Clinical Graft-versus-host Disease Scores Research Articles

Minnelide suppresses GVHD and enhances survival while maintaining GVT responses.

Allogeneic hematopoietic stem cell transplantation (aHSCT) can cure patients with otherwise fatal leukemias and lymphomas. However, the benefits of aHSCT are limited by graft-versus-host disease (GVHD). Minnelide, a water-soluble analog of triptolide, has demonstrated potent antiinflammatory and antitumor activity in several preclinical models and has proven both safe and efficacious in clinical trials for advanced gastrointestinal malignancies. Here, we tested the effectiveness of Minnelide in preventing acute GVHD as compared with posttransplant cyclophosphamide (PTCy). Strikingly, we found Minnelide improved survival, weight loss, and clinical scores in an MHC-mismatched model of aHSCT. These benefits were also apparent in minor MHC-matched aHSCT and xenogeneic HSCT models. Minnelide was comparable to PTCy in terms of survival, GVHD clinical score, and colonic length. Notably, in addition to decreased donor T cell infiltration early after aHSCT, several regulatory cell populations, including Tregs, ILC2s, and myeloid-derived stem cells in the colon were increased, which together may account for Minnelide's GVHD suppression after aHSCT. Importantly, Minnelide's GVHD prevention was accompanied by preservation of graft-versus-tumor activity. As Minnelide possesses anti-acute myeloid leukemia (anti-AML) activity and is being applied in clinical trials, together with the present findings, we conclude that this compound might provide a new approach for patients with AML undergoing aHSCT.

CRISPR/Cas9 Gene Editing of MIR155HG in Primary Human T Cells to Prevent Acute Graft-Versus-Host Disease

Abstract Introduction: Acute graft-versus-host disease (GVHD) mediated by donor alloreactive T cells is the leading cause of non-relapse mortality in patients post-allogeneic hematopoietic cell transplantation. Here we genetically engineered donor T cells by genomic deletion of microRNA-155 host gene (MIR155HG) to prevent GVHD. Methods: Using CRISPR/Cas9 genome editing, we targeted exon 3 (Ex3) of MIR155HG. Genomic deletion was evaluated by genomic PCR and RNA expression of mature miR-155 by qRT-PCR. In xenogeneic acute GVHD experiments, NSG mice were irradiated and transplanted with miR-155 Ex3 edited or control T cells to assess functionality in vivo. Histological analysis of target tissues (skin and liver) was performed at end of study. Weekly flow cytometric evaluation of human donor T cells in peripheral blood was performed. MOLM-13 leukemic target cells were cultured with miR-155 Ex3 edited or control CD8 +effector T cells for 72 hours and cytotoxic function was assessed by CD107a degranulation, intracellular IFN-γ production and leukemic cell death by flow cytometry. Results: Ex3 deletion was confirmed by genomic PCR resulting in downregulation of mature miR-155 expression. Recipients of Ex3 deleted human donor T cells display lower GVHD clinical scores, lower average histopathological scores and significantly improved survival compared to mice receiving control T cells. Ex3 T cells displayed potent CTL function comparable to control T cells resulting in leukemic cell death. Conclusion: CRISPR/Cas9 deletion of miR-155 in donor T cells protects against development of lethal GVHD while maintaining beneficial anti-leukemic effect. Our studies present genomic editing of donor T cells as a novel strategy to prevent GVHD. Funding for this project was provided through The Ohio State University Leukemia Research Program to PR and RG, NIH R01CA252469 to PR, and NIH T32CA090223 fellowship to KB.

A Novel Human OX40L-CAR-Treg Specifically Targets Activated Antigen-Presenting Cells and Effectively Controls T Cell Alloreactivity and Xeno-GvHD

Background: Graft-versus-host disease (GvHD) remains the leading cause of non-relapse mortality after allogeneic hematopoietic stem cell transplantation (HCT). Adoptive transfer of regulatory T cells (Tregs) has demonstrated promising results in controlling GvHD in both animal models and phase I/II clinical trials. However, strategies to increase the stability of ex-vivo expanded Tregs, and to optimize their potency, are needed. Recent efforts to accomplish these goals have focused on engineering Chimeric Antigen Receptor (CAR) Tregs, with the first attempts using CARs targeted against disparate HLA antigens. However, Tregs targeting HLA have several limitations, including the downregulation of these antigens that accompanies some diseases, as well as the lack of utility of this approach in the HLA-matched setting. In this study, we tested a novel GvHD prophylaxis approach consisting of infusing donor Tregs engineered to express an anti-OX40L CAR that, upon engagement with activated OX40L+ host antigen-presenting cells (APCs), increases their suppressor potency to dampen APC-mediated activation of alloreactive T cells. Methods and Results: We designed a CAR construct containing an scFv targeting OX40L, a CD28 costimulatory domain, along with CD3ζ, as well as the fluorescent reporter NeonGreen, all under the control of a synthetic FOXP3 promoter. This design enabled the selective and sustained expression of the anti-OX40L CAR on CD25+CD127- FoxP3+ Treg cells. We also created Tregs transduced with a non-targeting NeonGreen-only control construct (Neon-Tregs). Engineered OX40L CAR-Tregs maintained expression of the canonical Treg markers FOXP3, CD25, and CTLA-4, and demonstrated robust activation in the presence of OX40L-expressing cells. In co-culture assays of OX40L CAR-Tregs with OX40L-expressing K562 target cells, we demonstrate upregulation of Treg suppressive proteins CTLA-4, LAP, CD71, GARP and LAG3 without inducing expression of pro-inflammatory cytokines (no upregulation of IFNγ, TNFα, IL17a) (Figure 1A). Activation of Tregs via CAR recognition of OX40L+ cells resulted in 2.5-fold enhancement in their potency to suppress anti-CD3/CD28-driven T cell proliferation in vitro compared to Neon-Tregs at a 1:4 Treg-to-Teff ratio (p<0.05). Furthermore, OX40L-CAR-Tregs demonstrated a greater capacity to inhibit monocyte-derived dendritic cell activation, highlighting the superior suppressive activity of these OX40L-CAR-Tregs compared to non-targeting Neon-Tregs. Importantly, in a human xenograft model of GvHD, induced by transplantation of human PBMC into sublethally irradiated NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice, OX40L CAR-Treg demonstrated superior GvHD disease control compared to both PBMC alone and Neon-Tregs. Clinical scores were assessed by a combinatorial system including five parameters: weight loss, posture, mobility, skin, and fur conditions, and curves were compared using a 2-way ANOVA mixed-effects analysis, which demonstrated improvement in clinical xeno-GvHD with the OX40L CAR-Treg > Neon-Treg > PBMC alone over the entire length of analysis (p <0.001). An example of the substantial improvement in the clinical score on Day +14 is as follows: combined GvHD clinical score = 6.5 for PBMC vs 2.5 for Neon-Treg vs 0.15 for OX40L CAR-Treg, p <0.0001 for all comparisons. This control of clinical GvHD resulted in prolonged recipient survival when compared to both PBMC alone and to control Neon-Tregs (MST = 34 days for OX40L-CAR-Treg, 20 days for Neon-Treg, 14 days for PBMC alone, p<0.001, Figure 1B). Conclusions: Collectively, these results demonstrate a novel and efficacious approach using OX40L-CAR Tregs to enhance Treg suppressive function and to control GvHD. Because OX40L is upregulated on APCs in inflammatory environments, and its expression is limited to APCs and activated endothelial cells, this approach provides a unique strategy for the control of allo-immunity after HCT. Moreover, these OX40L-CAR Treg may be broadly applicable beyond HCT, to control allo-immunity after solid organ transplant and to suppress T cell activation in autoimmune diseases. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P1312: FORODESINE AMPLIFIES HOST INNATE IMMUNE RESPONSE THROUGH TOLL-LIKE RECEPTOR 7 ACTIVATION WHILE PREVENTING EXPERIMENTAL GRAFT-VERSUS-HOST DISEASE

Background: Forodesine, a novel inhibitor of purine nucleoside phosphorylase (PNP), causes nucleotide pool imbalances by accumulating intracellular deoxyguanosine, thereby resulting in selective apoptosis of T-cells. Recent evidence indicates that specific types of nuclear acids including guanosine and its derivatives act as natural ligands for toll-like receptor 7 (TLR7). So far, it is unclear whether and how forodesine can modulate host innate immunity, including antigen-presenting cells (APCs) such as macrophages and dendritic cells (DCs), which express TLR7 abundantly. Aims: The aim of this study is to investigate the role of forodesine in host APC function as well as donor T-cell activation both in vivo graft-versus-host disease (GVHD) models and in vitro cell assays. Methods: NOG mice were subjected to 250 cGy total body irradiation followed by intravenous injection of human isolated T-cells. Mice were treated with forodesine (20 mg/kg) or vehicle daily after transplantation. Results: Despite the mice without treatment developing severe GVHD after transplantation, guanosine and deoxyguanosine were hardly detected in the plasma, suggesting that purine nucleosides released from damaged tissues were rapidly degraded by PNP. In fact, inhibition of PNP activity by forodesine markedly increased plasma guanosine levels, whereas deoxyguanosine was still undetectable. This was also the case in GVHD target organs, including liver and lungs. At the same level of guanosine in the liver, forodesine promoted TNF-a production from in vitro alveolar macrophage cultures. Preincubation with a TLR7 antagonist, ODN20958, attenuated the TNF-a production from macrophages stimulated by guanosine and forodesine. Nuclease treatment did not reduce macrophage TNF-a production, suggesting that single-strand RNA, a well-known TLR7 ligand, is not involved in this response. Similar results were observed in Flt3-ligand-induced plasmacytoid DCs. Not only TNF-a but also IL-12 production was induced by the combination of guanosine and forodesine, and was comparable to that in pDCs pulsed with R848, a potent agonist of TLR7/8. These observations suggest that a sustained high level of guanosine caused by forodesine treatment lead to continuous activation of TLR7 signaling, resulting in cytokine production from APCs. Meanwhile, as reported previously, forodesine inhibited the proliferation of human T-cells activated with CD3 and CD28 antibodies in the presence of deoxyguanosine. Of note, however, this combination did not impair but rather stimulated IFN-g production from activated T-cells cocultured with pDCs. This suggests that T-cell priming capacity mediated by the interaction with pDCs is conserved through TLR7 activation. Finally, we investigated the in vivo effects of forodesine on survival, body weight change, and disease severity of GVHD mice. Forodesine treatment significantly prolonged survival of the mice compared with control mice, with a median survival of 17.5 days vs 14 days, respectively (p = 0.029). The mean body weight loss percentage at day 9 was 11.5% vs 19.0% in the forodesine group and control, respectively (p = 0.003). Furthermore, the clinical GVHD scores were also lower in the forodesine group vs control (4.9 vs 0.6 points, p = 0.001). Image:Summary/Conclusion: This study demonstrates that forodesine amplifies host innate immune response through TLR7 signaling, while ameliorating T-cell GVH reaction. Further studies are needed to investigate whether forodesine can provide host protection against infections in a transplant setting.

Post Transplantation Bilirubin Nanoparticles Ameliorate Murine Graft Versus Host Disease via a Reduction of Systemic and Local Inflammation.

Allogeneic stem cell transplantation is a curative immunotherapy where patients receive myeloablative chemotherapy and/or radiotherapy, followed by donor stem cell transplantation. Graft versus host disease (GVHD) is a major complication caused by dysregulated donor immune system, thus a novel strategy to modulate donor immunity is needed to mitigate GVHD. Tissue damage by conditioning regimen is thought to initiate the inflammatory milieu that recruits various donor immune cells for cross-priming of donor T cells against alloantigen and eventually promote strong Th1 cytokine storm escalating further tissue damage. Bilirubin nanoparticles (BRNP) are water-soluble conjugated of bilirubin and polyethylene glycol (PEG) with potent anti-inflammatory properties through its ability to scavenge reactive oxygen species generated at the site of inflammation. Here, we evaluated whether BRNP treatment post-transplantation can reduce initial inflammation and subsequently prevent GVHD in a major histocompatibility (MHC) mismatched murine GVHD model. After myeloablative irradiation, BALB/c mice received bone marrow and splenocytes isolated from C57BL/6 mice, with or without BRNP (10 mg/kg) daily on days 0 through 4 post-transplantation, and clinical GVHD and survival was monitored for 90 days. First, BRNP treatment significantly improved clinical GVHD score compared to untreated mice (3.4 vs 0.3, p=0.0003), and this translated into better overall survival (HR 0.0638, p=0.0003). Further, BRNPs showed a preferential accumulation in GVHD target organs leading to a reduced systemic and local inflammation evidenced by lower pathologic GVHD severity as well as circulating inflammatory cytokines such as IFN-γ. Lastly, BRNP treatment post-transplantation facilitated the reconstitution of CD4+ iNK T cells and reduced expansion of proinflammatory CD8α+ iNK T cells and neutrophils especially in GVHD organs. Lastly, BRNP treatment decreased ICOS+ or CTLA-4+ T cells but not PD-1+ T cells suggesting a decreased level of T cell activation but maintaining T cell tolerance. In conclusion, we demonstrated that BRNP treatment post-transplantation ameliorates murine GVHD via diminishing the initial tissue damage and subsequent inflammatory responses from immune subsets.

Graft-versus-host disease in the female genital tract: a prospective cohort study

Graft-versus-host disease (GVHD) is the main complication of allogeneic hematopoietic stem cell transplantation (HSCT). GVHD in the female genital tract can cause sinusorrhagia, dyspareunia, synechia, and even complete vagina occlusion. This prospective study aimed to evaluate the clinical characteristics and effects of preventive and prompt treatment for genital GVHD in females undergoing HSCT (n = 40). Genital GVHD was diagnosed in 11 of 40 patients (27.5%), and the most common complaint was vaginal dryness (54.6%). The majority of patients (63.6%) presented mild genital GVHD (clinical score 1), with interlabial fissures and lichen-like lesions, while a minority of patients (9.1%) presented advanced genital GVHD (clinical score 3) with the fusion of the small and large lips. The median time of onset of genital GVHD signs was 10months after HSCT, concomitant with GVHD in the skin and oral cavity. Personalized and topical therapy was effective in most cases (81.8%), and no patient required surgical intervention. We confirmed that female genital GVHD affects approximately one-third of females undergoing HSCT, highlighting the importance of periodic gynecological monitoring for early detection and treatment to improve care for these females.

TNFAIP8 (TIPE) Deficiency Exacerbates Acute Graft Versus-Host Disease in Murine Model

Background: Acute graft-versus-host disease (GVHD) occurs after allogeneic hematopoietic cell transplant (alloHCT) and presents a donor immune cell response against host tissues associated with significant morbidity and mortality. Up to 60% of alloHCT recipients will develop acute GVHD. Damage to the gastrointestinal tract from the conditioning is a key step in its pathogenesis by allowing for systemic translocation of lipopolysaccharide (LPS) and other danger signals, hereby promoting host APC activation. Tumor necrosis factor (TNF)–induced protein 8 (TNFAIP8) family is a newly identified group of proteins consisting of TNFAIP8 (TIPE), TIPE1 (TNFAIP8L1, TNF–induced protein 8-like 1), TIPE2 (TNFAIP8L2), and TIPE3 (TNFAIP8L3). TIPE acts as a negative mediator of apoptosis via inhibition of caspase-3 activation and also induces proliferation. TIPE signaling is involved in activation of NF-kappa-B and AP-1 pathways. TIPE-deficient mice have been shown to develop more severe colitis than wild type mice due to TIPE deficiency in non-hematopoietic cells along with higher levels of TNF and IL-6. Given the potential roles of TIPE in regulating these processes and considering the importance of these cytokines and of epithelial injury in the pathophysiology of GVHD, we hypothesized, that TIPE deficiency of allogeneic HCT recipients results in worsened GVHD.Methods: 9 - 12 week old naive C57BL/6 or TIPE knockout C57BL/6 mice were conditioned with 1000cGy single dose TBI, followed by transplantation of 10 million bone marrow cells and 20 million splenocytes from either syngeneic C57BL/6 or allogeneic BALB/c donors. Animals were monitored for clinical GVHD and survival and analyzed at week 6 after HCT for serum cytokines, T cell expansion, whole spleen cell chimerism and cell proliferation marker.Results: Allogeneic wild type recipients demonstrated significantly better survival when compared to allogeneic TIPE knockout recipients by week 6 (66.7% vs 40.0%, p =0.024). All syngeneic animals survived. Clinical GVHD scores were significantly higher in TIPE-deficient allogeneic recipients at Day +28 (p=0.0166), +35 (p=0.0349), +42 (p=0.0241) compared to allogeneic wild type recipients. Day +42 serum cytokine analysis revealed increased IL-17A (p=0.0027), TNF (p=0.0396), IL-6 (p=0.0064) levels in TIPE-deficient allogeneic recipients compared to allogeneic control. This was associated with increased splenic CD4+ (p=0.04) but not CD8+ (p= 0.0898) cell expansion when compared to allogeneic control. TIPE-deficient and allogeneic control mice showed no differences in splenic donor T cell chimerism (84.4 versus 89.4, p=0.178), indicating comparable donor T cell engraftment. Ki-67 expression was significantly decreased in epithelial cells of small intestine of TIPE knockout allogeneic recipients when compared to allogeneic controls, and a similar nonsignificant trend was seen in the colon as well. Active caspase-3 expression was significantly increased in the small intestine of TIPE knockout allogeneic recipients when compared to allogeneic controls and a similar nonsignificant trend was seen in the colon as well. This observation was associated with significantly increased GI tract pathology scores of TIPE-deficient allogeneic recipients compared to allogeneic control. No differences were observed in pathology of lung and liver between TIPE-deficient allogeneic recipients and allogeneic control. Histopathologic analysis of T cell infiltration in small intestine and colon by immunohistochemistry for CD3 antibody revealed no difference between allogeneic groups, suggesting a role of TIPE-deficient nonhematopoietic cells in GVHD pathophysiology.Conclusion TIPE deficiency in allogeneic C57BL/6 recipients resulted in increased mortality after transplant, which was associated with significant increase in pro-inflammatory cytokines expression and decreased proliferation and increased apoptosis of intestinal epithelial cells, suggesting not only exaggerated inflammatory damage but also decreased epithelial regeneration. Therefore, TIPE seems to be a TNF receptor family member with rather protective function in GVHD gut pathophysiology and overexpression may be a potential future target to prevent or treat this complication after allo HCT. 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.

Preclinical Studies of MSC-Derived Extracellular Vesicles to Treat or Prevent Graft Versus Host Disease: a Systematic Review of the Literature.

IntroductionTreating and preventing graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplant (HCT) remains a significant challenge. The use of mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) appears promising and a systematic review of preclinical studies is needed to accelerate the design of translational studies.MethodsWe identified 4 eligible studies from a systematic review performed on December 1, 2018. In brief, eligible studies included the treatment or prevention of GVHD in animal models and the use of MSC-EVs. Study design and outcome data were extracted and reporting was evaluated using the SYRCLE tool to identify potential bias.ResultsTwo studies assessed the efficacy of MSC-EVs in treatment of GVHD and 2 studies address prevention. Mice treated with MSC-EVs showed improved median survival, GVHD clinical scores and histology scores as compared to untreated mice with GVHD. Prophylactic treatment with MSC-EVs attenuated GVHD severity and improved median survival as compared to no treatment or saline.ConclusionOur systematic review provides important insight regarding the potential of MSC-EVs to treat or prevent GVHD. Although few studies were identified, improved survival and attenuated histologic findings of GVHD were observed in mice after MSC-EV administration for the treatment and prevention of GVHD. Dosing of EVs and route of administration remain inconsistent, however, and scalability of EV isolation for clinical studies remains a challenge. Standardized outcome reporting is needed to pool results for metanalysis. Graphical abstract

Genetic Manipulation Resulting in Decreased Donor Chondroitin sulfate Synthesis Mitigates Gvhd Following Allogeneic Hematopoietic Cell Transplantation in a Murine Model.

Introduction: Severe acute graft versus host disease (GVHD) represents a major risk associated with allogeneic hematopoietic cell transplantation (HSCT). Both acute and progressive GVHD following HSCT are in large part attributable to responses of donor T cells to host allo-antigens. Alloreactive T cell activation can be modulated by signals from the tissue microenvironment (TME). Glycosaminoglycans (GAGs) in the TME or modifying T cell surface proteoglycans are known to regulate T cell function. However, the roles of GAGs in acute GVHD following allogeneic HSCT remain to be elucidated, since a suitable murine model has not been available. We previously established a unique mouse model (T1KO), in which knockout of the chondroitin sulfate (CS) N-acetylgalactosaminyltransferase-1 (T1) gene - encoding the rate-limiting CS-synthesizing enzyme - decreases CS production. We also reported a role for CS in murine hematopoietic stem cells. In the present study, we focus on donor T cell CS levels to assess the role of T cell CS in acute GVHD following allogeneic HSCT. We were able to mitigate the clinical features of GVHD in a murine model using T1KO donors. Methods: Eight- to 12-week-old T1KO mice were generated from a C57BL/6N (WT) strain as donors (H-2b) for allogeneic bone marrow transplantation (BMT). Donor BM cells were then transplanted into eight- to 12-week-old BALB/c recipients (H-2d). Prior to transplantation, recipients were irradiated at a dose of 7 Gy. They then received 5 x 106 BM cells and splenocytes of identical genotype, as well as low (5 x 105), intermediate (1 x 106), or high (4 x 106) doses of CD90.2+ cells from either WT or T1KO donors. For five weeks following BMT, survival was monitored daily and clinical GVHD scores (incorporating body weight, activity, fur condition, alopecia, and tortoiseshell-like mucosae) were calculated three times per week. Results: The peripheral blood CD4+/CD8+ T cell ratio of T1KO mice was equivalent to that of WT mice. Following allogeneic BMT, histopathologic analysis confirmed onset of acute recipient GVHD on day 49 when donors were WT mice. As a result, significant lymphocyte infiltration was observed in target organs (liver, colon, and skin). High-dose transplantation of splenocytes from T1KO rather than WT mice significantly prolonged recipient median survival (27.0 versus 20.5 days, p = 0.02). A similar trend was observed for low- and intermediate-dose transplantation (low: 53.0 versus 29.0 days, p = 0.09; intermediate: 50.5 versus 30.5 days, p = 0.13. Similarly, a significant improvement in day 11 GVHD score was observed following high-dose splenocyte transplantation when donors were T1KO mice (mean scores: 1.00 versus 2.75, p &amp;lt; 0.01). However, no significant differences in GVHD score were observed following low- and intermediate-dose transplantation. Taken together, our results suggest that donor T cells producing lower CS levels alleviate acute GVHD following allogeneic HSCT in a murine model. Conclusion: Alloreactive T cell activation following allogeneic HSCT may be down-regulated by lower CS levels, thereby mitigating GVHD severity. Figure 1 Disclosures No relevant conflicts of interest to declare.

A single-nucleotide polymorphism in the human ENTPD1 gene encoding CD39 is associated with worsened graft-versus-host disease in a humanized mouse model.

Regulatory T cells (Tregs) protect against graft-versus-host disease (GVHD), a life-threatening complication of allogeneic hematopoietic stem cell transplantation. The ectoenzyme CD39 is important for increasing the immunosuppressive function of Tregs. The rs10748643 (A→G) single-nucleotide polymorphism (SNP) in intron 1 of the human ENTPD1 gene is associated with increased proportions of CD39+ Tregs. This study aimed to determine whether the rs10748643 SNP corresponded to increased proportions of CD39+ Tregs in an Australian donor population, and whether this SNP influences clinical GVHD in a humanized mouse model. Donors were genotyped for the rs10748643 SNP by Sanger sequencing, and the proportion of CD39+ T cells in donor peripheral blood was determined by flow cytometry. Donors encoding the G allele (donorsAG/GG ) demonstrated higher proportions of CD39+ CD3+ CD4+ CD25+ CD127lo Tregs, but not CD39+ CD3+ CD8+ T cells or CD39+ CD3+ CD4+ conventional T cells, compared with donors homozygous for the A allele (donorsAA ). NOD-SCID-IL2Rγnull mice were injected with human peripheral blood mononuclear cells from either donorsAA (hCD39AA mice) or donorsAG/GG (hCD39AG/GG mice). hCD39AG/GG mice demonstrated significantly greater weight loss and GVHD clinical scores, and significantly reduced survival, compared with hCD39AA mice. hCD39AG/GG mice showed significantly higher hCD4+ :hCD8+ T-cell ratios than hCD39AA mice, but displayed similar proportions of CD3+ hCD4+ hCD25+ hCD127lo Tregs and hCD39+ Tregs. However, the proportion of human Tregs corresponded to survival in hCD39AA mice, but not in hCD39AG/GG mice. This study demonstrates that donors encoding the G allele show higher percentages of CD39+ Tregs, but cause worsened GVHD in humanized mice compared with donors homozygous for the A allele.

TNFAIP8 Deficiency Exacerbates Acute Graft Versus Host Disease in a Murine Model of Allogeneic Hematopoietic Cell Transplantation.

Gastrointestinal acute graft-versus-host disease (GVHD) occurring after allogeneic hematopoietic cell transplant is an allo-reactive T cell and inflammatory cytokine driven organ injury with epithelial apoptosis as 1 of its hallmark findings and is associated with significant mortality. Tumor necrosis factor (TNF)-alpha-induced protein 8 (TNFAIP8 or TIPE) acts as a negative mediator of apoptosis via inhibition of caspase-3 activation, promotes cell proliferation and Tipe deficiency is associated with increased inflammation. To evaluate the role of TIPE in acute GVHD, naive C57BL/6 and Tipe C57BL/6 mice were conditioned with 1000 cGy single dose total body irradiation, followed by transplantation of 10 million bone marrow cells and 20 million splenocytes from either syngeneic C57BL/6 or allogeneic BALB/c donors. Allo TIPE-deficient mice developed exacerbated gut GVHD compared with allo controls and had significantly decreased survival (6 wk overall survival: 85% versus 37%; P < 0.05), higher clinical GVHD scores, more profound weight loss, increased serum proinflammatory cytokines (interleukin-17A, TNF, interleukin-6, and interferon-γ). T-cell infiltration into the ileum was increased; epithelial proliferation was decreased along with significantly higher levels of chemokines KC and monokine induced by gamma interferon. Using bone marrow chimeric experiments, TIPE was found to have a role in both hematopoietic and nonhematopoietic cells. Absence of TIPE results in excessive inflammation and tissue injury after allo-HCT, supporting that TIPE confers immune homeostasis and has tissue-protective function during the development of gut GVHD and may be a potential future target to prevent or treat this complication after allogeneic HCT.

Endothelial Protective Effects of Defibrotide Reduce Acute Graft Versus Host Disease after Experimental Allogeneic Hematopoietic Stem Cell Transplantation

Introduction Graft versus host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). The pathophysiology of acute GVHD involves donor T cell- and inflammatory cytokine-mediated injury to the patient's tissues and causes significant morbidity and mortality. Defibrotide is a mixture of single stranded nucleotides that has a number of pharmacological effects that contribute to its endothelial protective properties. We hypothesized that preventive treatment with defibrotide will reduce the incidence and severity of GVHD due to its endothelial protective and anti-inflammatory properties and potentially does not alter graft versus leukemia (GVL) responses. Methods Recipient B10.BR mice received Cyclophosphamide 120 mg/kg per day on day-3 and -2, and received lethal total body irradiation (750cGy) on day 0, followed by the infusion of donor C57BL/6 T-cell depleted bone marrow cells. To induce GVHD, respective groups were co-infused with splenocytes, while non-GVHD controls received BM cells only. Mice were treated with defibrotide at the dose of 800mg/kg per body weight or vehicle control for the first week daily and then three times/week thereafter. For GVL experiments, C57BL/6 recipients were lethally irradiated (12Gy) with TBI and transplanted with C3H.SW donor TCD bone marrow alone or in combination with 3 million T-cells along with C1498-luc and treated with defibrotide or vehicle controls. Results Allogeneic defibrotide treated recipients demonstrated significantly better survival when compared to control group (83.34% vs 54.55% on day +28). Clinical GVHD scores were significantly worse after day +14 in control group when compared to defibrotide treated recipients. Both at day +7 and on day +28 allogeneic defibrotide treated recipients showed significantly less organ GVHD in gut, lung and liver (p Conclusion Defibrotide treatment through its anti-inflammatory and endothelium-protective effects reduces the incidence and severity of acute GVHD, while not impairing GVL activity.

IL-2/IL-2R, TL1A/TNFRSF25 or Their Combined Stimulation Results in Distinct CD4+FoxP3+ Regulatory T Cell Phenotype and Suppressive Function

CD4+Foxp3+ regulatory T cells (Tregs) are non-redundant mediators of immunity and tolerance. Transfer of Tregs has emerged as a promising therapy for graft-versus-host disease (GVHD) following allogeneic HSCT (aHSCT). Our prior work demonstrated that a 2-pathway in vivo strategy targeting TNFRSF25 (with TL1A-Ig) and IL-2 receptor (with low-dose IL-2, IL-2LD) can elicit a strong increase in Treg numbers and function. In fact, very low numbers of these in vivo expanded donor Tregs suppressed GVHD post-aHSCT. Based on these findings and the success of IL-2 in pre-clinical and clinical studies, we asked: are there phenotypic/functional differences between Tregs expanded via IL-2 LD, TL1A-Ig or their combined application? Mice were administered IL-2LD, TL1A-Ig or TL1A-Ig+IL-2LD over 6 days. Splenic and lymph node (LN) Treg phenotype was determined by flow cytometry. Treg functionality was assessed with sorted populations using an in vivo MHC-mismatched aHSCT. Treatment of B6-FoxP3RFP mice with TL1A-Ig+IL-2LD vs. IL-2LD only resulted in significantly higher levels of Treg activation/differentiation and functional markers, i.e. KLRG1, CD103, Nrp1, ICOS (Fig. 1A). Ki67 expression was higher in 2-pathway vs. IL-2LD stimulation alone (Fig.1B). Notably, TL1A-Ig treatment alone induced the above phenotype. CD25 (IL-2Rα) expression was reduced after TL1A-Ig vs. IL-2-mediated expansion further emphasizing the difference between the TNFRSF25 and IL-2 receptor stimulation (Fig. 1A). Results were corroborated using a second independent mouse strain, BALB/c, following use of these protocols. To test if the observed phenotypic differences could be related to more potent Treg function in vivo, an MHC-mismatched aHSCT (B6→BALB/c) was performed using 200,000 sorted Tregs (>98% purity by CD4+FoxP3+ selection from C57BL/6-FoxP3RFP reporter mice) from donor unexpanded, IL-2LD, or TL1A-Ig+IL-2LD treated mice + T cells. As anticipated, transfer 200,000 TL1A-Ig+IL-2LD stimulated Tregs ameliorated acute GVHD (Fig. 1C). Interestingly, lower GVHD clinical scores were obtained using the same number of IL-2LD only vs.TL1A-Ig+IL-2LD stimulated Tregs (Fig. 1C). Moreover, early post-transplant, higher LN and splenic CD4/CD8 ratios were detected in aHSCT recipients treated with IL-2LD expanded Tregs vs. TL1A-Ig+IL-2LD (Fig. 1D). Overall, TL1A-Ig+IL-2LD promotes a more activated/differentiated and proliferative Treg phenotype accompanied by less effective long-term GVHD inhibition vs. IL-2LD only stimulated Tregs. We posit that these different 1- and/or 2-pathway driven Tregs may promote development of new strategies utilizing one or both populations dependent on the desire for GVHD prophylaxis or therapeutic treatment following aHSCT.

The Innate Immune Sensor Sting Promotes Donor CD8+ T Cell Activation and Recipient APC Death Early after Preclinical Allogeneic Hematopoietic Stem Cell Transplantation

Graft-versus-host disease (GVHD) remains a significant cause of morbidity and mortality in patients receiving allogeneic hematopoietic stem cell transplants (aHSCTs). Pre-HSCT chemoradiation results in the death of dividing cells and release of endogenous danger signals. These molecules drive the activation of antigen presenting cells (APCs) and the differentiation of allo-reactive donor T cells. We identified a role for Stimulator of Interferon Genes (STING), an innate immune sensor, in GVHD using pre-clinical MHC-matched unrelated donor (MUD) HSCT models. Here we show that STING rapidly promotes donor CD8⁺ T cell activation and recipient APC death early after aHSCT. Post MUD HSCT, we previously found a >2x reduction in IFNβ, TNFα and IL-6 mRNA in STING^−/− vs WT recipient colonic mRNA expression (48 hrs) as well as decreased weight loss, GVHD scores and skin pathology (6 wks) vs WT. Chimeric studies showed that STING loss in non-hematopoietic cells induced this effect. Conversely, we recently found a single early (<D14), but not late (>D100), dose of the STING agonist DMXAA increased GVHD scores and lethality in WT, but not STING^−/−, recipients. Thus, the activation of this pathway can promote GVHD following MUD HSCT. Furthermore, mice homozygous for a murine homologue of a human allele associated with diminished STING activity (STING^HAQ/HAQ) also exhibited reduced GVHD after MUD HSCT, suggesting potential clinical importance. Interestingly, our findings that STING deficiency ameliorates GVHD in MUD HSCT (Bader CS, et al, in revision <i>Sci. Transl. Med.</i>) contrasts reported observations that STING deficiency can exacerbate GVHD after MHC-mismatched (MMUD) HSCT. Since CD4⁺ and CD8⁺ T cells are central in MMUD and MUD GVHD, respectively, we transplanted MMUD BALB/c BM + CD8⁺ T cells into B6-WT and STING^−/− mice and notably, found that STING^−/- recipients now developed reduced GVHD clinical scores, skin pathology and frequencies of activated T cells 8 wks post-HSCT vs WT. We also found that STING^−/− mice had greater numbers of recipient splenic CD11b⁺CD11c⁺ APCs and these cells expressed reduced MHC I protein 1 day after MMUD B6 into BALB/c aHSCT vs WT (Fig. A, B). Moreover, STING^−/− recipient spleens contained lower numbers of donor CD8⁺ T cells producing IFNγ and TNFα (Fig. C), supporting the hypothesis that STING contributes to early activation of donor CD8⁺ T cells and loss of recipient APCs. Next, to identify if reduced host MHC II⁺ APCs affected donor CD4⁺ T cell activation, B6-Nur77^GFP T cells were used to explicitly monitor T cell receptor signaling. Indeed, STING^−/− spleens had greater numbers of donor Nur77^GFP CD4⁺ T cells expressing GFP, CD69 and IFNγ 6 days post-HSCT (Fig. D). Overall, these data provide a mechanism by which STING could promote CD8⁺ T cell-mediated GVHD yet diminish CD4⁺-mediated GVHD. Ongoing studies are exploring the use of STING agonists to augment GVL following aHSCT.

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.

The Innate Immune Sensor Sting Promotes Donor CD8+ T Cell Activation and Recipient APC Death Early after Preclinical Allogeneic Hematopoietic Stem Cell Transplantation

Graft-versus-host disease (GVHD) remains a significant cause of morbidity and mortality in patients receiving allogeneic hematopoietic stem cell transplants (aHSCTs). Pre-HSCT chemoradiation results in the death of dividing cells and release of endogenous danger signals. These molecules drive the activation of antigen presenting cells (APCs) and the differentiation of allo-reactive donor T cells. We identified a role for Stimulator of Interferon Genes (STING), an innate immune sensor, in GVHD using pre-clinical MHC-matched unrelated donor (MUD) HSCT models. Here we show that STING rapidly promotes donor CD8+ T cell activation and recipient APC death early after aHSCT. Post MUD HSCT, we previously found a >2x reduction in IFNβ, TNFα and IL-6 mRNA in STING−/− vs WT recipient colonic mRNA expression (48 hrs) as well as decreased weight loss, GVHD scores and skin pathology (6 wks) vs WT. Chimeric studies showed that STING loss in non-hematopoietic cells induced this effect. Conversely, we recently found a single early ( D100), dose of the STING agonist DMXAA increased GVHD scores and lethality in WT, but not STING−/−, recipients. Thus, the activation of this pathway can promote GVHD following MUD HSCT. Furthermore, mice homozygous for a murine homologue of a human allele associated with diminished STING activity (STINGHAQ/HAQ) also exhibited reduced GVHD after MUD HSCT, suggesting potential clinical importance. Interestingly, our findings that STING deficiency ameliorates GVHD in MUD HSCT (Bader CS, et al, in revision Sci. Transl. Med.) contrasts reported observations that STING deficiency can exacerbate GVHD after MHC-mismatched (MMUD) HSCT. Since CD4+ and CD8+ T cells are central in MMUD and MUD GVHD, respectively, we transplanted MMUD BALB/c BM + CD8+ T cells into B6-WT and STING−/− mice and notably, found that STING−/- recipients now developed reduced GVHD clinical scores, skin pathology and frequencies of activated T cells 8 wks post-HSCT vs WT. We also found that STING−/− mice had greater numbers of recipient splenic CD11b+CD11c+ APCs and these cells expressed reduced MHC I protein 1 day after MMUD B6 into BALB/c aHSCT vs WT (Fig. A, B). Moreover, STING−/− recipient spleens contained lower numbers of donor CD8+ T cells producing IFNγ and TNFα (Fig. C), supporting the hypothesis that STING contributes to early activation of donor CD8+ T cells and loss of recipient APCs. Next, to identify if reduced host MHC II+ APCs affected donor CD4+ T cell activation, B6-Nur77GFP T cells were used to explicitly monitor T cell receptor signaling. Indeed, STING−/− spleens had greater numbers of donor Nur77GFP CD4+ T cells expressing GFP, CD69 and IFNγ 6 days post-HSCT (Fig. D). Overall, these data provide a mechanism by which STING could promote CD8+ T cell-mediated GVHD yet diminish CD4+-mediated GVHD. Ongoing studies are exploring the use of STING agonists to augment GVL following aHSCT.

Multiple Pathways Targeting CD25 or TNFRSF25 Affect CD4+FoxP3+ Regulatory T Cell Phenotype and Suppressive Function

BACKGROUND: Regulatory T cells (Tregs) are non-redundant mediators of immunity and tolerance. Adoptive transfer of CD4+Foxp3+ Tregs has emerged as a promising therapy for graft-versus-host disease (GVHD) following allogeneic HSCT (aHSCT), solid organ transplantation and autoimmune diseases (Hanash AM, Blood 2005; Copsel S, Wolf D, Haematologica 2019; Marek-Trzonkowska N, Diabetes Care 2012; Tang Q, J Mol Cell Biol 2012). Our prior work was the first to demonstrate a two-pathway in vivo strategy targeting TNFRSF25 (with TL1A-Ig fusion protein) and CD25 (with low dose IL-2, IL-2LD) receptors can elicit a rapid and strong increase in Treg numbers and function (Wolf D, BBMT 2017). In fact, very low numbers of these in vivo expanded donor Tregs exhibited effective GVHD suppression in recipients following aHSCT (Copsel S, BBMT 2018). Based on these findings and the success of IL-2LD in pre-clinical and clinical studies, we asked: are there phenotypic/functional differences between Tregs expanded via IL-2LD, TL1A-Ig or their combined application (TL1A-Ig+IL-2LD)? METHODS: Mice were administered IL-2LD, TL1A-Ig or TL1A-Ig+IL-2LD over 6 days. Splenic and lymph node (LN) Treg phenotype was determined by flow cytometry. Treg functionality was assessed with sorted populations using an in vivo MHC-mismatched aHSCT. RESULTS: Treatment of C57BL/6-FoxP3RFP mice with TL1A-Ig+IL-2 LD versus IL-2LD only treatment resulted in significantly higher levels of activation/differentiation and functional markers on Tregs including KLRG1, CD103, Nrp1, ICOS (Fig. 1A). Ki67 expression was higher in two-pathway versus IL-2LD stimulation alone (Fig.1B). These data suggested a key role for TNFRSF25 stimulation. Notably, Treg stimulation with TL1A-Ig alone drove the above phenotype indicating a pathway difference between the TNFRSF25 and IL-2 receptors. This difference was further apparent as high affinity IL-2 receptor (CD25) expression was reduced after TL1A-Ig +/- IL-2LD -mediated expansion compared with IL-2 LD alone stimulated Tregs (Fig. 1A). Results were corroborated using a second independent mouse strain, BALB/c, following use of these protocols. To begin addressing if the observed phenotypic differences between CD25 vs. TNFRSF25 + CD25 expanded Tregs could be related to a more potent Treg in vivo suppressive activity, an initial MHC-mismatched aHSCT (donor/recipient = C57BL/6-BALB/c) was performed. We employed 200,000 sorted Tregs (&gt;98% purity by CD4+FoxP3+ selection from C57BL/6-FoxP3RFP reporter mice) from donor unexpanded, IL-2LD, or TL1A-Ig+IL-2LD treated mice combined with 1.0 x106 T cells. As anticipated, transfer 200,000 TL1A-Ig+IL-2LD stimulated Tregs ameliorated acute GVHD (Fig. 1C). Remarkably, lower GVHD clinical scores were obtained using the same number of IL-2LD only expanded Tregs compared with TL1A-Ig+IL-2LD stimulated Tregs (Fig. 1C). Moreover, early post-transplant, higher LN and splenic CD4/CD8 ratios were detected in aHSCT recipients treated with IL-2LD expanded Tregs vs. TL1A-Ig+IL-2LD (Fig. 1D). CONCLUSION: Our donor TL1A-Ig+IL-2LD Treg expansion protocol promotes a more activated/differentiated and proliferative phenotype versus IL-2LD stimulation alone. This finding may have accounted for their initial effectiveness - but less efficient long-term GVHD amelioration compared to IL-2LD only stimulated Tregs. Multiple variables are associated with the application of Tregs for therapy including numbers, persistence, and suppressive capacity. Our findings suggest a rationale that one-pathway and / or two-pathway stimulated Tregs may be beneficial for use in aHSCT recipients dependent on whether there is a perceived need for prolonged Treg presence and the stage of GVHD. Disclosures Levy: Heat Biologics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pelican Therapeutics: Consultancy, Research Funding.

The MEK Inhibitor Trametinib Enhances Diverse T Cell Reconstitution with Suppressing Xenogeneic Graft-Versus-Host-Disease

Introduction: Early immune reconstitution without severe graft-versus-host disease (GVHD) is required for the success of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We showed that MEK inhibitors suppress GVHD but retain antiviral immunity and graft-versus-tumor (GVT) effects (Shindo, Blood2013; Itamura, Shindo, JCI Insight2016). Furthermore, we have shown that they attenuate graft rejection but spare thymic function following rat lung transplantation (Takahagi, Shindo, Am J Respir Cell Mol Biol2019). Here we analyzed their effects on human polyclonal T cell reconstitution in xenogeneic transplant by evaluating T-cell receptor (TCR) repertoire diversity. Methods: As a xenogeneic GVHD model, human PBMCs were infused to NOD/Scid/JAK3null mice, immunodeficient mice lacking T/B/NK cells, after total body irradiation. Vehicle, tacrolimus, or the MEK inhibitor trametinib was administered from day 0 through 28 or day 15 through 28. Human TCR repertoire diversity was evaluated by an adapter ligation PCR method with next generation sequencing (Shindo, Oncoimmunol2018) in the liver, lung, and spleen. The assignment and frequencies of TCRαV/J clones were determined at the single-cell level. Their diversity and clonality were evaluated by Inv. Simpson's index 1/λ. Results: Trametinib prolonged their survival compared with vehicle (median survival: 88 vs 46 days, p&lt;0.05). It enhanced engraftment of human leukocytes in peripheral blood (human CD45+cells: 11.0 vs 2.5%), but prevented their infiltration into the lung (human CD45+cells on day 60: 1.5 vs 6.5%). Treatment with vehicle resulted in skewed TCR repertoire with limited clones in the spleen, liver and lung. Interestingly, expansion of one specific clone (TRAV20/J10) was commonly observed, which might reflect the GVHD-inducing pathological clone (Fig. 1: 3D graphs show the frequencies of TCRαV/J clones). However, trametinib enabled diverse and polyclonal T cell engraftment without the TRAV20/J10 clone. While CD4+and CD8+T cells within injected human PBMCs mainly consisted of naïve (CD45RA+CD27+) and central memory (CD45RA-CD27+) T cells, infiltrating T cells in each organ showed effector memory (CD45RA-CD27-) T cell phenotype. Of note, CD8+T cells in the bone marrow, spleen, and lung of trametinib-treated recipients showed reduced effector memory T cells (CD45RA-CD27-) compared with vehicle-treated mice at day 28 (bone marrow 21.7 vs 74.7%, p&lt;0.01; spleen 66.3 vs 88.7%, p&lt;0.05; lung 33.0 vs 72.5%, p&lt;0.05), which indicating that MEK inhibition suppresses functional differentiation of human T cells in vivo. Furthermore, trametinib treatment from day 14 to 28 still ameliorated clinical GVHD score, and maintained polyclonal T cell repertoire. Conclusions:GVHD can be characterized with skewed TCR repertoire diversity and expansion of pathological T cell clones in the target tissues. Trametinib suppresses GVHD but maintains polyclonal T cell reconstitution, even in established GVHD. These results explain the facts that MEK inhibitors separate GVHD from GVT effects/antimicrobial immunity. Furthermore, MEK inhibition enhances immune reconstitution after allo-HSCT, which would avoid post-transplant complications. Disclosures Shindo: Novartis: Research Funding. Kitaura:Repertoire Genesis Inc.: Employment. Okada:Bristol-Myers Squibb: Research Funding; Japan Agency for Medical Research and Development: Research Funding. Shin-I:BITS Co., Ltd: Equity Ownership. Suzuki:Repertoire Genesis Inc.: Equity Ownership. Takaori-Kondo:Celgene: Honoraria, Research Funding; Novartis: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Ono: Research Funding; Takeda: Research Funding; Kyowa Kirin: Research Funding; Chugai: Research Funding; Janssen: Honoraria; Pfizer: Honoraria. Kimura:Ohara Pharmaceutical Co.: Research Funding; Novartis: Honoraria, Research Funding.

Treatment with Apocynin Limits the Development of Acute Graft-versus-Host Disease in Mice.

Graft-versus-host disease (GVHD) is the most serious complication limiting the clinical utility of allogeneic hematopoietic stem cell transplantation (HSCT), in which lymphocytes of donors (graft) are activated in response to the host antigen. This disease is associated with increased inflammatory response through the release of inflammatory mediators such as cytokines, chemokines, and reactive oxygen species (ROS). In this study, we have evaluated the role of ROS in GVHD pathogenesis by treatment of recipient mice with apocynin (apo), an inhibitor of intracellular translocation of cytosolic components of NADPH oxidase complex. The pharmacological blockade of NADPH oxidase resulted in prolonged survival and reduced GVHD clinical score. This reduction in GVHD was associated with reduced levels of ROS and TBARS in target organs of GVHD in apocynin-treated mice at the onset of the mortality phase. These results correlated with reduced intestinal and liver injuries and decreased levels of proinflammatory cytokines and chemokines. Mechanistically, pharmacological blockade of the NADPH oxidase was associated with inhibition of recruitment and accumulation of leukocytes in the target organs. Additionally, the chimerism remained unaffected after treatment with apocynin. Our study demonstrates that ROS plays an important role in mediating GVHD, suggesting that strategies aimed at blocking ROS production may be useful as an adjuvant therapy in patients subjected to bone marrow transplantation.