8+ subsets in all four tissue compartments persisted at or above normal donor frequencies at days +7, +21, and +200 post-transplant. Persistence of Vβ6+ T cells in all four tissue compartments also was demonstrated in three other murine HCT models: B6→B6D2F1 (MHC-haploidentical; day +7), C3H→B6D2F1 (MHC-disparate; days +6 and +200), and C3H→AKR (MHC-matched; day +7). Using our B6C3F1→B6D2F1 model, we also created grafts in which 8% of donor CD8+ T cells expressed the 2C TCR, which is specific for H2-Ld (a major histocompatibility antigen in our model). 2C TCR+ T cells continued to proliferate highly and markedly expand from days +3 to +7 despite administration of PTCy 25 mg/kg on days +3 and +4, disproving selective alloreactive T-cell elimination after PTCy treatment in HCT. Additionally, PTCy was equally effective in our wild-type B6C3F1→B6D2F1 model when using thymectomized or non-thymectomized recipients, negating a necessary role for the thymus in GVHD prevention by PTCy.Compartmentalization could not explain GVHD amelioration despite persistence of alloreactive T cells as similarly normal Vβ6+ frequencies were found infiltrating the liver, a GVHD target organ, as were found in the blood, lymph nodes, or spleen. Indeed, 2C TCR+ T cells composed 70-80% of liver-infiltrating CD8+ T cells at day +7 in mice treated with or without PTCy. Yet, compared with vehicle-treated mice, liver-infiltrating wild-type T cells obtained at day +21 from PTCy-treated mice had slightly reduced proliferation but dramatically lower cytokine production when restimulated in vitro with DBA/2 (host parental) irradiated splenocytes. Similar results were seen when repeating these experiments using liver-infiltrating T cells that were flow cytometrically depleted of CD4+CD25+ T cells, suggesting impaired functionality of alloreactive T cells after PTCy.CD4+CD25+Foxp3+ T cells did preferentially expand in all four tissue compartments by day +21 post-transplant. A one-log reduction of these cells in the donor graft using an AutoMACS had minimal impact on transplant outcomes in our model. However, a two-log reduction of Foxp3+ cells immediately post-transplant in vivo using Foxp3-DTR-expressing donor cells resulted in marked worsening of the clinical scores and weights, which began to diverge starting approximately 30 days post-transplant. PTCy-treated mice also were resistant to reinfusion of 40 or 120 x 106 new donor splenocytes at +120-150 days, consistent with suppression being an active mechanism underlying PTCy's efficacy. Overall, our findings disprove the existing paradigm of understanding of how PTCy prevents GVHD, support our previously demonstrated role for regulatory T cells, and may have direct translational relevance towards improving outcomes for patients. DisclosuresNo relevant conflicts of interest to declare.

Oxidative Stress Regulates T Cell Pathogenicity in Gvhd

Allogeneic hematopoietic transplantation (allo-HCT) is a classic immunotherapy for hematologic malignances, but graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality.Oxidative stress is considered as an unavoidable consequence of allo-HCT and could contribute to GVHD pathogenicity significantly. Owing to the contributions of pre-existing disease conditions and the requirement for conditioning regimens that increases cellular reactive oxygen species (ROS), oxidative stress is elevated in all BMT recipients. Thioredoxin (Trx) is ubiquitously expressed enzymes, counteracts oxidative stress by scavenging ROS and regulating other enzymes metabolizing H2O2. In addition, it has been shown that regulatory T cells (Tregs) express and secrete higher levels of Trx than conventional CD4 T cells. Trx-1 is critical for the resilience of Tregs to oxidative stress and sustained expression of surface thiols at a high density. We hypothesis that oxidative stress plays a critical role in the pathology of GVHD development, and thus the maintenance of a redox balance would be an effective strategy to control GVHD after allo-BMT.To test this hypothesis, we used a strain of mice that express transgenic (Tg) human Trx-1 ubiquitously, refer as Trx-Tg mice. We first verified that Trx-Tg T cells demonstrated significantly reduced ROS accumulation after activation in vitro and in vivo. We next sought to test whether Trx1 affects T cell activation and function. Upon alloantigen stimulation in vitro, Trx-Tg T cells have substantially reduced ability to produce IFN-γ compared with WT counterparts. These results prompted us to evaluate T cell responses in vivo . Upon transfer of T cells into irradiated allogeneic recipients, we observed that the Trx-Tg T cells significantly reduced proliferation and IFN-γ production compared with WT counterparts. Given that Trx-Tg T cells displayed a reduced allogeneic response in vitro and in vivo, we further hypothesized that Trx plays an essential role in regulating allogeneic T cells in GVHD. We performed a MHC-mismatched B6→BALB/c BMT model, and found that recipients of WT T cells developed severe and lethal GVHD, whereas the majority of the recipients of Trx-Tg T cells survived long term and had significantly less weight loss and lower clinical score. Development of GVHD requires donor T cell expansion in lymphoid organs and migration into target organs. Hence, we asked whether TRX-1 is required for T cell infiltration and expansion into target organs. Three weeks after BMT, we observed that recipients of Trx-Tg T cells had significantly fewer pathological injuries in all GVHD target organs, which was consistent with the GVHD outcome. While comparable numbers of Trx-Tg T and WT T cells were found in recipient spleens, a significantly lower number of Trx-Tg T cells were found in recipient livers as compared with that of WT T cells. In addition, T cells that migrated into the liver expressed significantly lower levels of intercellular IFN-γ in the Trx-Tg group. These data suggest that Trx-Tg T cells may reduce expansion and/or migration. For translational purpose, we evaluated therapeutic potential of recombinant human Trx-1 (rhTrx-1), and found that administration of recombinant human Trx-1 significantly reduced ability of T cells to produce IFN-γ compared with control treatment upon alloantigen stimulation in vivo . Furthermore, we performed a MHC-mismatched B6→BALB/c BMT model and found that administration of rhTrx-1had significantly reduced the mortality and mobility of BLAB/c recipients. Theraputic effect of rhTrx-1 in the prevention of GVHD was validated in a haploidentical BMT model (B6→BD2F1). More importantly, administration of rhTrx-1 did not impair the graft-versus-leukemia (GVL) against p815 tumor. Taken together, the current work provides a strong rationale and demonstrates the feasibility to target ROS pathway, which can be readily translated into clinic. DisclosuresNo relevant conflicts of interest to declare.

Absence of Vasoactive Intestinal Peptide Synthesis in Recipients in Allogeneic Bone Marrow Transplant Increases Graft Versus Host Diseases

Introduction: Vasoactive intestinal polypeptide (VIP) is an immunosuppressive neuropeptide. Activation of donor T cells leading to graft-versus-host disease (GvHD) in allogeneic bone marrow transplant (allo-BMT) occurs via early post transplant interactions between donor T cells and host antigen presenting cells and leads to upregulation of immune co-inhibitory pathways, including expression of VIP in T cells and dendritic cells. We have previously shown VIP signaling favorably regulates the activation status and enhances the graft-versus-leukemia (GvL) activities of donor T cells (Li et al., Cancer Research 2016). The role of the VIP produced by host antigen presenting cells in regulating GvHD in allo-BMT is unknown. We tested the hypothesis that absence of VIP expression by host cells would augment donor T cell activation and increase GvHD.Methods and Results: We used the B10BR->B6 MHC fully mismatched murine model of allogeneic BMT. Recipient wildtype (WT) and VIP knockout B6 (VIP-KO) mice were lethally irradiated with 11Gy and injected i.v. with 5×106 T cell depleted (TCD) bone marrow cells (BM) plus 0, 1 or 3 ×106 splenocytes from WT B10BR donors. Fifteen mice (males and females) were assigned to each splenocytes dosage group. The survival of recipients after BMT was recorded daily and the average clinical GvHD scores were obtained by combining weight-loss, activity, posture, fur texture and skin integrity data following standard published procedures. All three groups of WT recipients had better survival than VIP-KO recipients. VIP-KO mice transplanted with TCD BM alone had only 33.3% survival at day 75 compared with 73.3% survival among the WT recipients. Chimerism studies on day 10 post-BMT showed equivalent level of donor chimerism: 80% donor in WT recipients vs 72% donor in VIP-KO recipients, indicating increased mortality in the VIP-KO recipients was not due to early graft rejection. The addition of donor splenocytes to the graft resulted in significantly more GvHD-related mortality in VIP-KO recipients compared with WT recipients (Figure 1), with 80% vs 100% day 75 survival among recipients of 1x106 splenocytes (p=NS) and 0% vs 40% survival (p<0.01) among recipients of 3x106 donor splenocytes, respectively.To determine how the absence of VIP production in KO recipients modulates immune effector mechanisms that contributes to the differential survival rate post allo-BMT, we analyzed the recipients' splenocytes on day 30 post transplant with flow cytometry and staining with CD3, CD4, CD8, CD62L, CD44 and PD1 antibodies. VIP-KO recipients transplanted with 5M TCD BM plus 1M splenocytes had more CD4+ central memory and effector T cells, and CD8+ central memory and effector T cells. VIP-KO recipients transplanted with 5×106 TCD BM alone in addition had significantly more CD4 and CD8 effector T cells (p<0.01). VIP-KO recipients transplanted with 5×106 TCD BM plus 1x106 splenocytes had more PD1 expression on donor CD4+ central memory and effector T cells, and CD8+ central memory effector T cells, consistent with enhanced antigen-mediated activation.To visualize VIP produced by the host cells, we transplanted C57/BL6 recipients expressing a GFP transgene under the control of the VIP promoter (n=10) with 5×106 TCD BM plus 3 ×106 splenocytes from WT B10BR donors, and visualized VIP gene promoter activity as GFP expression in recipient lymphoid tissues and GvHD target organs. Only small number of CD45+ VIP-GFP+ cells were detected from in host liver, spleen and lymph nodes on day 10 post transplant (Figure 2), suggesting residual host leukocytes, particularly dendritic cells, expressed VIP.Conclusion: VIP production by recipient antigen presenting cells is a key pathway in limiting the activation of donor T cells. Physiological expression of VIP by host leukocytes regulate the GvHD activity of donor T cells. Novel methods to enhance VIP production in transplant recipients or the parenteral administration of VIP receptor agonists may limit GvHD, and may be particularly important in allo-BMT where GvL is not necessarily critical for success, such as transplants for hemoglobinopathy. [Display omitted] DisclosuresWaller:Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding; PRA: Consultancy; AMGEN: Consultancy; National Institutes of Health: Research Funding; Coulter Foundation: Research Funding; Helocyte: Consultancy; Cambium Medical Technologies: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Katz Foundation: Research Funding; Chimerix: Equity Ownership; Cerus: Equity Ownership; Celldex: Consultancy.

Id3 Is Essential for Mediating Ezh2-Regulated Persistence of Alloreactive T Cells in Gvhd Target Tissues in Mice

Abstract: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is potentially a curative treatment for hematologic malignancies and non-malignant diseases. However, its application is limited by graft-versus-host disease (GVHD), which is induced by donor T cells that recognize and react to histocompatibilty differences. Despite pharmacological prophylaxis, acute and chronic GVHD still develop in 40-60% of allo-HSCT recipients. Data from our studies and others suggested critical roles of alloantigen-sensitized memory T cells in sustaining host tissue injury during GVHD. However, the molecular pathway(s) that are crucial for the development and maintenance of alloreactive memory T cells mediating persistent GVHD remain largely unknown. Id3 (Inhibitor of DNA Binding 3) is a transcription factor that negatively regulates the DNA-binding activity of E-protein. During response to acute viral infection, Id3 has been shown to regulate the generation of memory T cells, but is dispensable for primary effector response. Whether Id3 is responsible for persistent allo-immunity and tissue damage during GVHD has not been previously examined. Using experimental GVHD models, we demonstrate that Id3 functions as a key molecular gatekeeper for persistent generation of alloreactive T cells mediating tissue damage and GVHD. Infusion of donor T cells from wild-type (WT) B6 mice caused uniformly lethal GVHD in allogeneic BALB/C recipient mice. In contrast, transfer of donor T cells derived from Id3 conditional knockout (Id3-/-) B6 mice did not induce lethal GVHD. Notably, Id3-/- T cell recipients developed similar symptoms of GVHD (e.g. weight loss, hunched back and reduced mobility) as compared to WT T cell recipients within 20 days after allo-HSCT. However, while GVHD continually progressed in WT T cell recipients, which was evidenced by hair loss, severe diarrhea, skin ulceration, weight loss and death, it was gradually diminished in mice receiving allogeneic Id3-/- T-cells without apparent skin inflammation and tissue damage. As a result, all Id3-/- T-cell recipient mice survived over 70 days after allo-HSCT. We found that by 7 days and 14 days after transplantation, there were similar numbers of donor-derived CD4+ and CD8+ T cells in BALB/c recipients (e.g. spleen, liver, skin and gut) of Id3-/- T cells compared with WT T cells. Furthermore, during this acute alloreactive response phase both Id3-/- and WT T cells expressed high levels of activation markers (e.g. CD25, CD44, CD69, CD122) and had high IFN-g production. By 35 days after allo-HSCT, Id3-/- T cell recipient mice showed significantly increased number of donor T cells in the spleen compared to WT T cell recipients, but had 10-fold fewer numbers of alloreactve T cells in GVHD target organs including gut, skin and liver. Analysis of samples from human patients undergoing allo-HSCT revealed that GVHD patients (n=8) had significantly higher frequency of T cells expressing high levels of ID3 compared to time-matched non-aGVHD patients (n=6). These findings suggest that Id3 plays critical roles in mediating the persistence and accumulation of tissue-infiltrating alloreactive T cells, while being dispensable for early activation, proliferation and effector differentiation of alloreactive T cells. Our recent studies have discovered a central role of the epigenetic regulator Ezh2 in controlling allogeneic T cell proliferation, differentiation and function (Blood 2012, Blood 2013 and Blood 2017). Using genome-wide profiling analysis and functional assessment, we found that Ezh2 directly activates Id3 transcription, independent of its canonical role in repressing gene transcription. Retroviral engineering of Ezh2-deficient T cells to express Id3 significantly enhanced the persistence of antigen-activated T cells in vivo, suggesting a critical role of Id3 in mediating Ezh2 regulation of activated T cell survival and maintenance. In summary, our findings identify the importance of Id3 in regulating persistent allo-immunity and tissue damage, and illuminate a novel molecular pathway of Ezh2-Id3 that may be potentially targeted for GVHD therapy. DisclosuresNo relevant conflicts of interest to declare.

Complement Promotes Gvhd through Suppressing Autophagy in Recipient Dendritic Cells after Allogeneic Hematopoietic Cell Transplantation in Mice

Graft-versus-host disease (GVHD) limits the success of allogeneic hematopoietic stem cell transplantation (allo-HCT). Traditional therapies have targeted T cells, yet immunostimulatory dendritic cells (DCs) are critical in the pathogenesis of GVHD. Autophagy is a self-degradative process for cytosolic components and is required for maintaining homeostasis after allo-HCT. Complement receptors C3aR/C5aR can regulate immune responses by translating information gathered by complement fluid phase sensors into cellular responses. C3aR/C5aR have been reported to activate the autophagy inhibitor mTOR in cells. While depletion of autophagy in recipient DCs aggravates GVHD severity, and enhancing autophagy represents as a promising approach for treating immunological diseases, it is not known how escalation of autophagy regulates DC-functionality in GVHD pathogenesis. In the current study, we found that deficiency of C3aR/C5aR signaling significantly upregulated mTOR-independent autophagy in DCs (Figure 1B), which is associated with increased apoptosis, and reduced activation and antigen presenting capacity of C3aR/C5aR-/-DCs (Figure 1, A-C).To evaluate the role of C3aR/C5aR in modulating DC function in GVHD pathogenesis, we compared the severity of GVHD in WT versus C3aR/C5aR-/- recipients using murine models of allo-HCT. C3aR/C5aR-/- recipients developed less severe GVHD than their WT counterparts, reflected by a significantly higher rate of survival and lower clinical scores (Figure 1D). Donor T-cell survival and Th1-differentiation were significantly decreased, whereas Treg generation was increased in C3aR/C5aR-/- recipients. Glycolytic activity of donor T cells was significantly abrogated in the absence of recipient C3aR/C5ar (Figure 1E). Donor T-cells expressed lower levels of chemokine receptors, CXCR3 and CCR6, which were likely contributed to the decreased migration of the effector T cells into GVHD target organs of C3aR/C5aR-/- recipients. Using chimeric recipients in which either the hematopoietic or non-hematopoietic compartment is deficient for C3aR/C5aR, we found that C3aR/C5aR in the recipient hematopoietic cells was primarily responsible for donor T-cell response and pathogenicity in GVHD. Transfusion of C3aR/C5aR-/- DCs along with allo-HCT mitigated GVHD severity, indicating that C3aR/5aR signaling in DCs plays a predominant role in the pathogenesis of GVHD. Elevated autophagy was required for the suppressive effect of C3aR/C5aR-/- DCs on GVHD as autophagy inhibitor chloroquine (CQ) reverted the GVHD-suppressive effect of C3aR/C5aR-/-DCs (Figure 1F). For translational purpose, we evaluated the therapeutic potential of a combination of C3aR and C5aR antagonists, and found that pharmacological blockade of C3aR/C5aR could effectively reduce GVHD severity while sparing GVL effect through persevering CD8 cytolytic activity.The current study reveals for the first time that escalation of autophagy in DCs is an effective therapeutic approach for the control of GVHD, and establishes and interaction between complement and autophagy in allo-HCT. We also provide a novel strategy of enhancing autophagy as a DC-based vaccination strategy for the control of GVHD after transplantation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Targeting SLP76:ITK interaction separates GVHD from GVL in allo-HSCT

Abstract Allogeneic hematopoietic cell transplantation (allo-HCT) is a curative therapy for relapsed hematological malignancies due to graft-versus-leukemia (GVL) activity mediated by alloreactive donor T cells. However, graft-versus-host disease (GVHD) is also primarily mediated by the same donor T cells. Here we assessed the effect of attenuating TCR-mediated SLP76:ITK interaction in T cell-mediated GVL vs. GVHD effects after allo-HSCT. Neither CD8+ T cells nor CD4+ donor T cells from mice expressing a tyrosine to phenylalanine mutation at position 145 (Y145F) of the adapter protein SLP-76 caused GVHD, T cells cells preserved GVL effects after allogeneic transplantation. SLP76Y145FKI CD8+ and CD4+ donor T cells also produce less inflammatory cytokines and show decreased migration to GVHD target organs such as the liver and small intestine, while maintaining GVL efficacy against primary B-cell acute lymphoblastic leukemia (B-ALL). We also report the development of a novel peptide that can specifically inhibit SLP76 and ITK interactions, which results in decreased phosphorylation of PLCγ1 and ERK, and decreased cytokine production in human T cells. This peptide inhibited donor T cell-mediated GVHD while maintaining GVL effects. Altogether, our data suggest that inhibiting SLP76:ITK interaction could be a therapeutic strategy to reduce GVHD while retaining the beneficial GVL effects after allo-HSCT treatment.

Targeting SLP76:ITK interaction separates GVHD from GVL in allo-HSCT

SummaryAllogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapy for hematological malignancies, due to graft-versus-leukemia (GVL) activity mediated by alloreactive donor T cells. However, graft-versus-host disease (GVHD) is also mediated by these cells. Here, we assessed the effect of attenuating TCR-mediated SLP76:ITK interaction in GVL vs. GVHD effects after allo-HSCT. CD8+ and CD4+ donor T cells from mice expressing a Y145F mutation in SLP-76 did not cause GVHD but preserved GVL effects against B-ALL cells. SLP76Y145FKI CD8+ and CD4+ donor T cells also showed less inflammatory cytokine production and migration to GVHD target organs. We developed a novel peptide to specifically inhibit SLP76:ITK interactions, resulting in decreased phosphorylation of PLCγ1 and ERK, decreased cytokine production in human T cells, and separation of GVHD from GVL effects. Altogether, our data suggest that inhibiting SLP76:ITK interaction could be a therapeutic strategy to separate GVHD from GVL effects after allo-HSCT treatment.

Targeting Interleukin-2-Inducible T-Cell Kinase (ITK) Differentiates GVL and GVHD in Allo-HSCT.

Allogeneic hematopoietic stem cell transplantation is a potentially curative procedure for many malignant diseases. Donor T cells prevent disease recurrence via graft-versus-leukemia (GVL) effect. Donor T cells also contribute to graft-versus-host disease (GVHD), a debilitating and potentially fatal complication. Novel treatment strategies are needed which allow preservation of GVL effects without causing GVHD. Using murine models, we show that targeting IL-2-inducible T cell kinase (ITK) in donor T cells reduces GVHD while preserving GVL effects. Both CD8+ and CD4+ donor T cells from Itk-/- mice produce less inflammatory cytokines and show decrease migration to GVHD target organs such as the liver and small intestine, while maintaining GVL efficacy against primary B-cell acute lymphoblastic leukemia (B-ALL). Itk-/- T cells exhibit reduced expression of IRF4 and decreased JAK/STAT signaling activity but upregulating expression of Eomesodermin (Eomes) and preserve cytotoxicity, necessary for GVL effect. Transcriptome analysis indicates that ITK signaling controls chemokine receptor expression during alloactivation, which in turn affects the ability of donor T cells to migrate to GVHD target organs. Our data suggest that inhibiting ITK could be a therapeutic strategy to reduce GVHD while preserving the beneficial GVL effects following allo-HSCT treatment.

The role of TCF-1 in graft-versus-host disease and antitumor effects

Abstract Allogeneic hematopoietic stem cell transplants (HSCT) are used as a curative therapy for malignancies such as cancer. Residual tumor cells and recurrence are controlled by the graft-versus-tumor (GVT) effect, mediated by alloreactive T cells. However, these same cells also cause graft-versus-host disease (GVHD), a serious side effect. Naïve mature T cells in the graft recognize foreign HLA molecules on the host’s cells, and are alloactivated. The T cells then proliferate, produce cytokines, and migrate to the GVHD target organs (liver, skin, and small intestine), damaging host tissues. Separation of detrimental GVHD from beneficial GVT remains a serious unaddressed problem. Thus, the goal of this project is to achieve this separation through T cell signaling modulation. The transcription factors T-bet and Eomes impact GVHD and GVT. They are downstream of T Cell Factor-1 (TCF-1), which is important in thymocytes and T cell activation. To determine the role of TCF-1 in alloactivation, mice with T cell-specific deletion of TCF-1 were phenotyped. These mice had increased expression of Eomes, Tbet, CD8+ central memory T cells, and polarization of CD44 expression. They also showed reduced activating T cells, and reduced expression of CXCR5 (a major chemokine receptor) when alloactivated. Finally, GVHD severity was reduced in mice transplanted with TCF-1-deficient T cells, and survival was improved in these mice. These data suggest that loss of TCF-1 results in a more activated state, yet GVHD is still reduced. Therefore, these cells may still be capable of antitumor immunity, separating GVHD from GVT. This work will help to improve outcomes for HSCT patients and enhance understanding of how mature alloactivated T cells are regulated.

Graft vs. host disease leads to elimination of tissue-resident innate lymphoid cells following experimental and clinical allogeneic transplantation

Abstract Innate lymphoid cells (ILCs) are associated with protective mucosal responses after allogeneic bone marrow transplantation (allo-BMT). However, the specific in vivo roles of ILCs after BMT remain unclear. We thus sought to characterize the kinetics and significance of ILC reconstitution after allo-BMT. In a sex-mismatched acute graft-vs-host-disease (GVHD) model, depletion of recipient ILCs induced significantly worse systemic signs of GVHD, as well as increased thymic injury and intestinal pathology, indicating a contributory role for ILCs in tissue protection post-BMT. Analysis of ILCs in distinct GVHD target organs after T-cell-depleted BMT demonstrated a high frequency of NK cells and ILC1s in the liver, a higher proportion of ILC2s in lungs, and a higher proportion of ILC3s in the small intestine post-transplant in the absence of GVHD. For all tissues analyzed, the majority of ILCs were host-derived. In contrast, in the presence of GVHD, all ILC subsets were significantly reduced in all tissues analyzed. Furthermore, ILC precursors were significantly reduced in the bone marrow of mice with GVHD, suggesting that alloreactive immunity can impair ILC reconstitution by targeting both mature ILCs and their developmental niche. To examine the clinical relevance of these findings, human ILCs were analyzed by FACS of lamina propria lymphocytes isolated from patient duodenal biopsy specimens obtained to evaluate symptoms of acute GI GVHD. Consistent with experimental findings, duodenal ILC frequencies were significantly lower in patients with histologic evidence of GVHD. In conclusion, tissue-resident ILCs contribute to tissue protection after allo-BMT, but GVHD leads to ILC elimination and impairs their reconstitution.

CrkL is required for donor T cell migration to GvHD target organs.

The success of cancer therapies based on allogeneic hematopoietic stem cell transplant relies on the ability to separate graft-versus-host disease (GvHD) from graft-versus-tumor (GVT) responses. Controlling donor T cell migration into peripheral tissues is a viable option to limit unwanted tissue damage, but a lack of specific targets limits progress on this front. Here, we show that the adaptor protein CrkL, but not the closely related family members CrkI or CrkII, is a crucial regulator of T cell migration. In vitro, CrkL-deficient T cells fail to polymerize actin in response to the integrin ligand ICAM-1, resulting in defective migration. Using a mouse model of GvHD/GVT, we found that while CrkL-deficient T cells can efficiently eliminate hematopoietic tumors they are unable to migrate into inflamed organs, such as the liver and small intestine, and thus do not cause GvHD. These results suggest a specific role for CrkL in trafficking to peripheral organs but not the lymphatic system. In line with this, we found that although CrkL-deficient T cells could clear hematopoietic tumors, they failed to clear the same tumor growing subcutaneously, highlighting the role of CrkL in controlling T cell migration into peripheral tissues. Our results define a unique role for CrkL in controlling T cell migration, and suggest that CrkL function could be therapeutically targeted to enhance the efficacy of immunotherapies involving allogeneic donor cells.

Glutathione dynamics determine the therapeutic efficacy of mesenchymal stem cells for graft-versus-host disease via CREB1-NRF2 pathway.

Glutathione (GSH), the most abundant nonprotein thiol functioning as an antioxidant, plays critical roles in maintaining the core functions of mesenchymal stem cells (MSCs), which are used as a cellular immunotherapy for graft-versus-host disease (GVHD). However, the role of GSH dynamics in MSCs remains elusive. Genome-wide gene expression profiling and high-throughput live-cell imaging assays revealed that CREB1 enforced the GSH-recovering capacity (GRC) of MSCs through NRF2 by directly up-regulating NRF2 target genes responsible for GSH synthesis and redox cycling. MSCs with enhanced GSH levels and GRC mediated by CREB1-NRF2 have improved self-renewal, migratory, anti-inflammatory, and T cell suppression capacities. Administration of MSCs overexpressing CREB1-NRF2 target genes alleviated GVHD in a humanized mouse model, resulting in improved survival, decreased weight loss, and reduced histopathologic damages in GVHD target organs. Collectively, these findings demonstrate the molecular and functional importance of the CREB1-NRF2 pathway in maintaining MSC GSH dynamics, determining therapeutic outcomes for GVHD treatment.

Increased P2X7 expression in the gastrointestinal tract and skin in a humanised mouse model of graft-versus-host disease.

Allogeneic haematopoietic stem cell transplantation (HSCT) is a curative therapy for blood cancers; but results in the development of graft-versus-host disease (GVHD) in up to 70% of recipients. During GVHD, tissue damage results in ATP release into the extracellular compartment activating P2X7 on antigen-presenting cells, leading to the release of pro-inflammatory cytokines and subsequent activation of donor T cells. Therefore, the aim of the present study was to examine murine (m) P2rx7 and human (h) P2RX7 gene expression in GVHD target organs of humanised mice, and further characterise disease impact in these organs. NOD-scid IL2Rγnull (NSG) mice were injected with human peripheral blood mononuclear cells (hu-PBMC-NSG mice) or phosphate-buffered saline (PBS, control). Leucocytes were assessed by flow cytometry; gene expression was measured by quantitative polymerase chain reaction (qPCR), and tissue sections examined by histology. Compared with control mice, hu-PBMC-NSG mice had increased mP2rx7 and mP2rx4 expression in the duodenum, ileum and skin. hP2RX7 was expressed in all tissues examined. hu-PBMC-NSG mice also displayed increased mReg3g expression in the duodenum and ileum, despite limited histological gut GVHD. hu-PBMC-NSG mice showed histological evidence of GVHD in the skin, liver and lung. Compared with control mice, hu-PBMC-NSG mice displayed increased ear swelling. Combined data revealed that P2rx7 is up-regulated in gut and skin GVHD and that P2RX7 is present in target tissues of GVHD, corresponding to human leucocyte infiltration. Data also reveal increased mReg3g expression and ear swelling in hu-PBMC-NSG mice, offering new measurements of early-stage gut GVHD and skin GVHD, respectively.

ATG5 Dependent Autophagy in Graft-Versus-Host Disease Target Organs Mitigates Disease Severity Post Allogeneic Bone Marrow Transplant

Autophagy protects cells from a variety of stressors and is known to affect immune cell responses after allogeneic bone marrow transplant (allo-BMT). However, a definitive and exclusive role of target tissue pathways on mitigating graft-versus-host disease (GVHD) has yet to be demonstrated. Because GVHD target organs are under stress from alloreactive T cells, we sought to determine the role, if any, of target tissue autophagy in mitigating the severity of GVHD. Because the primary driver of morbidity and mortality after allo-BMT is gastrointestinal (GI) GVHD, we next hypothesized that autophagy protects the GI epithelium from alloimmune damage. To determine these effects in this target organ, we generated mice that lack a macroautophagy protein, ATG5, in the GI epithelium (Atg5D/IEC) and used them as recipients in the well-characterized MHC disparate GVHD murine model BALB/c→B6. As hypothesized, allo Atg5D/IEC mice showed significantly high mortality (P We next determined whether autophagy is critical for other GVHD target organs. To this end, we generated Atg5D/hep mice, which lack Atg5 expression in hepatocytes. Using the GVHD model described above, we observed dramatically shorter survival in the Atg5D/hep allo-recipients (P = 0.025) along with an increase in hepatocyte damage markers (bilirubin (P = 0.0001), alanine aminotransferase (P = 0.036), and alkaline phosphatase (P = 0.0002)) in Atg5D/hep allo-recipients. Again, there were similar numbers of splenic and liver-resident donor T cells, activation markers, and proinflammatory cytokines. To determine if autophagy is exclusively protective in GVHD target organs, we generated Atg5D/Myo mice, which lack Atg5 expression in cardiac myocytes, and Atg5D/Pod mice, which lack Atg5 expression in podocytes, in non-GVHD target organs. Using the same GVHD model as above, we monitored these mice and littermate controls for survival and severity of GVHD symptoms. In contrast to our findings in the Atg5D/hep and Atg5D/IEC recipients, we observed similar survival (P = 0.17, Myo; P=0.49, Pod) and GVHD severity between these allo-recipients. Collectively, our results demonstrated that autophagy is a tissue-intrinsic protective response that regulates tissue damage severity only in GVHD target organs.

Graft-versus-host disease targets R-spondin3-producing lymphatic endothelial cells in the small intestine

Graft-versus-host disease (GVHD) is a potentially fatal complication of allogeneic hematopoietic stem cell transplantation. The gastrointestinal tract is a major target organ of GVHD, and disruption of the barrier function of the intestinal mucosa leads to an influx of danger signals derived from intestinal microbiota, which may further exaggerate GVHD. We have shown that the recombinant human R-spondin1 protects intestinal stem cells against GVHD, improves intestinal dysbiosis, and ameliorates GVHD. However, the endogenous R-spondin-producing cells in the small intestine remained to be studied in greater detail. This study clarified that R-spondin3 is the predominant R-spondin protein produced in the mouse small intestine. We also found that R-spondin3 is predominantly produced by lymphatic endothelial cells. Furthermore, we found that GVHD targets lymphatic endothelial cells in the small intestine, leading to decreased R-spondin3 production. GVHD-induced reduction of endogenous R-spondin3 could delay intestinal epithelial regeneration, possibly resulting in GVHD deterioration.

S1PR5 regulates NK cell responses in preventing graft-versus-host disease while preserving graft-versus-tumour activity in a murine allogeneic haematopoietic stem cell transplantation model.

Graft-versus-host disease (GVHD) remains a major complication following allogeneic haematopoietic stem cell transplantation (allo-HSCT) leading to high transplant-related mortality. Natural killer (NK) cells have been found to mitigate GVHD without attenuating the graft-versus-tumour (GVT) activity in the murine model of haematopoietic stem cell transplantation. Sphingosine-1-phosphate receptor 5 (S1PR5) is a very important chemokine receptor on NK cells that governs NK cell distribution in vivo and trafficking at lesion sites. Our preliminary studies showed that the incidence of GVHD was negatively correlated with S1PR5 expression in the NK cells of patients after allo-HSCT. In the present study, we found that S1PR5 deficiency in murine NK cells blocked the migration of NK cells from the bone marrow to the GVHD target organs and attenuated the inhibitory effects on the alloreactive T cells, especially CD3+ CD8+ T cells, which may be the reason why the loss of S1PR5 in NK cells could aggravate GVHD in recipient mice. Furthermore, we also demonstrated that the absence of S1PR5 expression in NK cells did not interfere with the antitumour effects of NK cells and T cells in vivo. Taken together, our data indicate that S1PR5 plays an essential role in balancing GVHD and GVT activity.

ATG5 Dependent Autophagy in Graft-Versus-Host Disease Target Organs Mitigates Disease Severity Post Allogeneic Bone Marrow Transplant

In immune mediated diseases such as graft-versus-host disease (GVHD), research has focused almost entirely on hematopoietic cells. It has recently been posited that mechanisms within target tissues also play a significant role in regulating the severity of disease. Autophagy protects cells from a variety of stressors and is known to affect immune cell responses after allogeneic bone marrow transplant (allo-BMT). However, a definitive and exclusive role of target tissue pathways on mitigating GVHD has heretofore not been demonstrated. Because GVHD target organs are under stress from alloreactive T cells, we sought to determine the role of target tissue autophagy in mitigating the severity of GVHD. We hypothesized that the stress from alloreactive T cells would result in upregulation of target tissue autophagy. To test this in vivo, we utilized LC3-GFP-RFP reporter mice as transplant recipients in the well-characterized MHC disparate GVHD murine model BALB/c→B6. In these reporter mice, autophagosomes are marked with a dual GFP-RFP tag, and can be assessed for in vivo induction of autophagy by confocal microscopy through quantification of red punctal abundance. The recipient B6 reporter mice were lethally irradiated with split doses of 5Gy and transplanted with 5x106 BM and 3x106 splenic T-cells from either syngeneic B6 (syn) or allogeneic BALB/c (allo) donors. We assessed for induction of autophagy in GVHD target organs (gut, liver) and non-target organs (kidney, heart) and observed that autophagy was induced to a greater extent in target organs. Because the primary driver of morbidity and mortality after allo-BMT is gastrointestinal (GI) GVHD, we next hypothesized that autophagy protects the GI epithelium from alloimmune damage. To determine the effects of autophagy exclusively in the GI tract, we generated mice that lack a macroautophagy protein, ATG5, in the GI epithelium only (Atg5fl/flVil1-Cre) and utilized them as recipients in BALB/c→B6 allo-BMT. As hypothesized, survival of allo Atg5fl/flVil1-Cre mice was significantly reduced (P&lt;0.0001) compared to littermate controls. Importantly, all syn Atg5 KO mice survived the entire period of observation, demonstrating that protection from conditioning regimen toxicity did not require ATG5-dependent autophagy in the gut. We harvested both GVHD target and non-target organs and performed histopathological analysis at day 7 post-transplant and found that histological GVHD severity in the gut was significantly higher in allo Atg5fl/flVil1-Cre mice compared to Cre-null WT littermate controls (P=0.0063). All other organs were unaffected by loss of ATG5 in the GI epithelium, and we observed no difference between WT and Atg5 KO syn recipients in any organ. We harvested splenocytes and tissue-resident lymphocytes from the gut and found similar numbers of donor CD4 and CD8 T cells, activation markers CD69 and CD62L, and IFNγ and TNFα positive cells. Furthermore, similar levels of the proinflammatory serum cytokines IFNγ, TNFα, and IL-6 were observed in the sera of WT and Atg5 KO mice. We next determined whether autophagy is critical for protection in other GVHD target organs after allo-BMT. To this end, we generated Atg5fl/flAlb-Cre mice, which lack Atg5 expression in hepatocytes. Using a similar model of acute GVHD as above, we observed dramatically shorter survival in the Atg5fl/flAlb-Cre allo recipients (P=0.025) along with an increase in the hepatocyte damage markers bilirubin (P=0.0001), alanine aminotransferase (P=0.036), and alkaline phosphatase (P=0.0002) in Atg5 KO allo recipients. As before, there were similar numbers of splenic and liver-resident donor T cells, activation markers, and proinflammatory cytokines. To determine if autophagy is exclusively protective in GVHD target organs, we next generated Atg5fl/flMyh6-Cre mice, which lack Atg5 expression in cardiac myocytes, and Atg5fl/flAlb-Cre mice, which lack Atg5 expression in podocytes, as a control for lack of ATG5 in non-GVHD target organs. Using a similar GVHD model as above, we monitored these mice and littermate controls for survival and severity of GVHD symptoms. In contrast to our findings in the liver and gut, we observed similar survival (P=0.17, Myh6; P=0.49, Nphs2) and GVHD severity between allo recipients. Collectively, our results demonstrated that autophagy is a tissue-intrinsic protective response that regulates disease severity in GVHD target organs. Disclosures No relevant conflicts of interest to declare.

Transplanted Vasoactive Intestinal Peptide-Secreting Donor Plasmacytoid Dendritic Cells Migrate to Lymphoid Organs and GvHD Target Organs

Introduction: Donor plasmacytoid dendritic cells (pDCs) have important roles in the pathogenesis of graft versus host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Vasoactive intestinal peptide (VIP), a potent anti-inflammatory neuropeptide with pleiotropic effects, has shown promise in managing GvHD through induction of regulatory dendritic cells (Chorny, Blood 2006) and regulatory T cells (Delgado, JLB 2005). Our previous data showed that transplantation of hematopoietic stem cells (HSC) lacking VIP expression in enhanced Th1 polarization and antiviral immunity (Li, JI 2011). Whether VIP produced by pDCs plays a key role in preventing GvHD is the focus of the current study. Method: A reductive allo-HSCT model was established by transplanting purified populations of 5 x 103 hematopoietic stem cells (HSC), 5 x 104 pDCs and 106 T cells from B6 (H-2Kb) to MHC mismatched B10.BR (H-2Kk) recipients. Mice received either VIP knockout pDCs (experimental group) or wild-type pDCs (control). Survival and clinical GvHD scores were monitored for at least 60 days. Serum pro-inflammatory cytokines were measured by luminex on day 3 and day 8. Additional recipients were transplanted for interim analyses of GvHD pathology in histological sections of small intestine and colon on day 8 and day 15. To investigate pDC migration and proliferation status, 5 x 104 luciferase-expressing pDCs, together with 5 x 103 WT B6 HSC, were transplanted to MHC mismatched Balb/c (H2kd) recipients. Bioluminescent imaging was performed on day 14. We used mice carrying an eGFP reporter linked to a VIP promoter to measure VIP production by donor cells in vivo. The construct has the eGFP reporter gene, followed by a polyadenylation sequence, inserted into BAC clone RP23-25A8 at the initiating ATG codon of the first coding exon of the VIP gene, so that eGFP expression is driven by the regulatory sequences of the BAC gene. The BAC is inserted randomly in the genome. We used serial breeding to establish the eGFP-VIP reporter in B6 mice. Bone marrow pDCs were sorted from transgenic eGFP-VIP B6 mice and used in transplant experiments, allowing VIP gene activity from donor-derived pDCs to be detected as GFP expression using confocal microscopy. Results: Compared to recipients of WT pDCs, recipients of VIP-KO pDCs have a significantly lower survival rate with increased GvHD clinical score (Figure 1; *p&lt;0.05). Transplant recipients of VIP-KO donor pDC had higher serum pro-inflammatory cytokine levels on day 3 and day 8 respectively. (Figure 2). At 2 weeks post-transplant donor pDCs migrated primarily to the spleen, with limited presence of donor pDCs in the gastrointestinal tract and lung (Figure 3). Compared to mice transplanted with WT pDCs, recipients of VIP-KO pDCs had increased donor T cell migration to the lung and paw, which are chronic GvHD target organs after two weeks. (Figure 4; *p&lt;0.05). When stimulated with IFN-g, eGFP-VIP transgenic pDCs have activation of the VIP gene promoter in vitro, as detected using eGFP (Figure 5). Confocal microscopy data showed that pDCs have activated the VIP promoter and secreted more VIP following stimulation by IFN-g, consistent with VIP up-regulation by the inflammatory mileu in allo-transplant recipients. Conclusions: These data show that lack of VIP secretion by donor pDCs resulted in lower survival rates and higher GvHD scores in allo-BMT recipients. Recipients of VIP KO donor pDC had increased donor T cell proliferation in GvHD target organs and increased GvHD clinical signs. The major site of donor pDCs migration in recipients is spleen, suggesting that pDCs may interact with other lymphoid cells, such as T cells, inducing a more tolerogenic environment. Donor pDCs were also found, to a lesser extent, in the lung and gastrointestinal tract, which are both GvHD target organs. Thus, pDCs may protect recipients from GvHD by limiting the activity of alloreactive T cells at these sites. The mechanism by which VIP-secreting donor pDCs regulate GvHD will be discussed. Disclosures Waller: Amgen: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Other: Travel expenses, Research Funding; Cerus Corporation: Other: Stock, Patents &amp; Royalties; Chimerix: Other: Stock; Cambium Oncology: Patents &amp; Royalties: Patents, royalties or other intellectual property ; Kalytera: Consultancy.

Human CD83 Targeted Chimeric Antigen Receptor T Cell for the Prevention of Graft Versus Host Disease and Treatment of Myeloid Leukemia

Distinct from pharmacologic immunosuppression, we designed a programmed cytolytic effector T cell that prevents graft versus host disease (GVHD). CD83 is expressed on allo-activated conventional T cells (Tconv) and pro-inflammatory dendritic cells (DCs), which are implicated in GVHD pathogenesis. Therefore we developed a novel human CD83 targeted chimeric antigen receptor (CAR) T cell for GVHD prophylaxis. Here we demonstrate that human CD83 CAR T cells eradicate cell mediators of GVHD, significantly increase the ratio of regulatory T cells (Treg) to allo-activated Tconv, and provide lasting protection from xenogeneic GVHD. Further, we show human, acute myeloid leukemia (AML) expresses CD83 and can be targeted by CD83 CAR T cells. A 2nd generation CD83 CAR was generated with CD3ζ and 41BB costimulatory domain that was retrovirally transduced in human T cells to generate CD83 CAR T cells. The CD83 CAR construct exhibited a high degree of transduction efficiency of about 60%. The CD83 CAR T cells demonstrated robust IFN-γ and IL-2 production, killing, and proliferation when cultured with CD83+ target cells. To test whether human CD83 CAR T cells reduce alloreactivity in vitro, we investigated their suppressive function in allogeneic mixed leukocyte reactions (alloMLR). CD83 CAR T cells were added to 5-day alloMLRs consisting of autologous T cells and allogeneic monocyte-derived DCs at ratios ranging from 3:1 to 1:10. The CD83 CAR T cells potently reduced alloreactive T cell proliferation compared to mock transduced and CD19 CAR T cells. We identified that CD83 is differentially expressed on alloreactive Tconv, compared to Tregs. Moreover, the CD83 CAR T cell efficiently depletes CD83+ Tconv and proinflammatory DCs with 48 hours of engagement. To test the efficacy of human CD83 CAR T cells in vivo, we used an established xenogeneic GVHD model, where mice were inoculated with human PBMCs (25x106) and autologous CD83 CAR (1-10x106) or mock transduced T cells. The CD83 CAR T cells were well tolerated by the mice, and significantly improved survival compared to mock transduced T cells (Figure 1A). Mice treated with CD83 CAR T cells exhibited negligible GVHD target organ damage at day +21 (Figure 1B). Mice inoculated with CD83 CAR T cells demonstrated significantly fewer CD1c+, CD83+ DCs (1.7x106 v 6.2x105, P=0.002), CD4+, CD83+ T cells (4.8x103 v 5.8x102, P=0.005), and pathogenic Th1 cells (3.1x105 v 1.1x102, P=0.005) at day +21, compared to mice treated with mock transduced T cells. Moreover, the ratio of Treg to alloreactive Tconv (CD25+ non-Treg) was significantly increased among mice treated with CD83 CAR T cells (78 v 346, P=0.02), compared to mice injected with mock transduced T cells. Further, CD83 appears to be a promising candidate to target myeloid malignancies. We observed CD83 expression on malignant myeloid K562, Thp-1, U937, and MOLM-13 cells. Moreover, the CD83 CAR T cells effectively killed AML cell lines. Many AML antigens are expressed on progenitor stem cells. Thus, we evaluated for stem cell killing in human colony forming unit (CFU) assays, which demonstrated negligible on-target, off-tumor toxicity. Therefore, the human CD83 CAR T cell is an innovative cell-based approach to prevent GVHD, while providing direct anti-tumor activity against myeloid malignancies. Figure Disclosures Blazar: Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; Abbvie Inc: Research Funding; Leukemia and Lymphoma Society: Research Funding; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-Founder; Alpine Immune Sciences, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Fate Therapeutics, Inc.: Research Funding; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Davila:Atara: Research Funding; Celgene: Research Funding; Precision Biosciences: Consultancy; Bellicum: Consultancy; GlaxoSmithKline: Consultancy; Adaptive: Consultancy; Anixa: Consultancy; Novartis: Research Funding.