Abstract Introduction TGFβ−secreting, CD206+ profibrotic macrophages (macs) are key chronic graft-versus-host disease (cGVHD) drivers. Global depletion of macs could compromise protective immunity; therefore, we targeted CD206 + (mannose receptor) macs with mannosylated albumin nanoparticles (MANPs) containing TGFβ siRNA or non-targeting (NT) siRNA for control. A published bleomycin pulmonary fibrosis model showed disease prevention post TGFβ-MANPs internalization by CD206+ monocyte-derived alveolar macs. CD206+ TGFβ+ macs are also critical in sclerodermatous (Scl) GVHD (B10D2>Balb/c). We show that mice with established multiorgan cGVHD/bronchiolitis obliterans (BO) exhibit a progressive increase in lung tissue profibrotic macs (d49 vs. d28) highlighting their pathogenic role. Methods In our cGVHD/BO model, B10.BR recipients receive B6 bone marrow (BM) and a low dose of T cells (control = BM only). Co-culture of CD206 + (M2-like) and CD206neg (M1-like) macs confirmed that MANPs were preferentially internalized by CD206+ macs and effectively reduced TGFβ protein and mRNA with no effect on CD206neg macs. In vivo, MANPs are delivered intravenously 2x/week starting at d28 post transplantation. Results Compared to BM only controls, d49 cGVHD/BO mice had a significant increase (1.5-fold) in lung CD206+ macs frequency (p = 0.0010) and these cells had a 2.5-fold increase in TGFβ expression (p = 0.0070) by flow cytometry fluorescent intensity. TGFβ siRNA vs NT siRNA—loaded MANPs delivered i.v. to cGVHD/BO mice improved lung function to levels comparable to BM controls (n = 10/group): resistance (p < 0.0001), elastance (p < 0.05), compliance (p < 0.05). Early studies using MANP-based delivery of TGFβ siRNA in Scl GVHD improved skin scores (p < 0.0001) compared to NT siRNA delivery. Further studies with MANPs in Scl GVHD are ongoing. Conclusion These results identify CD206+ macs as a tractable therapeutic target with high selectivity and highlight MANP-based siRNA delivery as a promising strategy to limit cGVHD-driven pulmonary fibrosis. Funding Source NIH P01 HL158505 Topic Categories Transplantation Immunology (TRAN)

Abstract Introduction Chronic graft-versus-host disease (cGVHD) is a debilitating complication of allogenic hematopoietic stem cell transplantation (alloHSTC) characterized by immune dysregulation, autoimmunity, and fibrosis. Particularly devastating is scleroderma (Scl), a systemic sclerotic disease that targets the skin causing stiffness and delayed wound healing. Despite FDA approved therapies, complete responses in Scl-cGVHD are rare, highlighting an unmet need for more targeted therapies. Methods To delineate the inflammatory and profibrotic mechanisms underlying Scl disease, we used two distinct murine alloHSCT model in which hosts were lethally irradiated and given bone marrow (BM) ± T cells to induce cGVHD. We compared multiple minor antigen mismatch, B10.D2 into Balb/c to a semi-allogeneic, parent-into-F1 C57BL/6 (B6) into (B6D2) F1 models. Results Mice developed scleroderma signs at different rates facilitating the study of overlapping and diverging fibrosis mechanisms. Mice in the B10.D2into Balb/c model had visible skin lesions by d19 with immune cell infiltration seen as early as d3. In contrast, mice in the parent-into-F1 model had comparatively delayed Scl onset with visible lesions appearing ∼d35. In the B10.D2 into Balb/c model, a 20-fold increase in the frequency of IFNg producing CD4 conventional T cells (Tcon) as compared to BM controls was apparent by d21 (p = 0.0119) with very little IL-17 production (< 0.04% IL-17+ of Foxp3-CD4+ Tcon). Conversely, mice in the parent-into-F1 Scl-GVHD model had a strong signature of IL-17 production, with 60% of CD4 Tcon producing IL-17 at d21 (p = 0.0395). To begin to uncover Scl mechanisms, Scl and BM control skin biopsies (d3, 6, 9, 13 and 21; n = 3 mice/group/time) in the B10.D2into BALB/c model are being analyzed by Nanostring GeoMx along with multiplex immunofluorescence of skin from both models and will be presented. Conclusion Together, our data highlight the need for comprehensive studies of cGVHD/Scl models to develop improved translational therapeutics. Funding Source National Institutes of Health (grants P01 HL158505, P01 AI056299, and R37AI34495) Topic Categories Transplantation Immunology (TRAN)

Intestinal dysbiosis has been associated with both the effectiveness and toxicity of immunotherapy in cancer patients, inspiring multiple trials investigating fecal microbiota transplantation (FMT) in these patients. FMT restores microbial community structures damaged by antibiotics and enriches the microbiota with beneficial bacteria. However, the precise mechanism through which FMT exerts its effects and provides clinical benefits remains incompletely understood. Efforts to date have primarily focused on characterizing taxonomic changes following FMT. We hypothesized that FMT may also modify the functional pathways and metabolic capabilities of the gut microbiota, with possible clinical impact. To investigate this, we conducted a study involving 17 patients with blood disorders who received prophylactic FMT from one of the three healthy donors shortly after hematopoietic cell transplantation (HCT). By analyzing shotgun metagenomic profiles of the baseline, pre-FMT, and post-FMT gut microbiota, we demonstrate that FMT effectively restored pathways that had been depleted following HCT. However, it did not significantly reduce pathways that had expanded, indicating that FMT operates primarily through a restorative mechanism, reestablishing lost functional capabilities in the microbiota rather than suppressing overactive pathways. These findings highlight the potential for optimizing FMT protocols and identifying patient populations where FMT may be particularly beneficial.

BACKGROUND:Allogeneic peripheral-blood stem-cell transplantation (SCT) from a matched related donor after myeloablative conditioning is the preferred curative treatment for patients with high-risk blood cancers. The combination of a calcineurin inhibitor and an antimetabolite remains standard care for graft-versus-host disease (GVHD) prophylaxis in these patients. Data from two randomized trials have suggested that post-transplantation cyclophosphamide can reduce the risk of GVHD after SCT from a matched donor when it is added to or replaces the antimetabolite. However, the effects of post-transplantation cyclophosphamide specifically after SCT from a matched related donor remain uncertain, and effects in the context of myeloablative conditioning are unclear. METHODS:We randomly assigned adults who were undergoing SCT from a matched related donor after myeloablative or reduced-intensity conditioning to receive either post-transplantation cyclophosphamide-cyclosporin (experimental prophylaxis) or cyclosporin-methotrexate (standard prophylaxis). The primary end point was GVHD-free, relapse-free survival. RESULTS:Among 134 patients who underwent randomization, 66 were assigned to receive experimental prophylaxis and 68 to receive standard prophylaxis. GVHD-free, relapse-free survival was significantly longer with experimental prophylaxis (median, 26.2 months; 95% confidence interval [CI], 9.1 to not reached) than with standard prophylaxis (median, 6.4 months; 95% CI, 5.6 to 8.3; P<0.001 by a log-rank test). GVHD-free, relapse-free survival at 3 years was 49% (95% CI, 36 to 61) with experimental prophylaxis and 14% (95% CI, 6 to 25) with standard prophylaxis (hazard ratio for GVHD, relapse, or death, 0.42; 95% CI, 0.27 to 0.66). The cumulative incidence of grade III to IV acute GVHD at 3 months was 3% (95% CI, 1 to 10) in the experimental-prophylaxis group and 10% (95% CI, 4 to 19) in the standard-prophylaxis group. At 2 years, overall survival was 83% and 71%, respectively (hazard ratio for death, 0.59; 95% CI, 0.29 to 1.19). The incidence of serious adverse events was similar in the two groups in the first 100 days after SCT. CONCLUSIONS:The combination of post-transplantation cyclophosphamide and a calcineurin inhibitor led to longer GVHD-free, relapse-free survival than standard prophylaxis after transplantation from a matched related donor with either reduced-intensity or myeloablative conditioning in patients with blood cancers. (Funded by the Australian Government Medical Research Future Fund and others; ALLG BM12 CAST Australian-New Zealand Clinical Trials Registry number, ACTRN12618000505202.).

Gut microbiota disruptions after allogeneic hematopoietic cell transplantation (alloHCT) are associated with increased risk of acute graft-versus-host disease (aGVHD). We designed a randomized, double-blind placebo-controlled trial to test whether healthy-donor fecal microbiota transplantation (FMT) early after alloHCT reduces the incidence of severe aGVHD. Here, we report the results from the single-arm run-in phase which identified the best of 3 stool donors for the randomized phase. The primary and key secondary endpoints were microbiota engraftment and severe aGVHD, respectively. Three cohorts of patients (20 total) received FMT, each from a different donor. FMT was safe and effective in restoring microbiota diversity and commensal species. Microbiota engraftment, determined from shotgun sequencing data, correlated with larger microbiota compositional shifts toward donor and better clinical outcomes. Donor 3 yielded a median engraftment rate of 66%, higher than donors 1 (P = 0.02) and 2 (P = 0.03) in multivariable analysis. Three patients developed severe aGVHD; all 3 had received FMT from donor 1. Donor 3 was selected as the sole donor for the randomized phase. Our findings suggest a clinically relevant donor effect and demonstrate feasibility of evidence-based donor selection. FMT is a holistic microbiota restoration approach that can be performed as a precision therapeutic. ClinicalTrials.gov identifier NCT06026371 Here, in 3 cohorts of allogeneic stem cell transplant recipients, each receiving FMT from 1 of 3 stool donors for graft-versus-host disease prophylaxis, the authors show that microbiota and clinical outcomes are associated with the specific donor used, suggesting a donor effect with implications for FMT donor selection.

ABSTRACT:Chronic graft-versus-host disease (cGVHD) remains the leading cause of nonrelapse morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). Effective therapeutic agents targeting dysregulated cytokines including interleukin-17 (IL-17) and colony-stimulating factor 1 (CSF-1) have been defined in preclinical models of cGVHD, and efficacy in subsequent clinical trials has led to their recent US Food and Drug Administration approval. Despite this, these agents are effective in only a subset of patients, expensive, difficult to access outside the United States, and used in a trial-and-error fashion. The ability to readily discern druggable, dysregulated immunity in these patients is desperately needed to facilitate the selection of appropriate treatment and to potentially identify high-risk individuals for preemptive therapy. We used single-cell sequencing-based approaches in our informative preclinical cGVHD models to "reverse engineer" temporal IL-17 and CSF-1 signatures in mouse blood that could be used to interrogate patients. We defined distinct, nonintuitive IL-17 and CSF-1 signatures in mouse blood monocytes that could be identified in relevant monocyte populations within 70% of patients at diagnosis of cGVHD and in half of patients at day +100 after HCT who subsequently developed cGVHD. These signatures can now be evaluated prospectively in clinical studies to help delineate potential responder and nonresponders to relevant therapeutics targeting these pathways.

Chronic graft-versus-host disease (cGVHD) is the leading cause of morbidity and non-relapse associated mortality following allogeneic hematopoietic cell transplantation (aHSCT). Treating steroid resistant/refractory cGVHD remains challenging. Epigenetic regulators can have global transcriptional effects that control donor T-cell responses. We previously showed that inhibiting histone lysine motifs by chromatin-modifying enzymes can ameliorate murine cGVHD. Targeting donor T-cell DNA methyltransferases reduce acute GVHD. Here, we sought to investigate the DNA demethylase Tet (ten-eleven translocase) methylcytosine dioxygenases 2 (Tet2) and Tet3 in T follicular helper cell (TFH) dependent cGVHD. In a clinically relevant model of cGVHD that recapitulates pulmonary fibrosis from bronchiolitis obliterans, recipients of Tet2 deleted donor T-cells did not have improved pulmonary function tests in contrast to the markedly improved pulmonary function in Tet3 deleted donor T-cells. Tet3 deleted donor T-cells did not impair TFH-dependent germinal center (GC) formation. Unexpectedly, TET3 deficiency resulted in elevated GATA3 expression in and IL-4 production by TFH cells. TET3 deficient TFH cells supported GC B-cell immunoglobulin (Ig) class switching to nonpathogenic IgG1 but not pathogenic IgG2c allowing mice to escape cGVHD pulmonary fibrosis. Elevated GATA3 expression and disruption of IgG2c class switching was recapitulated in an in-vitro human GC culture system. These studies provide new insights into the function of Tet3 in TFH driven Ig class switching and suggest a new approach to mitigate cGVHD.

In 2020, the third NIH Consensus Development Project on Criteria for Chronic Graft-versus-Host Disease (GVHD) Clinical Trials was held with the goals of identifying gaps in understanding, prevention and treatment of chronic graft-versus-host disease (GVHD) and making actionable recommendations that would advance the field. An interim meeting was held in October 2024 to review progress on the 2020 recommendations. Each group was charged with reviewing their previous recommendations, assessing whether the field is on track to eventually achieve the goals, and considering whether recommendations should be modified in light of new data or insufficient progress. This manuscript summarizes the Working Groups' reports and helps define the research agenda for future studies in chronic GVHD. Overall, modest progress has been made on most initiatives. Some studies in progress will address key recommendations and results are eagerly anticipated.

Chimeric antigen receptor (CAR) T cells and bispecific antibodies targeting B-cell maturation antigen (BCMA) have significantly advanced the treatment of relapsed and refractory multiple myeloma. Resistance to BCMA-targeting therapies, nonetheless, remains a significant challenge. BCMA shedding by gamma- secretase is a known resistance mechanism, and preclinical studies suggest that inhibition may improve anti-BCMA therapy. Leveraging a phase 1 clinical trial of the gamma- secretase inhibitor (GSI), crenigacestat, with anti-BCMA CART cells (FCARH143), we used single-nuclei RNA sequencing and assay for transposase-accessible chromatin sequencing to characterize the effects of GSI on the tumor microenvironment. The most significant impacts of GSI involved effects on monocytes, which are known to promote tumor growth. In addition to observing a reduction in the frequency of nonclassical monocytes, we also detected significant changes in gene expression, chromatin accessibility, and inferred cell-cell interactions after exposure to GSI. Although many genes with altered expression are associated with gamma- secretase - dependent signaling, such as Notch, other pathways were affected, indicating GSI has farreaching effects. Finally, we detected monoallelic deletion of the BCMA locus in some patients with prior exposure to anti-BCMA therapy, which significantly correlated with reduced progression-free survival (PFS; median PFS, 57 vs 861 days). GSIs are being explored in combination with the full spectrum of BCMA-targeting agents, and our results reveal widespread effects of GSI on both tumor and immune cell populations, providing insight into mechanisms for enhancing BCMA-directed therapies.

Interleukin 10 (IL-10)-producing CD4+ type-1 regulatory T cells (Tr1) promote immune tolerance during chronic infection, autoimmunity, and transplantation. However, specific Eomesodermin (Eomes)-dependent stages of Tr1 differentiation and function remain unclear. Using preclinical models of bone marrow transplantation (BMT), we demonstrated a Tr1 differentiation trajectory in vivo from Eomes+IL-10- to Eomes+IL-10+ subsets with the acquisition of cytokine, cytolytic, and exhaustion features. The Eomes+CD4+ fraction represented the dominant cytotoxic subset after BMT, mediating graft-versus-leukemia effects while limiting inflammation. In CD19-targeted chimeric antigen receptor (CAR) T cell immunotherapy, Eomes drove the same CD4+ Tr1 phenotype that controlled cytolysis, while mitigating immune toxicity and promoting persistence. In individuals with high-grade B cell lymphomas that had long-term disease control after receiving commercial CD19-targeted CAR T cells, Eomes+ Tr1 cells represented a stable population comprising 40%-80% of the CD4+ CAR T cell population. Hence, Eomes controls both regulatory and cytotoxic programs in CD4+ T cells, essential for curative immunotherapy outcomes.