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Chronic graft-versus-host disease (cGVHD) is characterized as autoimmune-like fibrosis and antibody production mediated by pathogenic T cells and B cells. MicroRNA-17-92 (miR-17-92) influences the survival, differentiation, and function of lymphocytes in cancer, infections, and autoimmunity. To determine whether miR-17-92 regulates T- and B-cell responses in cGVHD, we generated mice conditionally deficient for miR-17-92 in T cells, B cells, or both. Using murine models of allogeneic bone marrow transplantation, we demonstrate that expression of miR-17-92 in donor T and B cells is essential for the induction of both scleroderma and bronchiolitis obliterans in cGVHD. Mechanistically, miR-17-92 expressed in T cells not only enhances the differentiation of pathogenic T helper 1 (Th1) and Th17 cells, but also promotes the generation of follicular Th cells, germinal center (GC) B cells, and plasma cells. In B cells, miR-17-92 expression is required for autoantibody production and immunoglobulin G deposition in the skin. Furthermore, we evaluated a translational approach using antagomirs specific for either miR-17 or miR-19, key members in miR-17-92 cluster. In a lupus-like cGVHD model, systemic administration of anti–miR-17, but not anti–miR-19, alleviates clinical manifestations and proteinuria incidence in recipients through inhibiting donor lymphocyte expansion, B-cell activation, and GC responses. Blockade of miR-17 also ameliorates skin damage by reducing Th17 differentiation in a scleroderma-cGVHD model. Taken together, our work reveals that miR-17-92 is required for T-cell and B-cell differentiation and function, and thus for the development of cGVHD. Furthermore, pharmacological inhibition of miR-17 represents a potential therapeutic strategy for the prevention of cGVHD.

Conditioning-induced damage of the intestinal tract plays a critical role during the onset of acute graft-versus-host disease (GVHD). Therapeutic interference with these early events of GVHD is difficult, and currently used immunosuppressive drugs mainly target donor T cells. However, not donor T cells but neutrophils reach the sites of tissue injury first, and therefore could be a potential target for GVHD prevention. A detailed analysis of neutrophil fate during acute GVHD and the effect on T cells is difficult because of the short lifespan of this cell type. By using a novel photoconverter reporter system, we show that neutrophils that had been photoconverted in the ileum postconditioning later migrated to mesenteric lymph nodes (mLN). This neutrophil migration was dependent on the intestinal microflora. In the mLN, neutrophils colocalized with T cells and presented antigen on major histocompatibility complex (MHC)-II, thereby affecting T cell expansion. Pharmacological JAK1/JAK2 inhibition reduced neutrophil influx into the mLN and MHC-II expression, thereby interfering with an early event in acute GVHD pathogenesis. In agreement with this finding, neutrophil depletion reduced acute GVHD. We conclude that neutrophils are attracted to the ileum, where the intestinal barrier is disrupted, and then migrate to the mLN, where they participate in alloantigen presentation. JAK1/JAK2-inhibition can interfere with this process, which provides a potential therapeutic strategy to prevent early events of tissue damage-related innate immune cell activation and, ultimately, GVHD.

Natural killer (NK) cells mediate surveillance for malignancy. In some chemotherapy refractory myeloid leukemia patients, adoptive transfer of NK cells from haploidentical donors can induce remission. We have previously shown that remission induction is linked to NK cell persistence at day +14, but the factors influencing NK cell persistence are unknown. To address this question, patient samples from a phase I trial of National Cancer Institute (NCI) IL-15 in whom either did or did not show NK cell expansion were compared with healthy donor control subjects. Before lymphodepleting chemotherapy, high absolute CD3+ count was predictive of patients who failed to expand their haploidentical NK cell graft. Interestingly, both groups had elevated expression of inhibitory receptors and decreased cytokine production compared with control subjects, suggestive of T cell exhaustion among all patients before haploidentical NK cell infusion. At day +14, however, haploidentical NK cell expanders had persistence of recipient CD8+ T cells with the most exhausted inhibitory phenotype (either PD-1high or dual PD-1+Tim-3+) and elevated expression of T-bet and Eomes compared with NK cell nonexpanders and control subjects. This suggested that maintenance of an exhausted T cell state at day +14 permits haploidentical NK cell expansion and supports further efforts to selectively deplete recipient T cells or modulate their dysfunction.

Background & Aims Fibrosis, the primary cause of morbidity in chronic liver disease, is induced by pro-inflammatory cytokines, immune cell infiltrates, and tissue resident cells that drive excessive myofibroblast activation, collagen production, and tissue scarring. Rho-associated kinase 2 (ROCK2) regulates key pro-fibrotic pathways involved in both inflammatory reactions and altered extracellular matrix remodelling, implicating this pathway as a potential therapeutic target. Methods We used the thioacetamide-induced liver fibrosis model to examine the efficacy of administration of the selective ROCK2 inhibitor KD025 to prevent or treat liver fibrosis and its impact on immune composition and function. Results Prophylactic and therapeutic administration of KD025 effectively attenuated thioacetamide-induced liver fibrosis and promoted fibrotic regression. KD025 treatment inhibited liver macrophage tumour necrosis factor production and disrupted the macrophage niche within fibrotic septae. ROCK2 targeting in vitro directly regulated macrophage function through disruption of signal transducer and activator of transcription 3 (STAT3)/cofilin signalling pathways leading to the inhibition of pro-inflammatory cytokine production and macrophage migration. In vivo, KDO25 administration significantly reduced STAT3 phosphorylation and cofilin levels in the liver. Additionally, livers exhibited robust downregulation of immune cell infiltrates and diminished levels of retinoic acid receptor-related orphan receptor gamma (RORγt) and B-cell lymphoma 6 (Bcl6) transcription factors that correlated with a significant reduction in liver IL-17, splenic germinal centre numbers and serum IgG. Conclusions As IL-17 and IgG–Fc binding promote pathogenic macrophage differentiation, together our data demonstrate that ROCK2 inhibition prevents and reverses liver fibrosis through direct and indirect effects on macrophage function and highlight the therapeutic potential of ROCK2 inhibition in liver fibrosis. Lay summary By using a clinic-ready small-molecule inhibitor, we demonstrate that selective ROCK2 inhibition prevents and reverses hepatic fibrosis through its pleiotropic effects on pro-inflammatory immune cell function. We show that ROCK2 mediates increased IL-17 production, antibody production, and macrophage dysregulation, which together drive fibrogenesis in a model of chemical-induced liver fibrosis. Therefore, in this study, we not only highlight the therapeutic potential of ROCK2 targeting in chronic liver disease but also provide previously undocumented insights into our understanding of cellular and molecular pathways driving the liver fibrosis pathology., Graphical abstract, Highlights • ROCK2 inhibition with the small-molecule inhibitor KD025 prevents and reverses hepatoxin-induced liver fibrosis. • ROCK2 inhibition attenuates profibrogenic immune function. • KD025 exerts direct effects on liver macrophages resulting in decreased TNF secretion and impeded migration. • KD025 administration attenuates T cell IL-17 production and B-cell IgG production, which indirectly contributes to downregulation of profibrogenic macrophage function.

Corticosteroids (CS) represent first-line treatment for gastrointestinal graft vs host disease (GI GVHD), and CS failure is associated with severe morbidity and mortality. While the immune system is the intended target of CS treatment, the glucocorticoid receptor (GR) is widely expressed, and there is limited understanding of the direct effects of CS on intestinal epithelium following immune-mediated damage. We thus investigated how CS treatment could impact intestinal homeostasis and regeneration following experimental bone marrow transplantation (BMT). In healthy C57BL/6 (B6) mice, in vivo administration of clinically relevant CS doses reduced Ki67 + epithelial proliferation in the ileum (p We next investigated CS effects on epithelial cells during immune-mediated damage. Pre-treatment of mice with 2 mg/kg MP x 7 days in vivo prior to crypt harvest and organoid culture increased organoid sensitivity to T-cell-mediating killing ex vivo (p Despite potential harmful side effects, CS are frequently necessary for treatment of clinical GVHD. We hypothesized that CS-mediated epithelial suppression could be mitigated by concurrent administration of agents capable of inducing tissue regeneration. Interleukin-(IL)-22 has been shown to promote epithelial proliferation and recovery following GI damage. We thus investigated whether IL-22 treatment could counterbalance CS-induced impairment of epithelial recovery in GVHD. Indeed, addition of IL-22 to MP-treated organoids promoted organoid growth without inducing toxicity/organoid loss in both murine and human SI organoid cultures (p In summary, these findings indicate that CS treatment can suppress epithelial proliferation in the intestines and exacerbate GI damage if it fails to control the pathologic immune response. However, deleterious CS side effects can be counterbalanced by promotion of epithelial regeneration, providing rationale for combining immunosuppression with tissue-supporting therapeutics such as IL-22 to optimize intestinal recovery in GVHD. Figure 1 Figure 1. Disclosures Blazar: Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Rheos Medicines: Research Funding; Equilibre Pharmaceuticals Corp: Research Funding; Carisma Therapeutics, Inc: Research Funding; Tmunity Therapeutics: Other: Co-founder. Hanash: Evive Biotech: Ended employment in the past 24 months.

Galectin-3 (Gal-3) is a unique member of the galectin family of lectins. Gal-3 possesses immune-regulatory functions depending on the immune cell and the immunologic situation. There are no studies that specifically delineate the role of Gal-3 in the setting of acute GvHD but mounting research suggests that dysregulation of pathways involving the galectin family may contribute to the pathogenesis of other immune disorders. Gal-3 is expressed by many types of immune cells, including T-cells. It suppresses signaling downstream of the TCR, decreases effector T-cell cytokine production, but increases the development and differentiation of memory T cells, myeloid cells, and macrophages. We investigated the mechanisms and downstream events of Gal-3 signaling in donor T cells after Allo-HCT, using Gal-3 knockout (Gal-3 -/-) mice. We further studied the effect of Gal-3 in controlling aGvHD incidence and severity while preserving the Graft-versus Leukemia (GvL) effect by overexpressing Gal-3 in human T cells. We utilized both a major MHC-mismatch (C57B/6 (H-2 b) into BALB/c (H-2 k) model and a MHC-matched, multiple minor histocompatibility antigen (miHA) mismatched B6 (H-2 b) into C3H/SW (H-2 b) model. Lethally irradiated recipient BALB/c and C3H/SW WT animals were injected with T cell depleted bone marrow alone (3 ×10 6) or with splenic T cells derived from allogeneic WT or Gal-3 -/- B6 donors (0.7 × 10 6 T cells in B6 → BALB/c and 1.5 × 10 6 in B6 → C3H/SW). We found that donor T cells express Gal-3 after Allo-HCT and that Gal-3 expression in WT T cells plays an important role in controlling GvHD, as evidenced by less severe weight loss, decreased clinical GvHD scores, and longer survival when compared to mice receiving Gal-3 -/- donor T cells (Figure 1A). We studied the mechanisms by which Gal-3 signaling controls the severity of aGvHD. Using flow cytometry analysis, we determined that Gal-3 plays a critical role in T cell proliferation and exhaustion. Gal-3 -/- T cells have a cytotoxic T phenotype with increased IFN-ℽ and GM-CSF production in T cells from the spleen and liver tissues on days 7 and 14 after Allo-HCT when compared to WT T cells (Figure 1B). There was a significant increase in T cell proliferation in Gal-3 -/- CD4 +T cells with a significantly higher level of IFN- ℽ mediated activation induced cell death (AICD) when compared to WT T cells. Gal-3 expression in T cells significantly increased the expression of exhaustion markers evidenced by a higher percentage of Slamf6 + Tim-3 + in WT T cells when compared to Gal-3 -/- T cells (Figure 1B). Gal-3 induced T cell exhaustion by through overactivation of NFAT signaling (data not shown). We sought to determine whether overexpression of Gal-3 in human T cells could control GvHD without affecting GVL. Gal-3 was overexpressed in human T cells using retrovirus containing Gal-3, vector alone and control T cells: Gal-3 T cells (T RV-Gal-3), GFP T cells (T RV-GFP) and control T cells were injected in irradiated NSG-HLA-A2 mice. All human cells expressed HLA-A2. Gal-3 overexpression in T cells effectively controlled the severity and mortality of GvHD after Allo-HCT in this humanized murine model of GvHD, evidenced by decreased body weight loss and decreased GvHD clinical scores in recipients transplanted with Gal-3 T cells when compared to control or GFP T cells (Figure 1C). Gal-3 overexpression did not impair the GvL effect when T cells cultured with Raji and THP-1 cell lines in vitro (data not shown). Gal-3 overexpression in T cells increased the frequencies of exhausted CD4 + T cells, and central memory CD4 + T cells while decreasing the percentage of effector CD4 T cell and INF-ℽ + CD4 + T cells. Clinical GI colon biopsies from patients undergoing allo-HCT were evaluated for Gal-3 expression in T cells using the multi-color Vectra 3 Automated Quantitative Pathology Imaging System. T cells in the colon biopsies expressed Gal-3. There was a significant correlation between Gal-3 MFI in CD4+ T cells, and GI histopathology score when analyzing Gal-3 intensity on Gal-3-expressing T cells. The Gal-3 MFI in CD4+ T cells was significantly lower in biopsies with higher colon GI histopathology scores (III-IV) compared to with lower colon GI histopathology scores I-II. In conclusion, these data reveal how Gal-3 can influence donor T cell proliferation and function in preclinical aGvHD models and point to the feasibility of manipulation of Gal-3 signaling to ameliorate aGvHD in the clinical setting. Figure 1 Figure 1. Disclosures Blazar: Rheos Medicines: Research Funding; Carisma Therapeutics, Inc: Research Funding; Equilibre Pharmaceuticals Corp: Research Funding; Tmunity Therapeutics: Other: Co-founder; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees. McCarthy: Magenta Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bluebird: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Juno: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Regulatory T cells are a critical population for the maintenance of self-tolerance and the use of these cells for therapeutic purpose in transplantation has emerged based on their capacity to control immune activation, exhibiting positive therapeutic potential to ameliorate acute graft-versus-host disease (GvHD), one of the major complications after allogeneic bone marrow transplantation (allo-BMT). One of the remaining technical challenges in the clinical practice of Treg administration is the number of Treg required to effectively prevent GvHD. In this study, we engineered murine Treg cells to express an altered IL-2 receptor β subunit that selectively interacts with an orthogonal mutant IL-2 (oIL2) without interacting with the natural cytokine or receptor counterparts, thus preventing acute -GvHD. For investigation of the suppressive function of Treg orthogonal IL-2/IL-2 receptor system in a murine major mismatch of acute GvHD model, the BALB/c mice were lethally irradiated (8.8 Gy) and transplanted with 5x10 6 T cell-depleted bone marrow cells (TCD-BM) from C57Bl/6 mice alone or together with fresh 1x10 6 C57Bl/6 FoxP3 DRT-GFP-luc+ Treg cells (fTreg) or with 0.2x10 6 and 1x10 6 oTreg (transduced with the construct for murine oIL-2 receptor β chain) on day 0. On day 2, C57Bl/6 Tcon cells (1x10 6) were injected to induce GvHD. Recipients were treated with PBS or oIL-2 (25K IU/daily) for 14 days and then 3 times a week for another 14 days. Using bioluminescence imaging, we observed that ortho IL2Rβ +Treg not only maintains migration ability to the gut, mLN and LN but also exhibited significant expansion ability treated with oIL2 at day 7 post-transplant. The expansion of oTreg with PBS and fTreg was observed on day 14 after allo-BMT. The administration of oTreg-oIL2 and fTreg (1:1 Tcon:Treg ratio) significantly increased the survival of the mice compared to Tcon group (P oIL-2 protein injection was able to selectively expand the oTreg in vivo, with a significantly increased percentage of Foxp3 GFP+ in CD4 + T cells (PBS;14.96±2.4%, oIL-2; 27.76±23.6%, P=0.02) in the peripheral blood (PB) and spleen after 7 days post-transplant. We also observed a significant decrease in the percentage of effector T cells in the LN, mLN and spleen of the mice transplanted with oTreg and oIL-2 administration after 7 days post transplantation compared with GvHD group. We further demonstrated that ortho-IL2 administration-maintained graft-versus-leukemia (GvL) responses against lymphoma cells (A20). Engineered orthogonal cytokine receptor improves the suppressing abilities of Treg potential in a murine model of acute GvHD with the ability to maintain GvL, representing a novel approach to the utilization of Treg in Allogeneic Hematopoietic Stem Cell Transplant. Disclosures Garcia: Synthekine: Membership on an entity's Board of Directors or advisory committees. Blazar: Tmunity Therapeutics: Other: Co-founder; Equilibre Pharmaceuticals Corp: Research Funding; Carisma Therapeutics, Inc: Research Funding; Rheos Medicines: Research Funding; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees.

ABSTRACT Introduction: Belumosudil (KD025) is a novel oral selective rho-associated coiled-coil-containing kinase-2 (ROCK2) inhibitor specifically designed for the treatment of cGVHD following an allogeneic hematopoietic cell transplant. It has been evaluated in the phase 2a dose-finding (KD025-208) and the pivotal phase 2 ROCKstar trials. Patients with cGVHD in the KD025-208 trial received belumosudil after failure of 1 to 3 prior systemic lines of therapy (LOTs), and those in the ROCKstar trial received belumosudil after failure of 2 to 5 prior systemic LOTs. The best overall response rate (95% CI) achieved, which was the primary end point, was 72% (64%-78%) across the 3 dosages (belumosudil 200 mg QD, 200 mg BID and 400 mg QD) that were studied across the 2 trials. This led to the recent FDA approval of the 200-mg QD dose for the treatment of adult and pediatric patients aged ≥12 years with cGVHD after failure of ≥2 prior systemic LOTs. FFS was a secondary end point in both trials. FFS is an established composite indicator for treatment success in cGVHD, as it incorporates recurrent malignancy, nonrelapse mortality (NRM) and the absence of subsequent cGVHD therapy. Historic rates of FFS in a prior large observational study of patients with cGVHD after second-line systemic therapy were 56% at 6 months, 45% at 12 months and 31% at 24 months (Inamoto, Blood 2013). Given limited data from contemporaneous clinical trials on FFS and prognostic factors of treatment failure, we studied pooled FFS and its determinants from the KD025-208 and ROCKstar trials. Methods: A total of 186 patients from the KD025-208 (n=54) and ROCKstar (n=132) trials treated with belumosudil 200 mg QD, 200 mg BID or 400 mg QD were analyzed . The median duration of belumosudil treatment was 9.9 months (range, 0.4-44.7 months). Prior therapies included tacrolimus (58%), sirolimus (46%), extracorporeal photopheresis (42%), mycophenolate mofetil (27%), ibrutinib (27%), ruxolitinib (21%) and cyclosporine (5%). Results: At enrollment, 70% of patients had severe cGVHD according to NIH global score, 52% had ≥4 organs involved and 37% received >3 prior LOTs. Overall, belumosudil was well tolerated, with drug discontinuations occurring in 10% of patients due to possible drug-related adverse events. The median FFS was 14 months. The estimated overall FFS (95% CI) rates were 75% (68%-81%), 54% (47%-61%) and 38% (29%-47%) at 6, 12 and 24 months, respectively (Table 1 and Figure). Reasons for failure included recurrent malignancy (6%), NRM (7%) and the addition of a new systemic cGVHD therapy (43%). Factors associated with increased risk of treatment failure (1-FFS) in both univariate and multivariate analyses (Table 2) included progressive onset of cGVHD (multivariate hazard ratio [HR]=2.1 [1.2-3.4]), absence of glucocorticoids in upfront therapy for cGVHD (HR=2.2 [1.2-4.0]) and ≥2 prior LOTs (HR=3.7 [1.2-12.2]). Conclusion: Treatment with belumosudil resulted in high FFS rates compared with historic benchmarks in cGVHD refractory to prior LOTs. Both NRM and relapse rates were low with the use of belumosudil. We identified distinct prognostic factors for FFS that can inform risk stratification and prognostication of patients being treated with belumosudil. Figure 1 Figure 1. Disclosures Lazaryan: Humanigen: Membership on an entity's Board of Directors or advisory committees; Avrobio: Membership on an entity's Board of Directors or advisory committees; Kadmon: Consultancy. Cutler: Mesoblast: Consultancy; Syndax: Consultancy; Omeros: Consultancy; Incyte: Consultancy; CareDx: Consultancy; Mallinckrodt: Consultancy; Pfizer: Consultancy; Kadmon: Consultancy; Editas: Consultancy; Cimeio: Consultancy; Deciphera: Consultancy; Jazz: Consultancy. Yang: Kadmon: Current Employment. Ieyoub: Kadmon: Current Employment. Eiznhamer: Kadmon: Current Employment. Blazar: BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Carisma Therapeutics, Inc: Research Funding; Rheos Medicines: Research Funding; Tmunity Therapeutics: Other: Co-founder; Equilibre Pharmaceuticals Corp: Research Funding; Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees. Lee: Amgen: Research Funding; Incyte: Research Funding; Novartis: Other: clinical trials, Research Funding; Pfizer: Research Funding; Kadmon: Research Funding; Takeda: Research Funding; Syndax: Research Funding; AstraZeneca: Research Funding; JANSSEN: Other; National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees. Pavletic: Center for Cancer Research: Research Funding; National Cancer Institute: Research Funding; National Institutes of Health: Research Funding; Celgene: Research Funding; Actelion: Research Funding; Eli Lilly: Research Funding; Pharmacyclics: Research Funding; Kadmon: Research Funding.

Summary The existence of a dysfunctional CD8+ T cell state in cancer is well established. However, the degree to which CD8+ T cell fates are influenced by the context in which they encounter cognate tumor antigen is less clear. We previously demonstrated that CD8+ T cells reactive to a model leukemia antigen were deleted by antigen cross-presenting type 1 conventional dendritic cells (cDC1s). Here, through a study of T cell receptor (TCR) transgenic CD8+ T cells (TCRTg101) reactive to a native C1498 leukemia cell antigen, we uncover a different mode of T cell tolerance in which TCRTg101 undergo progressive expansion and differentiation into an exhausted state. Antigen encounter by TCRTg101 requires leukemia cell major histocompatibility complex (MHC)-I expression and is independent of DCs, implying that leukemia cells directly mediate the exhausted TCRTg101 phenotype. Collectively, our data reveal that leukemia antigens are presented to CD8+ T cells via discrete pathways, leading to distinct tolerant states.

Summary Monocytic-lineage inflammatory Ly6c+CD103+ dendritic cells (DCs) promote antitumor immunity, but these DCs are infrequent in tumors, even upon chemotherapy. Here, we examined how targeting pathways that inhibit the differentiation of inflammatory myeloid cells affect antitumor immunity. Pharmacologic inhibition of Bruton’s tyrosine kinase (BTK) and the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) or deletion of Btk or Ido1 allowed robust differentiation of inflammatory Ly6c+CD103+ DCs during chemotherapy, promoting antitumor T cell responses and inhibiting tumor growth. Immature Ly6c+c-kit+ precursor cells had epigenetic profiles similar to conventional DC precursors; deletion of Btk or Ido1 promoted differentiation of these cells. Mechanistically, a BTK-IDO axis inhibited a tryptophan-sensitive differentiation pathway driven by GATOR2 and mTORC1, and disruption of the GATOR2 in monocyte-lineage precursors prevented differentiation into inflammatory DCs in vivo. IDO-expressing DCs and monocytic cells were present across a range of human tumors. Thus, a BTK-IDO axis represses differentiation of inflammatory DCs during chemotherapy, with implications for targeted therapies.

We report a novel phase 2 clinical trial in patients with poor prognosis refractory non-Hodgkin lymphoma (NHL) testing the efficacy of haploidentical donor natural killer (NK) cell therapy (NK dose 0.5-3.27 × 107 NK cells/kg) with rituximab and IL-2 (clinicaltrials.gov NCT01181258). Therapy was tolerated without graft-versus-host disease, cytokine release syndrome, or neurotoxicity. Of 14 evaluable patients, 4 had objective responses (29%; 95% CI 12-55%) at 2 months: 2 had complete response lasting 3 and 9 months. Circulating donor NK cells persisted for at least 7 days after infusion at the level 0.6-16 donor NK cells/µl or 0.35-90% of total CD56 cells. Responding patients had lower levels of circulating host-derived Tregs (17 ± 4 vs. 307 ± 152 cells/µL; p = 0.008) and myeloid-derived suppressor cells at baseline (6.6 ± 1.4% vs. 13.0 ± 2.7%; p = 0.06) than non-responding patients. Lower circulating Tregs correlated with low serum levels of IL-10 (R 2 = 0.64; p

When T cells from a bone marrow donor begin to attack the host within 3 months after hematopoietic-cell transplantation, acute graft-versus-host disease results. The disease mainly affects the skin, liver, and gastrointestinal tract and is treated with immunosuppressive drugs.

Follistatin is an angiogenic factor elevated in the circulation after allogeneic hematopoietic cell transplantation (HCT). Elevations in follistatin plasma concentrations are associated with the onset of and poor survival after acute GvHD (aGvHD). Using data from the Blood and Marrow Transplant Clinical Trials Network 0402 study (n=247), we sought to further quantify the longitudinal associations between plasma follistatin levels in transplant recipients, as well as baseline HCT donor follistatin levels, and allogeneic HCT outcomes. Higher recipient baseline follistatin levels were predictive of development of aGvHD (P=0.04). High donor follistatin levels were also associated with the incidence of aGvHD (P 1088 pg/mL at day 28 had a 4-fold increased risk for NRM (relative risk (RR)=4.3, 95% confidence interval (CI) 1.9–9.9, P

Maturation of human natural killer (NK) cells as defined by accumulation of cell-surface expression of CD57 is associated with increased cytotoxic character and TNF and IFNγ production upon target-cell recognition. Notably, multiple studies point to a unique role for CD57+ NK cells in cancer immunosurveillance, yet there is scant information about how they mature. In this study, we show that pharmacologic inhibition of GSK3 kinase in peripheral blood NK cells expanded ex vivo with IL15 greatly enhances CD57 upregulation and late-stage maturation. GSK3 inhibition elevated the expression of several transcription factors associated with late-stage NK-cell maturation including T-BET, ZEB2, and BLIMP-1 without affecting viability or proliferation. When exposed to human cancer cells, NK cell expanded ex vivo in the presence of a GSK3 inhibitor exhibited significantly higher production of TNF and IFNγ, elevated natural cytotoxicity, and increased antibody-dependent cellular cytotoxicity. In an established mouse xenograft model of ovarian cancer, adoptive transfer of NK cells conditioned in the same way also displayed more robust and durable tumor control. Our findings show how GSK3 kinase inhibition can greatly enhance the mature character of NK cells most desired for effective cancer immunotherapy. Cancer Res; 77(20); 5664–75. ©2017 AACR.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that affects the function and development of immune cells. Here, we show that recipient mice receiving AhR-/- T cells have improved survival and decreased acute graft-versus-host disease (aGVHD) in 2 different murine allogeneic bone marrow transplant (BMT) models. We also show that CD4+ T cells lacking AhR demonstrate reduced accumulation in secondary lymphoid tissue because of low levels of proliferation 4 days after BMT. Additionally, we found a significant increase in the quantity of peripherally induced regulatory donor T (pTreg) cells in the colon of recipients transplanted with AhR-/- T cells 14 days after transplant. Blockade of AhR using a clinically available AhR antagonist greatly enhanced the in vitro generation of inducible Treg (iTreg) cells from naive CD4+ human T cells. We have identified AhR as a novel target on donor T cells that is critical to the pathogenesis of aGVHD.

Substantial preclinical and clinical research into chronic graft-versus-host disease (cGVHD) has come to fruition in the last five years, generating a clear understanding of a complex cytokine-driven cellular network. cGVHD is mediated by naive T cells differentiating within IL-17–secreting T cell and follicular Th cell paradigms to generate IL-21 and IL-17A, which drive pathogenic germinal center (GC) B cell reactions and monocyte-macrophage differentiation, respectively. cGVHD pathogenesis includes thymic damage, impaired antigen presentation, and a failure in IL-2–dependent Treg homeostasis. Pathogenic GC B cell and macrophage reactions culminate in antibody formation and TGF-β secretion, respectively, leading to fibrosis. This new understanding permits the design of rational cytokine and intracellular signaling pathway–targeted therapeutics, reviewed herein.

Allogeneic hematopoietic stem cell transplantation is hampered by chronic graft-versus-host disease (cGVHD), resulting in multiorgan fibrosis and diminished function. Fibrosis in lung and skin leads to progressive bronchiolitis obliterans (BO) and scleroderma, respectively, for which new treatments are needed. We evaluated pirfenidone, a Food and Drug Administration (FDA)-approved drug for idiopathic pulmonary fibrosis, for its therapeutic effect in cGVHD mouse models with distinct pathophysiology. In a full major histocompatibility complex (MHC)-mismatched, multiorgan system model with BO, donor T-cell responses that support pathogenic antibody production are required for cGVHD development. Pirfenidone treatment beginning one month post-transplant restored pulmonary function and reversed lung fibrosis, which was associated with reduced macrophage infiltration and transforming growth factor-β production. Pirfenidone dampened splenic germinal center B-cell and T-follicular helper cell frequencies that collaborate to produce antibody. In both a minor histocompatibility antigen-mismatched as well as a MHC-haploidentical model of sclerodermatous cGVHD, pirfenidone significantly reduced macrophages in the skin, although clinical improvement of scleroderma was only seen in one model. In vitro chemotaxis assays demonstrated that pirfenidone impaired macrophage migration to monocyte chemoattractant protein-1 (MCP-1) as well as IL-17A, which has been linked to cGVHD generation. Taken together, our data suggest that pirfenidone is a potential therapeutic agent to ameliorate fibrosis in cGVHD.

Acute graft-versus-host disease (aGVHD) continues to be a frequent and devastating complication of allogeneic hematopoietic stem cell transplantation (HSCT), posing as a significant barrier against the widespread use of HSCTs as a curative modality. Recent studies suggested serum/plasma microRNAs (miRs) may predict aGVHD onset. However, little is known about the functional role of circulating miRs in aGVHD. In this article, we show in two independent cohorts that miR-29a expression is significantly upregulated in the serum of allogeneic HSCT patients at aGVHD onset compared with non-aGVHD patients. Serum miR-29a is also elevated as early as 2 wk before time of diagnosis of aGVHD compared with time-matched control subjects. We demonstrate novel functional significance of serum miR-29a by showing that miR-29a binds and activates dendritic cells via TLR7 and TLR8, resulting in the activation of the NF-κB pathway and secretion of proinflammatory cytokines TNF-α and IL-6. Treatment with locked nucleic acid anti–miR-29a significantly improved survival in a mouse model of aGVHD while retaining graft-versus-leukemia effects, unveiling a novel therapeutic target in aGVHD treatment or prevention.

Although stable mixed-hematopoietic chimerism induces robust immune tolerance to solid organ allografts in mice, the translation of this strategy to large animal models and to patients has been challenging. We have previously shown that in MHC-matched nonhuman primates (NHPs), a busulfan plus combined belatacept and anti-CD154-based regimen could induce long-lived myeloid chimerism, but without T cell chimerism. In that setting, donor chimerism was eventually rejected, and tolerance to skin allografts was not achieved. Here, we describe an adaptation of this strategy, with the addition of low-dose total body irradiation to our conditioning regimen. This strategy has successfully induced multilineage hematopoietic chimerism in MHC-matched transplants that was stable for as long as 24 months posttransplant, the entire length of analysis. High-level T cell chimerism was achieved and associated with significant donor-specific prolongation of skin graft acceptance. However, we also observed significant infectious toxicities, prominently including cytomegalovirus (CMV) reactivation and end-organ disease in the setting of functional defects in anti-CMV T cell immunity. These results underscore the significant benefits that multilineage chimerism-induction approaches may represent to transplant patients as well as the inherent risks, and they emphasize the precision with which a clinically successful regimen will need to be formulated and then validated in NHP models.

In the past decade, the power of harnessing T cell co-signaling pathways has become increasingly understood to have significant clinical importance. In cancer immunotherapy, the field has concentrated on two related modalities: First, targeting cancer antigens through highly activated chimeric antigen T cells (CAR-Ts) and second, re-animating endogenous quiescent T cells through checkpoint blockade. In each of these strategies, the therapeutic goal is to re-ignite T cell immunity, in order to eradicate tumors. In transplantation, there is also great interest in targeting T cell co-signaling, but with the opposite goal: in this field, we seek the Yin to cancer immunotherapy’s Yang, and focus on manipulating T cell co-signaling to induce tolerance rather than activation. In this review, we discuss the major T cell signaling pathways that are being investigated for tolerance-induction, detailing pre-clinical studies and the path to the clinic for many of these molecules. These include blockade of co-stimulation pathways and agonism of coinhibitory pathways, in order to achieve the delicate state of balance that is transplant tolerance: a state which guarantees lifelong transplant acceptance without ongoing immunosuppression, and with preservation of protective immune responses. In the context of the clinical translation of immune tolerance strategies, we discuss the significant challenge that is embodied by the fact that targeted pathway modulators may have opposing effects on tolerance based on their impact on effector versus regulatory T cell biology. Achieving this delicate balance holds the key to the major challenge of transplantation: lifelong control of alloreactivity while maintaining an otherwise intact immune system.

Background: Chronic graft-versus-host disease (CGVHD) is the leading cause of long-term morbidity and mortality following hematopoietic stem cell transplant (HCT) and occurs in over 50% of patients undergoing unrelated donor HCT. Despite its frequency, the mechanisms driving this disease remain incompletely understood, making its prevention and successful treatment challenging. To address this issue, we have undertaken a transcriptomic analysis of T cell reconstitution after unrelated donor HCT, to dissect mechanisms driving CGVHD. Methods: The patients studied were enrolled on a Phase 2, randomized, placebo-controlled trial of abatacept for GVHD prevention in patients receiving 8/8 unrelated-donor HCT for hematologic malignancies (NCT01743131). All immune analyses in the current study were performed on patients randomized to standard GVHD prophylaxis with calcineurin inhibition + methotrexate alone (placebo cohort, n =69), and thus provide insights into the drivers of CGVHD during standard unrelated donor HCT. On Day +100, CD4+ T cells were purified from the peripheral blood of these patients, and then analyzed by RNASeq. To determine the transcriptomic drivers of CGVHD without the confounder of significant prior acute GVHD (AGVHD) or exposure to steroids, we focused on profiling the CD4+ transcriptome of de novo CGVHD (CGVHD which develops in the absence of prior grade II-IV AGVHD, n = 7) and compared these patients to those who were 'operationally tolerant' and never developed either grade II-IV AGVHD or any CGVHD (n= 4). Gene expression from the resulting transcriptomes was quantified using kallisto. Differentially expressed (DE) genes were identified using DESeq2 (threshold for DE, adjusted (for multiple testing) p Results: DE analysis identified 101 genes that were significantly upregulated in CD4+ T cells from de novo CGVHD group and 54 genes that were significantly upregulated in the 'operationally tolerant' group (Figure 1A). GSEA identified that the mostly highly enriched signatures in patients with de novo CGVHD encompassed naïve CD4+ transcriptional programing (Figure 1B-C), in agreement with flow cytometric analysis, which also demonstrated expansion of CD4+ naïve T cells at Day +100 in patients developing de novo CGVHD compared to those demonstrating operational tolerance (Figure 1D). Importantly, the naïve CD4+ T cell signatures that were identified were distinct from those defining CD4+ stem cell memory T cells (which did not enrich in the de novo CGVHD cohort). In contrast, the gene signature of the operationally tolerant patients were enriched for regulatory gene sets (Figure 1C), consistent with a large body of evidence demonstrating that Treg expansion can be protective against CGVHD. Discussion: This study represents, to our knowledge, the first interrogation of the transcriptomic features of patients developing de novo CGVHD versus those operationally tolerant patients who develop neither significant AGVHD nor CGVHD after HCT. These patients may represent a particularly effective cohort in which to study immunologic drivers of CGVHD, given their freedom from prior treatment with corticosteroids, which can confound downstream transcriptomic analyses. Our data provide compelling evidence for a prominent naïve CD4+ T cell signature in patients who develop moderate-to-severe CGVHD despite their lack of antecedent AGVHD. These results are provocative, as they implicate a cell subset that is often considered more quiescent (naïve T cells) as associated with patients who develop immune pathology associated with CGVHD. These results suggest that naïve CD4+ T cells may represent a potent reservoir for alloreactivity, that, once activated, can cause significant disease. This would be in agreement with the implications of previously reported trials of naïve T cell depletion, which resulted in significant control of CGVHD. These results suggest that strategies to restrain naïve T cell pathogenic activation after Day +100 may improve CGVHD outcomes, and that the CD4+ T cell transcriptomic signature at this timepoint could be developed into a robust immunologic biomarker for the risk of developing CGVHD versus operational tolerance after HCT. Figure 1 Disclosures Watkins: Bristol Myers Squib: Honoraria. Qayed:Novartis: Consultancy; Mesoblast: Consultancy. Blazar:Tmunity: Other: Co-founder; KidsFirst Fund: Research Funding; BlueRock Therapeutics: Research Funding; Childrens' Cancer Research Fund: Research Funding; BlueRock Therapeuetic: Consultancy; Magenta Therapeutics: Consultancy; Fate Therapeutics Inc.: Research Funding. Horan:Bristol Myers Squib: Honoraria, Research Funding. Langston:Kadmon Corporation: Research Funding; Astellas Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Research Funding; Incyte: Research Funding; Bristol Myers Squib: Research Funding; Chimerix: Research Funding; Takeda: Research Funding. Kean:fortyseven: Consultancy; regeneron: Research Funding; hifibio: Consultancy; kymab: Consultancy; Bristol Meyers Squibb: Research Funding; gilead: Research Funding; novartis: Consultancy; bluebird bio: Research Funding; magenta: Research Funding.

Introduction: Belumosudil (KD025) is a novel oral selective rho-associated coiled-coil kinase 2 (ROCK2) inhibitor specifically designed for the treatment of cGVHD, an immune-mediated inflammatory and fibrotic disorder. In a previous dose-finding study (KD025-208, N=54), two-thirds of patients, including those with fibrotic and inflammatory manifestations, achieved a partial or complete response with belumosudil. Herein, we report on the top-line results (6 months after the last patient in) from the pivotal phase 2 trial (ROCKstar [KD025-213], N=132). Methods: This phase 2, open-label, randomized, multicenter study evaluated belumosudil 200 mg QD (n=66) and BID (n=66) in patients with cGVHD who received 2 to 5 prior lines of therapy (LOT). Treatment continued until clinically significant progression of cGVHD. The primary end point was overall response rate (ORR), defined per the 2014 National Institutes of Health Consensus Criteria. Additional end points included duration of response (DOR), Lee Symptom Scale (LSS) score, failure-free survival (FFS), corticosteroid (CS) dose reductions and overall survival. The study was powered such that the lower bound of the 95% confidence interval (CI) excludes 30%, with appropriate multiplicity adjustment. Results: At enrollment, the median age was 56 years, the median time from cGVHD diagnosis to enrollment was 29 months, 67% of patients had severe cGVHD, 52% had ≥4 organs involved, 72% had received ≥3 prior LOT (including ibrutinib [n=46] or ruxolitinib [n=38]) and 73% were refractory to their last LOT. The baseline characteristics of both arms were well balanced. With a median follow-up of 8 months, the ORR (95% CI) with belumosudil 200 mg QD and BID was 73% (60%-83%) and 74% (62%-84%), respectively (Table 1). In patients who previously received ruxolitinib (29%), the ORR with belumosudil 200 mg QD and BID was 65% (41%-85%) and 72% (47%-90%), respectively. In patients who previously received ibrutinib (35%), the ORR with belumosudil 200 mg QD and BID was 73% (50%-89%) and 71% (49%-87%), respectively. High ORRs were seen in all patient subgroups, regardless of length of time from diagnosis to treatment, including those with severe cGVHD, involvement of ≥4 organs and a refractory response to prior LOT (Figure 1). The response rate was similar across all affected organs. The median time to response was 4 weeks. Of responders, 49% have maintained response for ≥20 weeks. The median DOR has not yet been reached. Clinically meaningful improvement (≥7-point reduction) in LSS score on consecutive assessments was observed in 39% and 33% of patients in the QD and BID groups, respectively. Both responders (43%) and nonresponders (17%) experienced a clinically meaningful improvement in LSS score. FFS was 77% (69%-84%) at 6 months. CS and calcineurin inhibitor discontinuations were seen in 18% and 13% of patients, respectively. Belumosudil was well tolerated, with >95% relative dose intensity in 83% of patients. Drug discontinuation occurred in 10% of patients due to possible drug-related adverse events (AEs), 3% due to progression of underlying disease and 12% due to progression of cGVHD. AEs were consistent with those expected in patients with cGVHD receiving CS and other immunosuppressants (Table 2). Common AEs included fatigue (32%), diarrhea (29%), nausea (26%), cough (24%), dyspnea (24%), upper respiratory tract infection (23%), peripheral edema (21%) and headache (20%). At least 1 serious AE occurred in 34% of patients. Twenty-three percent of patients had at least 1 liver-related investigation; the most common was increased gamma-glutamyltransferase (11%), and only 1 patient showed an increase in bilirubin. Eight patients died during the study; 5 due to AEs (1 possibly related to belumosudil) and 3 during long-term follow-up (>28 days after last dose). There were no reports of cytomegalovirus reactivation or infection. Conclusion: Treatment with belumosudil at both doses resulted in high ORRs across key subgroups, meeting the primary end point of this pivotal randomized trial in cGVHD. Responses were durable and clinically meaningful, irrespective of patient and cGVHD characteristics, and were seen in patients who previously received ruxolitinib and ibrutinib. Belumosudil was well tolerated, with limited and manageable AEs. Further studies will evaluate its use earlier in disease management. The 12-month data analysis will be presented at ASH 2020. Disclosures Cutler: Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kadmon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Medsenic: Consultancy, Membership on an entity's Board of Directors or advisory committees; Generon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Mesoblast: Consultancy, Membership on an entity's Board of Directors or advisory committees. Lee:Pfizer: Consultancy, Research Funding; Incyte: Consultancy, Research Funding; Amgen: Research Funding; Kadmon: Research Funding; AstraZeneca: Research Funding; Novartis: Research Funding; Takeda: Research Funding; Syndax: Research Funding. Rotta:Merck: Speakers Bureau; Jazz Pharma: Speakers Bureau. Ramakrishnan:Amgen: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Cigna: Honoraria. Eiznhamer:Kadmon Corporation, LLC: Current Employment, Current equity holder in publicly-traded company. Schueller:Kadmon Corporation, LLC: Current Employment, Current equity holder in publicly-traded company. Yang:Kadmon Corporation, LLC: Current Employment, Current equity holder in publicly-traded company. Green:Kadmon Corporation, LLC: Current Employment, Current equity holder in publicly-traded company. Aggarwal:Kadmon Corporation, LLC: Consultancy; Angiocrine Bioscience, Inc: Current Employment, Other: stock options. Blazar:BlueRock Therapeutics: Research Funding; BlueRock Therapeuetic: Consultancy; Fate Therapeutics Inc.: Research Funding; Magenta Therapeutics: Consultancy; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-founder. Jagasia:Ocugen: Other; Mallinckrodt: Research Funding; Janssen: Research Funding.

Background: Chronic graft versus host disease (cGVHD) is a major cause of morbidity and late non-relapse mortality after allogeneic hematopoietic cell transplantation and is commonly associated with prolonged immune suppression. Patients (pts) with inadequate response to steroids have few effective therapeutic options and represent an unmet medical need. Available therapies are associated with significant toxicity, immunosuppression, and increased risk of infections. Preclinical studies demonstrate that CSF-1/CSF-1R is a key regulatory pathway involved in the expansion and infiltration of donor-derived macrophages that mediate cGVHD. Axatilimab (SNDX-6352, axa) is a humanized, full-length IgG4 antibody with high affinity to CSF-1R. Axa affects the migration, proliferation, differentiation, and survival of monocytes and macrophages by binding to CSF-1R and blocking its activation by its two known ligands, CSF-1 and IL-34. It offers a novel therapeutic option for treatment of these pts Methods: SNDX-6352-0503 is a Phase 1/2 dose-escalation and dose-expansion study evaluating safety, tolerability, pharmacokinetics (PK)/pharmacodynamics (PD), and efficacy of axa in pts >6 years of age with active symptomatic cGVHD despite ≥2 prior lines of therapy. The Phase 1 endpoints were safety, tolerability, PK and PD with the primary objective of defining optimal biologic dose; the primary endpoint of the Phase 2 study is overall response rate (CR+PR) by 6 months. Pts were dosed in 28-day cycles. The data cutoff was 22 July 2020. Results: Twelve pts have been enrolled in the Phase 1 study. Median age at enrollment was 58y (range, 29-73y), 8 pts were male. Pts had failed a median of 5 prior lines of treatment (range 4-9). Doses included 0.15 mg/kg (n=1), 0.5mg/kg (n=1), 1mg/kg (n=3), 3 mg/kg (n=6) every 2 weeks (q2w), and 3mg/kg q4w (n=1). Of these, 5 pts (42%) are still receiving axa. The median number of cycles for all pts is 5 (range 1-12). Of the 3 pts whose starting dose was 3 mg/kg q2w and remain on study, 2 dose reduced; one to 2 mg/kg q4w and one to 1 mg/kg q2w. Seven pts (58%) discontinued due to: adverse events (3 mg/kg q2w, n=2); death due to traumatic fall (1 mg/kg q2w, n=1); investigator decision (0.5 mg/kg q2w, n=1); progressive cGVHD (1 and 0.15 mg/kg q2w, n=1 each); and non-compliance (3 mg/kg q2w, n=1). Two of 6 pts (17%) at a dose of 3 mg/kg q2w reported a treatment emergent adverse event that was considered a dose limiting toxicity (DLT): 1 with CTCAE Grade 4 creatine kinase increase with symptoms of myositis after dose 1, and the 2nd with an elevation in amylase/lipase that delayed the 3rd dose for >2 weeks. The latter pt restarted therapy at 1 mg/kg q2w and remains on treatment after 5 cycles. Four pts (1 at 0.15 mg/kg and 3 at 3 mg/kg q2w, 33%) had a related treatment emergent adverse event that was ≥Grade 3: increase in aspartate aminotransferase (n=2); increase in creatine phosphokinase (n=2); and increase in gamma-glutamyl transferase (n=2). Such biochemical elevations may be a consequence of CSF-1R blockade on Kupffer cells leading to an inhibition in the clearance of these enzymes, consistent with the mechanism of action of axa and when asymptomatic have not been associated with clinical manifestations of hepatitis, pancreatitis, or rhabdomyolysis. Periorbital edema was observed in 2 pts (≤Grade 2); no additional CSF-1Ri class-effect associated TEAEs were observed Clinical responses as defined by the 2014 NIH cGVHD Consensus Criteria have been observed in 7 pts (58%) across all dose levels; median time to response was 12 weeks. Organ-specific responses have been observed in esophagus (n=1/1), eyes (n=3/10), joints/fascia (n=5/9), mouth (n=1/7), and skin (n=3/8). Prior therapies received by the responders included ibrutinib (6 pts), ruxolitinib (5 pts), and KD025 (3 pts); 3 of the responding pts had received all of these. Six pts (50%) reported at least a 7-point improvement in the Lee Symptom Score. Preliminary PK profiles and pharmacodynamic endpoints, including circulating CD14+CD16+ nonclassical and CD14++CD16+ intermediate monocyte kinetics, are consistent with those observed in healthy volunteers and pts with solid tumors see figure below. Conclusions: These data demonstrate that axatilimab is clinically active with acceptable safety profile and responses observed in heavily pretreated pts with active cGVHD. Enrollment continues in the Phase 1 study at 3 mg/kg q4w and Phase 2 study at a dose of 1 mg/kg q2w. Figure Disclosures Arora: Syndax: Research Funding; Fate Therapeutics: Consultancy; Pharmacyclics: Research Funding; Kadmon: Research Funding. Jagasia:Janssen: Research Funding; Mallinckrodt: Research Funding; Ocugen: Other. Meyers:Syndax Pharmaceuticals, Inc: Current Employment. Quaranto:Syndax Pharmaceuticals, Inc: Current Employment. Schmitt:Syndax Pharmaceuticals, Inc: Current Employment. Sankoh:Syndax Pharmaceuticals, Inc: Current Employment. Abu Zaid:Syndax: Research Funding. Hill:Roche: Research Funding; Generon: Consultancy; CSL: Research Funding; Implicit Bioscience: Research Funding; Pharmacyclics: Research Funding; Compass Pharmaceuticals: Research Funding. Weisdorf:Incyte: Research Funding; FATE Therapeutics: Consultancy. Blazar:BlueRock Therapeuetic: Consultancy; Magenta Therapeutics: Consultancy; BlueRock Therapeutics: Research Funding; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-founder; Fate Therapeutics Inc.: Research Funding. Ordentlich:Syndax Pharmaceuticals, Inc: Current Employment. Lee:Takeda: Research Funding; AstraZeneca: Research Funding; Pfizer: Consultancy, Research Funding; Amgen: Research Funding; Novartis: Research Funding; Kadmon: Research Funding; Incyte: Consultancy, Research Funding; Syndax: Research Funding. OffLabel Disclosure: The drug (Axatilimab) is a humanized antibody against CSF-1R. The study is a phase I trial testing this drug in patients with chronic graft versus host disease

Background: Graft vs. host disease (GVHD) remains a major complication of allogeneic stem cell transplantation (alloSCT). To create space for donor stem cells and prevent their rejection, alloSCT protocols rely on conditioning regimens involving chemotherapy, frequently combined with radiation. Interleukin (IL)-33 is a nuclear protein that is found in the secondary lymphoid organs and barrier tissues. IL-33 is augmented in recipient barrier tissues by conditioning. We have established that IL-33 is a clinically-relevant signal. While we have further elucidated that IL-33 is acting directly on donor T cells to augment GVHD, neither the critical place nor timing of IL-33 stimulation of donor T cells during the alloimmune response mediating GVHD have been defined. Methods: We used both IL-33 knockout mice as recipients and ST2 (IL33 receptor) knockout mice as T cell donors in MHC mismatch alloSCT models to define the spatial and temporal importance of IL-33 in GVHD tissue damage. More specifically, to establish the role of IL-33 in T cell effector function in the small intestine during GVHD, irradiated Il33-/- and Il33+/+ C57BL/6 (B6) recipients were given BALB/c bone marrow (BM) and T cells. Additionally, irradiated BALB/c mice were given St2+/+ B6 BM and Cd4 Cre x St2fl/fl or St2-/- and St2+/+ T cells. To determine the role of IL-33 on T cell persistence in GVHD, we used an MHCII-disparate model. Irradiated Il33-/- and Il33+/+ bm12 recipients were reconstituted with B6 BM, with or without CD90.1+ B6 T cells. Results: When donor T cell responses were characterized by flow cytometry at days 7, 14, and 28 post alloSCT, the bulk of these data suggest that IL-33 sustains CD4+ T helper Type 1 (Th1) cells in the barrier tissues at late points following GVHD initiation. Specifically, B6 Il33-/- recipients had fewer BALB/c CD4+Tbet+ cells in the small intestine lamina propria (SI LP; WT 15.9 ± 1.31, KO 9.53 ± 1.32; p=0.03; n=3/group), but not in the spleen (WT 54.8 ± 7.37, KO 57.68 ± 4.90; p=0.75; n=4/group) at day 7. Additionally, we found that when we used therapeutic doses of anti-IL-12p40 to neutralize IL-12 there was no impact on the CD4+Tbet+ cells in the SI of B6 il33-/- recipients (KO control IgG 7.29 ± 0.63, KO anti-IL-12p40 6.61 ± 0.75; p=0.52; n=4/group) suggesting that the decrease in the frequency of CD4+Tbet+ T cells in Il33-/- in the SI is independent of IL-12. In the CD4-dependent B6 to bm12 GVHD model, we find that IL-33-deficient recipients have reduced overall CD4+ donor T cells in the SI LP at both day 14 and day 28 (d14: WT 16.15 ± 2.55, KO 0.35 ± 0.07; p=0.003; n=3/group; d28: WT 33.07 ± 7.17, KO 6.27 ± 1.59; p=0.022; n=3/group). Consistent with our B6 recipients of BALB/c T cells above, this decrease was not observed in the spleen or lymph nodes. Reduced clusters of CD3+ cells were evident in immunohistochemistry of bm12 Il33-/- relative to bm12 Il33+/+ at day 28 post-alloSCT. In corroboration with our IL-33-deficient recipient results, B6 CD4+ T cells lacking the ST2 receptor when co-transferred with ST2 competent T cells were not sustained in the SI LP at day 7 (ST2 WT 18.55 ± 0.77, ST2 KO 6.35 ± 1.42; p=0.0003; n=4/group). ST2 deficient CD4+ donor cells were of equal frequency to the ST2 competent donor cells in the spleen of BALB/c recipients (ST2 WT 54.58 ± 1.914, ST2 KO 50 ± 2.53; p=0.19; n=4/group). Conclusions: Our data reveal that IL-33 is not required for donor Th1 responses in the spleen and LN, but IL-33 is critical for Th1 cells to persist in barrier tissues like the small intestine after alloSCT in the absence of IL-12 stimulation. These data suggest that targeting IL-33 signaling may be an effective therapy to diminish GVHD in target tissues. Disclosures Blazar: Fate Therapeutics Inc.: Research Funding; Childrens' Cancer Research Fund: Research Funding; BlueRock Therapeutics: Research Funding; BlueRock Therapeuetic: Consultancy; Magenta Therapeutics: Consultancy; KidsFirst Fund: Research Funding; Tmunity: Other: Co-founder. Shlomchik:Bluesphere Bio: Consultancy, Other: shareholder.

Approximately 19,940 new cases of acute myeloid leukemia (AML) will be diagnosed in 2020 with an estimated 11,180 deaths, which has remained steady for 3 decades. We established that allogeneic natural killer (NK) cell infusions have an antileukemic effect and induce remission in 25-40% of relapsed/refractory AML patients. After hundreds of individual donor product infusions, we noted therapeutic limitations to include effector potency, cell numbers,persistence, specificity and exportability. To overcome these barriers, we developed a robust genetic editing and manufacturing platform for the uniform engineering and expansion of high-quality NK cells derived from induced pluripotent stem cells (iPSCs). This manufacturing system allows efficient production of iPSC-derived NK (iNK) cells engineered to enhance persistence and potency, and enables distribution of highly homogenous iNK cells to multiple clinical sites and multi-dosing to treat patients on-demand with advanced cancer. We hypothesized that iNK cells could be combined with the anti-CD38 monoclonal antibody daratumumab (dara) to directly target various tumors and harness their antibody-dependent cellular cytotoxicity (ADCC) potential. Previously, we reported on our ability to effectively target multiple myeloma with our FT538 program (the iNK defined below), now FDA approved for clinical trials. Here we discuss applying these same engineered iNK cells to specifically target and kill AML blasts induced to upregulate CD38 by retinoic acid exposure. We utilized iNK cells expressing a high-affinity, non-cleavable version of CD16 (the Fc receptor that mediates ADCC) termed hnCD16 alone or combined with CD38 knockout (KO) to prevent dara-mediated NK cell fratricide (Fig. 1A). CD38 KO iNK cells had intact calcium flux in response to ionomycin or CD16 crosslinking (Fig. 1B). Adding dara to peripheral blood NK cells or hnCD16 iNK cells triggered ADCC-mediated fratricide, while hnCD16/CD38 KO iNK cells that cannot be targeted by dara were unaffected (Fig 1C). CD38, expressed intracellularly and on the plasma membrane, functions as an NADase, degrading nicotinamide adenine dinucleotide (NAD+) into ADP-ribose and nicotinamide. Because NAD+ levels influence several key metabolic pathways, we assessed the impact of CD38 KO on iNK cell metabolism. We found that, relative to hnCD16 iNK cells, hnCD16/CD38 KO iNK cells had significantly higher intracellular NAD+, NADH, and ATP levels (Fig. 1D). Additionally, hnCD16/CD38 KO iNK cells exhibited elevated mitochondrial oxidative phosphorylation (Fig. 1E) and marked resistance to oxidative stress to hydrogen peroxide exposure (Fig. 1F). Together, these results demonstrate a significant enhancement of NK cell mitochondrial oxidative phosphorylation and redox homeostasis in iNK CD38 KO cells. To enhance hnCD16/CD38 KO iNK cells further, we incorporated a third modification consisting of an IL-15 receptor signaling complex (IL-15RF). This receptor complex is expressed on the cell surface and provides IL-15 signals required for NK cell survival and proliferation independent of exogenous cytokine. To determine the efficacy of hnCD16/CD38 KO/IL-15RF iNK cells combined with dara to target AML, we first assessed CD38 expression on THP-1 cells (an AML cell line) and primary AML blasts incubated with or without retinoic acid. We observed high CD38 expression on both THP-1 cells and primary AML cells further elevated with retinoic acid treatment (Fig. 1G). THP-1 and primary AML cells were labeled with CellTrace dye and used as targets for killing assays using hnCD16/CD38 KO/IL-15RF iNK cells. Compared to hnCD16/CD38 KO/IL-15RF iNK cells cultured with targets alone, adding dara led to higher target cell killing, especially after retinoic acid exposure (Fig. 1H). In 12-hour live imaging experiments testing iNK cell cytotoxicity against THP-1 cells, similar results were observed (Fig. 1I). Collectively, our results show that utilizing the iNK cell platform to uniformly express hnCD16 and IL15RF combined with complete CD38 KO is an effective strategy to promote effective ADCC against CD38+ cells in the absence of fratricide, and that CD38 KO reprograms NK cells for higher oxidative metabolic fitness for improved persistence and anti-tumor function. Furthermore, we have generated proof-of-concept data supporting triple gene-modified iNK cells combined with dara as a novel AML immunotherapy. Disclosures Cichocki: Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding. Bjordahl:Fate Therapeutics: Current Employment. Bonello:Fate Therapeutics, Inc: Current Employment. Mahmood:Fate Therapeutics, Inc: Current Employment. Rogers:Fate Therapeutics, Inc: Current Employment. Ge:Fate Therapeutics, Inc: Current Employment. Lee:Fate Therapeutics, Inc.: Current Employment. Felices:GT Biopharma: Consultancy. Walcheck:Fate Therapeutics: Consultancy, Research Funding. Blazar:Fate Therapeutics Inc.: Research Funding; KidsFirst Fund: Research Funding; Magenta Therapeutics: Consultancy; Childrens' Cancer Research Fund: Research Funding; BlueRock Therapeutics: Research Funding; BlueRock Therapeuetic: Consultancy; Tmunity: Other: Co-founder. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company. Miller:GT Biopharma: Consultancy, Patents & Royalties, Research Funding; Onkimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; Vycellix: Consultancy. OffLabel Disclosure: FT538 is a genetically modified induced pluripotent stem cell derived NK cell product that can be combined with daratumumab to target CD38 on AML after retinoid acid induction.

Introduction: Chronic Graft-versus-Host Disease (cGvHD) frequently requires prolonged immune suppressive therapy (IST) with > 50% still on IST at 5 years. The IST typically involves a slow taper of steroids often with flare of cGvHD, necessitating augmentation of previous therapy or addition of new IST. Studies describing cGvHD flares are limited. We analyzed patients with cGvHD who flared during the treatment with systemic IST, their overall survival (OS) and non-relapse mortality (NRM). Methods: This study included all adult patients with cGvHD (n=145) following an allogeneic transplant (2010 - 2017) from a matched sibling donor peripheral blood stem cell transplant (MSD, n=104 (72%) or double/single umbilical cord blood transplant (UCBT, n=41 (28%). The 2014 NIH Consensus Criteria were used to classify organ/overall cGvHD severity. Flare of cGvHD was defined as progression in cGvHD manifestations (after initial response), which was less severe than at diagnosis. Multivariate regression of flares was based on the Prentice, Williams and Peterson model for ordered multiple events (flares). Time-dependent effects on OS and NRM were analyzed by Cox and Fine and Gray regression with propensity scoring to control for confounding. Results: Flares occurred in 87 patients; the cumulative incidence of flares was 60% (95% CI: 51-70%) at a median of 188 days (range 16-751) after diagnosis of cGvHD. The median dose of prednisone was 1 mg/kg/day (range 0-4.2) at diagnosis of cGvHD. At the diagnosis of flare, 36 (41%) of the patients were off prednisone, 50 (57%) were receiving 0.1-0.5 mg/kg /day, and 2 patients > 0.5 mg/kg /day. Thirty two of the 87 (36%) patients experienced multiple flares (2 to 4). The most common organs involved at cGvHD flare were skin (n=45; 51%), mouth (n=27; 31%), GI tract (n=22; 25%) and liver (n=12; 14%); often in combinations of skin/mouth in 11 cases (13%), skin/GI in 6 (7%) and liver/mouth in 4 (5%) cases. Treatment for flare was mostly increase in dose of prednisone to 0.5 mg/kg/day (range 0.3-1.0) in 77 patients (88%) plus the addition of another line of IST in 48 patients (55%). In multiple regression analysis, only donor type was significant predictor of flare in cGvHD. UCBT was associated with 2-fold lower probability of flaring (HR 0.5; 95% CI: 0.3-0.9; p=0.03) compared to MSD. cGvHD severity, organ involvement, platelet count at diagnosis and type of onset were not significant predictors of cGvHD flares. At 2 years after the initial flare, the OS was 77% (95% CI: 66-84%) and NRM 19% (95% CI: 11-28%). Multiple regression analysis evaluating OS and NRM from onset of cGvHD comparing flare to non-flare were performed using flare as a time dependent variable. Compared to cGvHD patients without flare at 2 years, those with flare of cGvHD had a similar risk of NRM (HR 1.2; 95% CI: 0.2-6.1, p=0.86) and OS (HR 0.9; 95% CI: 0.4-2.3, p=0.85). At 2 years from cGvHD onset, the cumulative incidence of resolved cGvHD (durable discontinuation of steroids for ≥ 6 consecutive months) was 31% (95% CI: 21-41%) in those who flared vs. 86% (95% CI: 75-96%) in those without flare. Conclusions: Though cGvHD patients with flare had similar risk of NRM and OS as those without a flare, patients with flare required extended steroids, along with clinical monitoring and intensified IST. cGvHD after UCBT was associated with significantly lower risk of flaring compared to MSD. The ongoing burden of IST, risk of infection and morbidity of cGvHD is substantial and needs better approaches than chronic slow taper of steroids. Disclosures Weisdorf: Incyte: Research Funding; FATE Therapeutics: Consultancy. Wagner:Novartis: Research Funding; Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company; Magenta Therapeutics: Consultancy, Research Funding; BlueRock: Research Funding; Gadeta: Membership on an entity's Board of Directors or advisory committees. Blazar:Fate Therapeutics Inc.: Research Funding; Childrens' Cancer Research Fund: Research Funding; BlueRock Therapeutics: Research Funding; BlueRock Therapeuetic: Consultancy; Magenta Therapeutics: Consultancy; KidsFirst Fund: Research Funding; Tmunity: Other: Co-founder. MacMillan:Mesoblast: Consultancy; Angiocrine Biosciences, Inc.: Consultancy; Equillium, Inc.: Consultancy; Talaris Therapeutics, Inc: Consultancy; Fate Therapeutics, Inc.: Consultancy. Holtan:Generon: Consultancy; BMS: Consultancy; CSL Behring: Other: Clinical trial data adjudication; Incyte: Consultancy. Brunstein:AlloVir: Other: Advisory board; Gamida: Research Funding; Astex: Research Funding; Magenta: Research Funding. Betts:Patent Pending: Patents & Royalties: Dr. Betts has a pending patent WO2017058950A1: Methods of treating transplant rejection. This includes the use of JAK inhibitors. Neither he nor his institution have received payment related to claims described in the patent.. Bachanova:FATE: Research Funding; Karyopharma: Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding; Gamida Cell: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding. Rashidi:Synthetic Biologics: Other: DSMC member (1 trial) and related honorarium. Arora:Fate Therapeutics: Consultancy; Kadmon: Research Funding; Pharmacyclics: Research Funding; Syndax: Research Funding.

Mixed hematopoietic cell chimerism induction is a promising approach to establish transplantation tolerance, though achievement of donor cell engraftment in patients given non-myeloablative conditioning remains challenging. Adoptive transfer of regulatory T cells (Tregs) has been shown to improve alloengraftment in animal models. However, ex vivo Treg expansion for the purpose of infusing large numbers is technically challenging. An innovative approach is to engineer an orthogonal IL-2 (oIL-2) cytokine-receptor pair that selectively interacts with each other and transmits native IL-2 signals, but does not interact with the natural cytokine or receptor counterparts, thereby enabling selective stimulation of target cells in vivo. Here, we introduced this ortho cytokine-receptor combination into Treg with the goal of improving donor hematopoietic cell engraftment upon adoptive transfer in a murine mixed chimerism model to induce tolerance to a transplanted organ allograft. CD4+CD25hiFoxp3GFP+cells were isolated from Foxp3GFP-BALB/c mice. Purified Tregs were preactivated and transduced with the construct for murine oIL-2 receptor β chain (oIL-2Rβ) through a retroviral vector. Transduction efficiency was approximately 40%. Transduced oIL-2Rβ+Tregs (oTreg) were injected at 1 × 106 /mouse together with 15 × 106 bone marrow cells (BMCs) that were obtained from luciferase transgenic C57BL/6 donor mice into WT BALB/c recipient mice that received 3.3-Gy total body irradiation and anti-CD40L mAb (0.3 mg, ip on d0) as preconditioning. Recipients were treated with PBS, WT IL-2, or oIL-2 (both 25K IU/day) for 14 days. Without oTreg transfer, donor cells were rejected by d10 as measured by bioluminescence. Improvement of engraftment by oTreg transfer was not statistically significant without IL-2 injection (P = 0.13 compared to BMC + PBS group on d30). WT IL-2 injection increased CD8+T cells and resulted in rapid graft rejection. On the other hand, oIL-2 injection significantly increased %Foxp3GFP+ in CD4+T cells (PBS; 4.3±1.6%, oIL-2; 9.3±4.7%, each n = 10, P = 0.007) without increasing CD8+T cells. Consequently, donor cell engraftment was significantly improved in this group (P Our data indicates that selective IL-2 stimulation improves Treg potential for facilitation of engraftment and for the induction of transplantation tolerance. Cytokine receptor engineering could be a powerful option to manipulate target cells such as Treg in vivo.

Introduction Immune suppressive donor regulatory T cells (Tregs) can be expanded to prevent or treat graft-versus-host disease (GVHD). In human induced Tregs (iTregs), we demonstrated that inhibiting STAT3 phosphorylation (pSTAT3) with S3I-201 increases demethylated Foxp3 and significantly improves their suppressive potency in vitro, mitigating phenotypically plastic iTreg de-differentiation. Objectives To investigate whether human pSTAT3-inhibited iTregs 1) prevent GVHD and 2) maintain donor immunity against leukemia. Methods Our human skin/NSG mouse xenograft model is relevant to GVHD and permits preclinical testing of human pSTAT3-inhibited iTregs in vivo. NSG mice were transplanted with a 1cm2 human skin graft, followed by 5 × 106 allogeneic peripheral blood mononuclear cells with or without 1 × 105 iTregs, dendritic cell-expanded for 5 days with S3I-201 or DMSO. Results We now show human pSTAT3-inhibited iTregs prevent skin graft rejection by alloreactive T cells and spare the anti-leukemia activity of donor cytotoxic T lymphocytes (CTL) (Figure 1A-C). Compared to DMSO-treated iTregs, pSTAT3-inhibited iTregs upregulate skin-homing CLA, as well as immune suppressive IL-9, GARP, and PD-1 (P Conclusion We demonstrate that human pSTAT3-inhibited iTregs can prevent GVHD-mediated tissue damage, preserve anti-leukemia CTL function, and that metabolic reprogramming with coQ10 significantly enhances their suppressive potency. Based upon these data, we will clinically test the safety and efficacy of human pSTAT3-inhibited iTregs in GVHD prevention.

α‐defensin‐5 (HD5) accounts for 70% of the bactericidal peptide activity of Paneth cells and regulates gut microbial homeostasis. We previously reported that the G allele of rs4415345, a single nucleotide polymorphism (SNP) in the gene for HD5, was associated with approximately 40% lower risk of grade II-IV aGVHD (PMID: 30004111). Considering the role of microbiota disruptions in aGVHD, we hypothesized that rs4415345G may have a specific gut microbiota signature. First, we analyzed linked host-microbiota genetic data from 248 subjects in the Human Microbiome Project to test our hypothesis in healthy individuals. With the assumption that many microbial taxa have no association with a given SNP, we used 10-fold cross-validated Lasso regression as a test of association between rs4415345 and taxon relative abundance. A single taxon (Odoribacter splanchnicus) was retained in 99% of the simulations, always with a positive association with the G allele. Next, we evaluated whether O. splanchnicus is associated with less aGVHD in 226 patients with a 16S rRNA gene-sequenced research stool sample collected between days -30 and -6 of allo-HCT. O. splanchnicus was present in 65 (29%) patients. The univariate hazard ratio (HR) of O. splanchnicus detectability for grade II-IV aGVHD was 0.70, with a 95% confidence interval (95%CI) of 0.44-1.10 (P = 0.12). Multivariable analysis, adjusted for anti-anaerobic antibiotic exposure between days -7 and +14 yielded a similar result (HR: 0.70, 95%CI: 0.44-1.11, P = 0.13). In summary, the G allele of rs4610776 is associated with the higher relative abundance of O. splanchnicus in healthy individuals and less aGVHD in allo-HCT recipients. In an independent dataset, O. splanchnicus presence was associated with a 30% reduction in risk for aGVHD, though this association did not reach statistical significance. O. splanchnicus is a weakly butyrogenic bacterium also capable of producing indole, and depleted in patients with IBD. Both butyrate and indole can limit aGVHD. In addition, O. splanchnicus correlates with less TNFα production in response to inflammatory stimuli. We propose that O. splanchnicus may reduce aGVHD risk in rs4415345G individuals.

Background HSC dose and HLA match are risk factors that impact mortality using cord blood units (CBU) in transplant. Low CD34+ doses result in prolonged cytopenia and higher graft failure risk. A minimum cell dose of 3.0 × 107 total nucleated cells (TNC)/kg has generally been required in CBU selection; however, cell dose limits often require the use of a 2nd unit and markedly limits the availability of 7-8/8 HLA-matched units in larger patients. MGTA-456 is a cell therapy product utilizing an aryl hydrocarbon receptor antagonist for expansion of CD34+ HSCs in vitro. In prior studies with fresh MGTA-456, 36 patients with hematologic malignancies demonstrated rapid neutrophil recovery and 100% engraftment. This study (NCT03674411) will evaluate the safety and efficacy of cryopreserved MGTA-456 and the effectiveness of lowering the TNC threshold of the selected CBU to 1.0 × 107 TNC/kg before expansion to improve HLA match. Methods 13 patients aged 2-47 years (12-159 kg) with high-risk hematologic malignancy were enrolled with 11 transplanted to date. Patients received cyclophosphamide 120 mg/kg, fludarabine 75 mg/m2 and total body irradiation 1320 cGy or a busulfan-based regimen for children ≤3 years of age prior to MGTA-456 with cyclosporine/mycophenolate mofetil prophylaxis. G-CSF began the day after infusion until the neutrophil count exceeded 2500/uL for 3 days. Results MGTA-456 contained a median 2.3 × 109 CD34+ cells (range, 0.65-8.0) after expansion (422-fold expansion of CD34+ cells [range, 219-1313]). Neutrophil recovery occurred in all patients with a median of 15 days (range, 0-31), similar to recipients of fresh MGTA-456 in a prior study (median 14 days) and significantly faster than in recipients of unmodified CBUs (median 25 days). Median platelet recovery was 41 days (range 24-49) vs 64 days with unmodified CBUs. MGTA-456 CD34+CD90+ content strongly correlated with speed of neutrophil recovery (Fig. 1), consistent with preclinical murine data showing CD34+CD90+ cells represent the true engraftable HSC population. Lowering the TNC requirement to 1.0 × 107 TNC/kg for CBU selection pre-expansion improved HLA match and/or eliminated the need for double CBU transplant in all 5 patients weighing >80 kg. Three patients received 8/8 grafts who would have otherwise received 1 or 2 6/8 units (n=2) or two 7/8 units (n=1) which may account for the low incidence of aGVHD overall (3 cases of Grade 1-2 and no Grade 3-4). With a follow-up of 5.2 months (0.4-8.5 months), all patients are alive. Conclusion Cryopreserved MGTA-456 cell therapy resulted in rapid and 100% engraftment with speed of neutrophil recovery correlating with CD34+CD90+ cell dose in patients with high risk hematologic malignancy. Expansion of smaller CBUs increases the chance of finding a better HLA match, particularly for adults, thus reducing the barriers associated with low cell dose and poor HLA match in CBU transplantation.

Introduction cGVHD exhibits both autoimmune and fibrotic features across multiple organ systems. KD025 is an orally available Rho-associated coiled-coil kinase 2 (ROCK2) selective inhibitor. Methods KD025-208 enrolled 3 cohorts (C1: 200 mg QD, C2: 200 mg BID, and C3: 400 mg QD) of cGVHD patients (pts) after 1-3 prior lines of therapy. Treatment was until progression or toxicity. Primary endpoint is overall response rate (ORR) per 2014 NIH response criteria. Additional endpoints include Duration of Response (DOR), corticosteroid (CS) dose reductions, Failure Free Survival (FFS) and Lee Symptom Scale (LSS) score. Results 17, 16, and 21 pts enrolled in C1, C2, and C3. As of 8Mar19, median duration of follow up was 112, 97 and 64 weeks (wks) respectively. Median age was 52 yrs, median time from cGVHD diagnosis was 20 mos, and median prior lines of therapy was 2. Median duration of treatment was 37, 33, and 39 wks, respectively. 14 pts remained on KD025. Reasons for discontinuation: cGVHD progression (18), voluntary withdrawal (7), relapse of underlying disease (5), investigator decision (5), AE (3), and death (2). ORR [95% CI] was 65% [38, 85] in C1, 69% [41, 89] in C2, and 62% [38, 82] in C3, 65% [51, 77] across all 3 cohorts. Responses were achieved across key subgroups with ORRs of 62% (24/39) in pts with ≥2 prior lines of therapy, 70% (19/27) in pts with ≥4 organs involved and 60% (25/42) in pts with severe cGVHD, and 65% (22/34) in pts refractory to their previous line. CRs were observed in all affected organs except lung; PRs were observed in lung. Median Time to Response was 8.14 wks (IQR 8-12); 4/35 responses occurred after 24 wks. Responses were durable with a Kaplan-Meier median DOR of 34 wks across all cohorts. 57% of responders sustained a response for ≥20 wks. FFS at 6, 12 and 24 mos was 76%, 47% and 33% respectively. Overall survival was 94%, 91% and 83%. Baseline median CS dose was 0.21 mg/kg/day (prednisone eq). During treatment, median CS dose was reduced by 50%. 20% pts discontinued CS. 52% of pts reported a ≥7-point reduction in LSS with median time to improvement 9 wks. AEs were consistent with those expected in cGVHD pts receiving CS. Common AEs were URI 35%, diarrhea 31%, nausea 31%, fatigue 30%, dyspnea 28%, increased LFTs 24%, and peripheral edema 22%. 63% had a Grade ≥3 AE, the most common was dyspnea, 13%. 2% were dyspnea (7%), lung infection (6%), hypoxia (4%) and flu-like illness (4%). Three pts discontinued KD025 due to possibly related AEs (C1: diarrhea, headache; C3: fatigue). 3 pts died on study (C3: relapse of leukemia; lung infection; cardiac arrest), all considered unrelated to KD025. Conclusions Durable and clinically meaningful responses have been seen across all 3 cohorts. KD025 was well tolerated, with sustained responses and encouraging FFS in patients with advanced cGVHD.

Graft-versus-host disease (GvHD) remains a significant complication of allogeneic hematopoietic cell transplantation (HCT), associated with significant morbidity and mortality. GVHD is characterized by a dysregulated immune response with resulting tissue damage of target organs. Recent investigations have focused on Foxp3+ regulatory T cells (Tregs) as a therapeutic tool, based on its regulatory functions in GvHD pathogenesis and their instrumental role in mitigating GvHD severity while preserving graft-versus leukemia (GvL) activity. There are several challenges to its clinical application, including their paucity, impaired suppressive activity, and instability in vivo. Herein, we report that IL-27 pre-stimulation enhances suppressive functions to both mouse and human Tregs. In a complete MHC mismatched murine bone marrow transplant model, IL-27 pre-stimulated polyclonal iTregs diminish acute (a)GvHD lethality, while preserving the GvL effect. Allo-antigen specificity further improves suppressive functions when combined with IL-27 pre-stimulation. In a xenogeneic (human to mouse) GvHD model, IL-27 pre-stimulated human iTregs are superior in protecting recipients from GvHD. Lastly, we compared gene expression profiles of circulating Tregs isolated from HCT recipients with and without aGvHD and found that Tregs from aGvHD patients express distinct gene signatures enriched in immune activation and inflammation. Therefore, these results highlight a novel function of IL-27 in enforcing Treg functions to prevent aGvHD mediated lethality, proposing the hypothesis that dysregulated Treg functions may account for the potential mechanisms underlying GvHD development.

In vitro induced human regulatory T cells (iTregs) have demonstrated in vivo therapeutic utility, but pathways regulating their function have not been elucidated. Here, we report that human iTregs generated in vitro from naïve cord blood cells preferentially recruit Disc large homolog 1 (Dlgh1) and exclude protein kinase C (PKC)-θ from immunological synapses formed on supported lipid bilayers with laterally mobile ICAM-1 and anti-CD3 mAb. Also, iTregs display elevated Dlgh1 overall and Dlgh1-dependent p38 phosphorylation, higher levels of phosphatase and tensin homolog (PTEN), and diminished Akt phosphorylation. Pharmacological interruption of PKC-θ increases and Dlgh1 silencing decreases the ability of iTregs to suppress interferon-γ production by CD4

A VISTA on naïve T cell fate T cell quiescence and tolerance restrain the immune system from becoming overactive and attacking healthy tissue. Negative checkpoint regulators normally limit T cell responses to help safeguard against conditions such as autoimmunity. ElTanbouly et al. report that the checkpoint regulator VISTA (V-type immunoglobulin domain-containing suppressor of T cell activation) restricts early stages of T cell activation by shaping the inherent heterogeneity of the naïve CD4 + T cell compartment to one that is more uniformly quiescent and silent (see the Perspective by Brown and Rudensky). Therapeutic targeting of VISTA using an agonistic antibody in mice curbed the development of graft-versus-host disease and promoted the death of naïve T cells abnormally activated by self-antigen. VISTA thus represents a distinctive immunoregulatory molecule that controls naïve T cell function by maintaining quiescence and peripheral tolerance. Science , this issue p. eaay0524 ; see also p. 247

Immunosuppressive donor Tregs can prevent graft-versus-host disease (GVHD) or solid-organ allograft rejection. We previously demonstrated that inhibiting STAT3 phosphorylation (pSTAT3) augments FOXP3 expression, stabilizing induced Tregs (iTregs). Here we report that human pSTAT3-inhibited iTregs prevent human skin graft rejection and xenogeneic GVHD yet spare donor antileukemia immunity. pSTAT3-inhibited iTregs express increased levels of skin-homing cutaneous lymphocyte-associated antigen, immunosuppressive GARP and PD-1, and IL-9 that supports tolerizing mast cells. Further, pSTAT3-inhibited iTregs significantly reduced alloreactive conventional T cells, Th1, and Th17 cells implicated in GVHD and tissue rejection and impaired infiltration by pathogenic Th2 cells. Mechanistically, pSTAT3 inhibition of iTregs provoked a shift in metabolism from oxidative phosphorylation (OxPhos) to glycolysis and reduced electron transport chain activity. Strikingly, cotreatment with coenzyme Q10 restored OxPhos in pSTAT3-inhibited iTregs and augmented their suppressive potency. These findings support the rationale for clinically testing the safety and efficacy of metabolically tuned, human pSTAT3-inhibited iTregs to control alloreactive T cells.

Acute graft-versus-host disease (GVHD) is a life-threatening complication after allogeneic hematopoietic cell transplantation (allo-HCT). Although currently used GVHD treatment regimens target the donor immune system, we explored here an approach that aims at protecting and regenerating Paneth cells (PCs) and intestinal stem cells (ISCs). Glucagon-like-peptide-2 (GLP-2) is an enteroendocrine tissue hormone produced by intestinal L cells. We observed that acute GVHD reduced intestinal GLP-2 levels in mice and patients developing GVHD. Treatment with the GLP-2 agonist, teduglutide, reduced de novo acute GVHD and steroid-refractory GVHD, without compromising graft-versus-leukemia (GVL) effects in multiple mouse models. Mechanistically GLP-2 substitution promoted regeneration of PCs and ISCs, which enhanced production of antimicrobial peptides and caused microbiome changes. GLP-2 expanded intestinal organoids and reduced expression of apoptosis-related genes. Low numbers of L cells in intestinal biopsies and high serum levels of GLP-2 were associated with a higher incidence of nonrelapse mortality in patients undergoing allo-HCT. Our findings indicate that L cells are a target of GVHD and that GLP-2–based treatment of acute GVHD restores intestinal homeostasis via an increase of ISCs and PCs without impairing GVL effects. Teduglutide could become a novel combination partner for immunosuppressive GVHD therapy to be tested in clinical trials.

Despite graft-versus-host disease (GVHD) prophylactic agents, the success and wider utilization of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is limited by GVHD. Increasing donor graft regulatory T cell (Treg):effector T cell (Teff) ratios can substantially reduce GVHD in cancer patients, but pre-HSCT conditioning regimens and GVHD create a challenging inflammatory environment for Treg stability, persistence, and function. Metabolism plays a crucial role in T cell and Treg differentiation, and development of effector function. Although glycolysis is a main driver of allogeneic T cell-driven GVHD, oxidative phosphorylation is a main driver of Treg suppressor function. This review focuses on recent advances in our understanding of Treg metabolism in the context of GVHD, and discusses potential therapeutic applications of Tregs in the prevention or treatment of GVHD in cancer patients.

Acute graft-versus-host disease (aGVHD) is a potentially fatal complication of allogeneic hematopoietic cell transplantation that fails to improve with intense immunosuppression in some patients. We hypothesized that urinary-derived human chorionic gonadotropin (uhCG) could help facilitate resolution of life-threatening aGVHD when added as supportive care via 2 potential mechanisms: immunomodulation (akin to its role in pregnancy) and supplementation of epidermal growth factor (EGF; to aid in epithelial repair). In a phase 1 study, 26 participants received subcutaneous injections of uhCG in addition to standard immunosuppression (13 receiving initial therapy for high-risk aGVHD [according to the Minnesota criteria] and 13 receiving second-line therapy). Participants underwent serial blood testing for biomarkers of hormone response, immune modulation, and aGVHD activity on study. uhCG was well tolerated, with no dose-limiting toxicities. Sixty-two percent of patients in the high-risk cohort and 54% of patients in the second-line cohort had a complete response at study day 28. Plasma EGF was elevated sixfold (from 4 to 24 pg/mL; P = .02) at 6 hours postdose in the high-risk cohort, in contrast to no peak in plasma EGF in the more severe second-line cohort. After 1 week of uhCG, patients reported a twofold increase in the regulatory T cell to conventional T-cell ratio, suggesting immune modulation despite high-dose steroids. Responding patients reported significantly lower plasma amphiregulin and higher plasma butyrate levels at study completion, suggesting improvement in mucosal damage over time. uhCG is a novel, safe, supportive therapy, proceeding to phase 2 testing at 2000 units/m2 in high-risk aGVHD. This study was registered at www.clinicaltrials.gov as #NCT02525029.

Expression of programmed cell death protein 1 (PD-1) on natural killer (NK) cells has been difficult to analyze on human NK cells. By testing commercial clones and novel anti-PD-1 reagents, we found expression of functional PD-1 on resting human NK cells in healthy individuals and reconstituting NK cells early after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Peripheral blood samples from healthy individuals and transplant recipients were stained for PD-1 expression using the commercial anti-PD-1 clone PD1.3.1.3, fluorescein isothiocyanate (FITC)–labeled pembrolizumab, or an FITC-labeled single-chain variable fragment (scFv) reagent made from pembrolizumab. These reagents identified low yet consistent basal PD-1 expression on resting NK cells, a finding verified by finding lower PD-1 transcripts in sorted NK cells compared with those in resting or activated T cells. An increase in PD-1 expression was identified on paired resting NK cells after allo-HSCT. Blockade of PD-1 on resting NK cells from healthy donors with pembrolizumab did not enhance NK function against programmed death-ligand 1 (PD-L1)–expressing tumor lines, but blocking with its scFv derivative resulted in a twofold increase in NK cell degranulation and up to a fourfold increase in cytokine production. In support of this mechanism, PD-L1 overexpression of K562 targets suppressed NK cell function. Interleukin-15 (IL-15) activity was potent and could not be further enhanced by PD-1 blockade. A similar increase in function was observed with scFv PD-1 blockade on resting blood NK cells after allo-HSCT. We identify the functional importance of the PD-1/PD-L1 axis on human NK cells in which blockade or activation to overcome inhibition will enhance NK cell–mediated antitumor control.

As regulatory T cell (Treg) adoptive therapy continues to develop clinically, there is a need to determine which immunomodulatory agents pair most compatibly with Tregs to enable persistence and stabilize suppressor function. Prior work has shown that mechanistic target of rapamycin (mTOR) inhibition can increase stability of thymic Tregs. In this study we investigated the transcriptomic signatures of ex-vivo expanded Tregs after adoptive transfer in the setting of clinically relevant immunosuppression using a non-human primate (NHP) model as a prelude to future transplant studies. Here, we found that adding interleukin-2 (IL2) to rapamycin in vivo supported a logarithmic increase in the half-life of adoptively transferred CFSE-labeled, autologous NHP Tregs, effectively doubling the number of cells in the peripheral blood Treg compartment compared to Treg infusion with rapamycin alone. Using single cell transcriptomics, we found that transferred ex-vivo expanded Tregs initially exhibit a gene expression signature consistent with an activated state. Moreover, those cells with the highest levels of activation also expressed genes associated with p53-mediated apoptosis. In contrast, transferred Tregs interrogated at Day +20 post-transfer demonstrated a gene signature more similar to published profiles of resting Tregs. Together, these preclinical data further support combining IL2 and rapamycin in vivo as adjunctive therapy for ex-vivo expanded adoptively transferred Tregs and suggest that the activation status of ex-vivo expanded Tregs is critical to their persistence.

B7-H4 is a negative regulatory B7 family member. We investigated the role of host and donor B7-H4 in regulating acute graft-versus-host disease (GVHD). Allogeneic donor T cells infused into B7-H4(–/–) versus WT recipients markedly accelerated GVHD-induced lethality. Chimera studies pointed toward B7-H4 expression on host hematopoietic cells as more critical than parenchymal cells in controlling GVHD. Rapid mortality in B7-H4(–/–) recipients was associated with increased donor T cell expansion, gut T cell homing and loss of intestinal epithelial integrity, increased T effector function (proliferation, proinflammatory cytokines, cytolytic molecules), and reduced apoptosis. Higher metabolic demands of rapidly proliferating donor T cells in B7-H4(–/–) versus WT recipients required multiple metabolic pathways, increased extracellular acidification rates (ECARs) and oxygen consumption rates (OCRs), and increased expression of fuel substrate transporters. During GVHD, B7-H4 expression was upregulated on allogeneic WT donor T cells. B7-H4(–/–) donor T cells given to WT recipients increased GVHD mortality and had function and biological properties similar to WT T cells from allogeneic B7-H4(–/–) recipients. Graft-versus-leukemia responses were intact regardless as to whether B7-H4(–/–) mice were used as hosts or donors. Taken together, these data provide new insights into the negative regulatory processes that control GVHD and provide support for developing therapeutic strategies directed toward the B7-H4 pathway.

Oncogenic Ras mutations occur in various leukemias. It was unclear if, besides the direct transforming effect via constant RAS/MEK/ERK signaling, an inflammation-related effect of KRAS contributes to the disease. Here, we identify a functional link between oncogenic KrasG12D and NLRP3 inflammasome activation in murine and human cells. Mice expressing active KrasG12D in the hematopoietic system developed myeloproliferation and cytopenia, which is reversed in KrasG12D mice lacking NLRP3 in the hematopoietic system. Therapeutic IL-1-receptor blockade or NLRP3-inhibition reduces myeloproliferation and improves hematopoiesis. Mechanistically, KrasG12D-RAC1 activation induces reactive oxygen species (ROS) production causing NLRP3 inflammasome-activation. In agreement with our observations in mice, patient-derived myeloid leukemia cells exhibit KRAS/RAC1/ROS/NLRP3/IL-1β axis activity. Our findings indicate that oncogenic KRAS not only act via its canonical oncogenic driver function, but also enhances the activation of the pro-inflammatory RAC1/ROS/NLRP3/IL-1β axis. This paves the way for a therapeutic approach based on immune modulation via NLRP3 blockade in KRAS-mutant myeloid malignancies., Oncogenic Ras mutations are common drivers in myeloid leukemia. Here, the authors show in patient cells and in mice that oncogenic K-Ras activates NLRP3 inflammasome to drive myeloproliferation, which can be reversed by genetic or pharmacologic NLRP3 blockade.

Steroid-refractory intestinal acute graft-versus-host disease (aGVHD) is a frequently fatal condition, with little known about mechanisms driving failed steroid responses in gut mucosa. To uncover molecular insights in steroid-refractory aGVHD, we compared gene expression profiles of rectosigmoid biopsies from patients at diagnosis of clinical stage 3–4 lower intestinal aGVHD with repeat biopsies when the patients became steroid refractory and normal controls. We also performed single-gene analyses of factors associated with tolerance (programmed cell death ligand 1 [PDL1], indoleamine 2,3-dioxygenase [IDO1], and T cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibition motif domains [TIGIT]) and found that significantly higher expression levels of these aGVHD inhibitory genes (PDL1, IDO1, TIGIT) at aGVHD onset became decreased in the steroid-refractory state. We examined genes triggered by microbial ligands to stimulate gut repair, amphiregulin (AREG) and the aryl hydrocarbon receptor (AhR), and found that both AREG and AhR gene expression levels were increased at aGVHD onset and remained elevated in steroid-refractory aGVHD. We also identified higher expression levels of metallothioneins, metal-binding enzymes induced in stress responses, and M2 macrophage genes in steroid-refractory aGVHD. We observed no differences in T cell subsets between onset and steroid-refractory aGVHD. Patients with a rapidly fatal course showed greater DNA damage and a distinct microbial signature at aGVHD onset, whereas patients with more prolonged survival exhibited a gene expression profile consistent with activation of Smoothened. Our results extend the paradigm beyond T cell–centric therapies for steroid-refractory gastrointestinal aGVHD and highlight potentially new mechanisms for therapeutic exploration.

The inclusion of in vitro derived T cell progenitor (proT) therapy with hematopoietic stem cell transplant (HSCT) aids in the recovery of the thymus damaged by total body irradiation and improves de novo thymopoiesis. To understand the interaction between proTs and the thymic microenvironment, wildtype (WT) mice were lethally irradiated and given T cell deficient donor (Rag1−/−) marrow along with in vitro generated proT from WT donors, limiting mature T cell development to infused proT. Donor proTs within the host thymus led to a significant increase in thymic epithelial cell (TEC) numbers by day 21 post-transplant, and increased actively cycling TECs as measured by Ki67 expression and BrdU uptake. However, that gain was temporary and lost by day 28, suggesting that continued signaling from proT cells is required to sustain TEC cycling and mass. We also find a significant improvement in total thymocyte number by day 21 followed by a significant increase in the total mature T cell number in the secondary lymphoid organs by day 28. This protective surge is also temporary, receding by day 60. In this time period, infused DN2 proTs selectively increased thymocyte number while DN3 proTs preferentially led to a greater TEC numbers. Interestingly, an exception in persistence occurs when DN3 proTs are used and the increase in mature T cells in the spleen persists at day 60. As a result of the lack of competition for thymic niches by cells from the Rag1−/− graft, a subpopulation of the infused proT persisted in the thymus in an immature state at day 60. These findings highlight the importance of the interaction between developing T cells and TECs in the proliferation and survival of these critical components of the thymic microenvironment.

Summary How cells adapt metabolism to meet demands is an active area of interest across biology. Among a broad range of functions, the polyamine spermidine is needed to hypusinate the translation factor eukaryotic initiation factor 5A (eIF5A). We show here that hypusinated eIF5A (eIF5AH) promotes the efficient expression of a subset of mitochondrial proteins involved in the TCA cycle and oxidative phosphorylation (OXPHOS). Several of these proteins have mitochondrial targeting sequences (MTSs) that in part confer an increased dependency on eIF5AH. In macrophages, metabolic switching between OXPHOS and glycolysis supports divergent functional fates stimulated by activation signals. In these cells, hypusination of eIF5A appears to be dynamically regulated after activation. Using in vivo and in vitro models, we show that acute inhibition of this pathway blunts OXPHOS-dependent alternative activation, while leaving aerobic glycolysis-dependent classical activation intact. These results might have implications for therapeutically controlling macrophage activation by targeting the polyamine-eIF5A-hypusine axis., Graphical Abstract, Highlights • The polyamine synthesis pathway and hypusinated eIF5A modulate mitochondrial OXPHOS • Hypusinated eIF5A maintains TCA cycle and ETC integrity in macrophages • Some mitochondrial enzymes depend on eIF5AH for efficient expression • Inhibition of hypusinated eIF5A blunts macrophage alternative activation, Puleston et al. show that polyamine biosynthesis modulates mitochondrial metabolism through eIF5A hypusination (eIF5AH). They find that inhibiting the polyamine-eIF5A-hypusine pathway blocks OXPHOS-dependent macrophage alternative activation, while leaving aerobic glycolysis-dependent macrophage classical activation intact. These results might have implications for therapeutically controlling macrophage activation by targeting the polyamine-eIF5A-hypusine axis.

BACKGROUND: Stromal laminins α4 and α5 are differentially regulated in transplant tolerance and immunity, respectively, resulting in altered T cell trafficking. We hypothesized laminins directly regulated T cell activation and polarization. METHODS: Human and mouse CD4 T cells were activated in Th1, Th2, Th17 or Treg environments with/without laminin α4 and/or α5. Laminin α5 receptors were blocked with anti-α6 integrin or anti-α dystroglycan mAbs, and T cell polarization determined. TCR transgenic TEa CD4 cells that recognized donor alloantigen were transferred into C57BL/6 mice that received alloantigen or cardiac allografts. Laminin receptors were blocked, and TEa T cell migration and differentiation were assessed. Laminin expression was measured in several models of immunity and tolerance. RESULTS: In diverse models, laminins α4 and α5 were differentially regulated. Immunity was associated with decreased laminin α4:α5 ratio, while tolerance was associated with an increased ratio. Laminin α4 inhibited CD4+ T cell proliferation and Th1, Th2 and Th17 polarization, but favored Treg induction. Laminin α5 favored T cell activation and Th1, Th2, and Th17 polarization; and inhibited Treg. Laminin α5 was recognized by T cell integrin α6 and important for activation and for inhibition of Treg. Laminin α5 was also recognized by T cell α-dystroglycan and required for Th17 differentiation. Anti-α6 integrin or anti-α-dystroglycan prolonged allograft survival. CONCLUSIONS: Laminins α4 and α5 are, respectively, co-inhibitory and co-stimulatory ligands for human and mouse CD4 T cells. Laminins and their receptors modulate immune responses by acting as one of the molecular switches for immunity or suppression.