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Supplementary S1: PLZF expression affects CD4+ driven alloreactivity. Supplementary S2: PLZF-TG T cells undergo allo-activation that is similar to donor B6-WT T cells. Supplementary S3: Fewer deaths from GVHD are seen in BMT recipients of PLZF-TG T cells compared to the BMT recipients of B6-WT T cells.

Introduction: Graft-versus-host-disease (GVHD) arises from the inflammatory cascade triggered by alloreactive T cell following allogeneic hematopoietic cell transplantation (allo-HCT). The tissues most frequently involved by GVHD include the small and large intestines, skin, and liver. Prior studies of T cell receptor (TCR) sequencing in diagnostic biopsies and blood have identified dominant T cell clones in GVHD-affected tissues that were not abundant in circulation or shared across multiple patients, but this has not been studied in sites poorly accessible to biopsy nor in lymphoid tissues in humans. We hypothesized that the GVHD-affected tissues have distinct TCR repertoires reflecting the differential expression of allo-antigens at different anatomic sites. Methods: We performed rapid autopsies on patients whose post allo-HCT course was complicated by GVHD to profile the TCR repertoire in tissues inaccessible to biopsy. Tissues were obtained from seven patients (HLA-identical allografts with a variety of graft sources and GVHD-prophylaxis regimens), all with active GVHD and/or on immunosuppression for GVHD control. Spleen, liver, skin, and multiple sites along the gastrointestinal (GI) tract were sampled, when possible, from the esophagus to the rectum. When available, blood and bone marrow mononuclear cells were also viably preserved. T cell receptors were sequenced from 38 different snap-frozen tissues from five patients via genomic DNA based next-generation sequencing of the TCR-beta CDR3 (ImmunoSeq, Adaptive Biotechnologies). Four out of five subjects sequenced had skin and GI GVHD, one only GI. In parallel, TCRs were sequenced from GVHD-affected tissues from a major and minor mouse model of GVHD: BALB/c 7-14 days after HCT with C57BL/6 T cells and C57BL/6 mice > 30 days after HCT with LP/J T cells. Results: Sequences representing 264,678 productive TCRs were recovered from the human autopsy samples with over 100 unique clones for nearly all tissues (mean 1539; standard deviation 1626). We found virtually no TCR clones defined by nucleotide sequence shared across patients, even in the context of shared HLA haplotypes (4/5 patients shared HLA-A*02-01). This is consistent with prior studies of diagnostic biopsies describing a paucity of clones shared across patients. We observed greater repertoire overlap between sites within the GI tract compared to other tissues in a given patient, as measured by the Jensen Shannon Diversity (JSD) index, especially compared to the skin, another GVHD-affected tissue. Despite differences in global repertoires across tissues, some clones were shared across all samples for a given patient, with at least one clone present among the top twenty clones by frequency for each tissue with sufficient sampling. A strikingly similar pattern of repertoire sharing across tissues was observed in the TCR repertoires of a major and minor mouse model of GVHD. While tissues within the GI tract and mesenteric lymph nodes shared the most clones in the mice, there were also dominant clones shared across tissues outside the GI tract. Individual mice with GVHD had highly divergent repertoires from each other in spite of the markedly controlled setting including inbred mice with the same donor T cell pool for each transplant, consistent with the notion that T cells that mediate GVHD may be present at very low frequency in the pool of naïve T cells in the allograft. Conclusion: This work characterizes T cell repertoire in GVHD in human tissues that have not been previously available, including lymphoid tissues and small intestine samples. Combined with the mouse data, this study reveals the relationships between TCR repertoires across different tissues, highlighting the striking diversity of clones driving the GVHD process across tissues and individuals. Although some even dominant clones are shared across all tissues within an individual, each sampled site had a tissue-specific clonal composition. While GVHD-directed therapy focuses primarily on the inhibition of the T cells themselves, additional attention must be devoted to understanding the pattern of target tissue-specific antigen expression in order to identify the key drivers of GVHD. Disclosures Giardina: Seres Therapeutics: Other: salary support. Slingerland:Seres Therapeutics: Other: salary support. Jenq:Kaleido Biosciences: Membership on an entity's Board of Directors or advisory committees; Karius Dx: Speakers Bureau; Merck: Consultancy; MicrobiomeDx: Consultancy; Prolacta: Consultancy; Seres Therapeutics: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: January 1, 2040. Giralt:Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; PFIZER: Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees, Research Funding; Actinuum: Other: Advisory board, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Other: Advisory Board, Research Funding; KITE: Membership on an entity's Board of Directors or advisory committees; SPECTRUM Pharma: Membership on an entity's Board of Directors or advisory committees; Jensenn: Membership on an entity's Board of Directors or advisory committees, Research Funding; JAZZ Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; MILTENYI: Research Funding; TAKEDA: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; OMEROS: Research Funding. Perales:Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Omeros: Honoraria, Membership on an entity's Board of Directors or advisory committees; Medigene: Membership on an entity's Board of Directors or advisory committees, Other; Celgene: Honoraria; Bellicum: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotec: Research Funding; Kite/Gilead: Honoraria, Research Funding; Incyte Corporation: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; MolMed: Membership on an entity's Board of Directors or advisory committees; NexImmune: Membership on an entity's Board of Directors or advisory committees; Cidara Therapeutics: Other; Nektar Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy, Honoraria; Servier: Membership on an entity's Board of Directors or advisory committees, Other; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Iacobuzio-Donahue:BMS: Research Funding. van den Brink:Seres Therapeutics: Consultancy, Patents & Royalties, Research Funding; DKMS Medical Council: Membership on an entity's Board of Directors or advisory committees; Forty-Seven, Inc: Consultancy; Juno Therapeutics: Patents & Royalties; WindMIL Therapeutics: Honoraria; Rheos: Honoraria; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Magenta: Honoraria; Kite Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Peled:Seres Therapeutics: Patents & Royalties, Research Funding; Davolterra: Consultancy.

Chronic graft-versus-host disease (GVHD) can be associated with significant morbidity, in part because of nonreversible fibrosis, which impacts physical functioning (eye, skin, lung manifestations) and mortality (lung, gastrointestinal manifestations). Progress in preventing severe morbidity and mortality associated with chronic GVHD is limited by a complex and incompletely understood disease biology and a lack of prognostic biomarkers. Likewise, treatment advances for highly morbid manifestations remain hindered by the absence of effective organ-specific approaches targeting “irreversible” fibrotic sequelae and difficulties in conducting clinical trials in a heterogeneous disease with small patient numbers. The purpose of this document is to identify current gaps, to outline a roadmap of research goals for highly morbid forms of chronic GVHD including advanced skin sclerosis, fasciitis, lung, ocular and gastrointestinal involvement, and to propose strategies for effective trial design. The working group made the following recommendations: (1) Phenotype chronic GVHD clinically and biologically in future cohorts, to describe the incidence, prognostic factors, mechanisms of organ damage, and clinical evolution of highly morbid conditions including long-term effects in children ; (2) Conduct longitudinal multicenter studies with common definitions and research sample collections ; (3) Develop new approaches for early identification and treatment of highly morbid forms of chronic GVHD, especially biologically targeted treatments, with a special focus on fibrotic changes ; and (4) Establish primary endpoints for clinical trials addressing each highly morbid manifestation in relationship to the time point of intervention (early versus late). Alternative endpoints, such as lack of progression and improvement in physical functioning or quality of life, may be suitable for clinical trials in patients with highly morbid manifestations. Finally, new approaches for objective response assessment and exploration of novel trial designs for small populations are required.

Summary Interleukin-22 (IL-22) acts on epithelial cells to promote tissue protection and regeneration, but can also elicit pro-inflammatory effects, contributing to disease pathology. Here, we engineered a high-affinity IL-22 super-agonist that enabled the structure determination of the IL-22–IL-22Rα–IL-10Rβ ternary complex to a resolution of 2.6 A. Using structure-based design, we systematically destabilized the IL-22–IL-10Rβ binding interface to create partial agonist analogs that decoupled downstream STAT1 and STAT3 signaling. The extent of STAT bias elicited by a single ligand varied across tissues, ranging from full STAT3-biased agonism to STAT1/3 antagonism, correlating with IL-10Rβ expression levels. In vivo, this tissue-selective signaling drove tissue protection in the pancreas and gastrointestinal tract without inducing local or systemic inflammation, thereby uncoupling these opposing effects of IL-22 signaling. Our findings provide insight into the mechanisms underlying the cytokine pleiotropy and illustrate how differential receptor expression levels and STAT response thresholds can be synthetically exploited to endow pleiotropic cytokines with enhanced functional specificity.

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.

Crypt apoptosis and regeneration are characteristic findings in GI-GVHD. Intestinal stem cells (ISCs) are critical for maintaining the intestinal epithelium, but their frequencies are reduced in experimental GVHD, and the mechanisms driving crypt regeneration in this context are poorly understood. To better understand the impact of GVHD on individual epithelial components, we performed single cell RNA sequencing (scRNAseq) of purified small intestine crypts from B6 mice during homeostasis and five days after B6 into B6 syngeneic (syn) or B10.Br into B6 allogeneic (allo) BMT. Sequenced cells were first partitioned into distinct clusters using PhenoGraph, and the cluster identities were annotated based on their gene expression profiles. Unsupervised clustering indicated multiple subpopulations within the various recognized crypt components (Fig 1A). While secretory lineages clustered similarly across experimental conditions, there was highly distinct clustering among ISC populations and striking dissimilarity between allo and syn ISCs. Quantification by computing the subpopulation's phenotypic distance, a composite score integrating both the number and amplitude of differentially expressed genes, indicated that ISCs demonstrated the greatest transcriptional change in response to GVHD among all crypt lineages (Fig 1B). Gene Set Enrichment Analysis (GSEA) highlighted activation of the interferon-γ (IFNγ) pathway in allo ISCs, and differential gene expression indicated that STAT1 was their most highly upregulated transcription factor. We then performed MHC-mismatched allo-BMT into STAT1-floxed x villin-Cre recipients. Consistent with a role in IFNγ-related toxicity, STAT1-deficient recipients initially demonstrated reduced GVHD pathology, as well as a reduction in proliferating Ki67 + cells (Fig 2A). However, the pathology reduction in STAT1-deficient recipients was transient, while reduction in crypt proliferation persisted. STAT1-deficient recipients ultimately demonstrated increased mortality after allo-BMT, indicating a complex response to epithelial STAT1 signaling in GVHD. While IFNγ can induce epithelial apoptosis and kill organoid cultures, organoid exposure to IFNγ augmented size even at IFNγ concentrations that did not impair viability in a STAT1-dependent manner, and co-culture with allo T cells augmented organoid size (Fig 2B, C). Moreover, cell cycle analysis showed augmentation of cell cycle in ISCs after IFNγ treatment in association with upregulation of cyclin D1 (Fig 2D), and human organoids also showed increased size in response to IFNγ treatment, further suggesting that this growth promotion was not simply a secondary response to tissue injury. In addition to the IFNγ signaling, GSEA of allo ISCs indicated activation of the Myc pathway. scRNAseq data showed specific upregulation of Myc in allo ISCs (Fig 3A). Myc expression within individual ISCs indicated that greater STAT1 expression and IFNγ signaling directly correlated with Myc expression in the same ISCs, providing a potential direct link between T-cell-derived IFNγ and ISC-dependent regeneration (Fig 3B). Additionally, intestinal organoid qPCR showed that Myc expression was upregulated after IFNγ treatment, and scRNAseq of IFNγ-treated organoids indicated this Myc upregulation was restricted to the ISC/progenitor compartment. Although Myc is downstream of Wnt signaling, which is critical for ISC maintenance, expression of the representative Wnt target gene Axin2 was downregulated after IFNγ treatment, and Irf1, a representative IFNγ/STAT1 target gene, was upregulated, suggesting that IFNγ/STAT1 signaling could replace Wnt/β-catenin as a driver of ISC Myc expression. We next examined Myc function and found that treatment with the Myc inhibitor 10058-F4 suppressed IFNγ-dependent organoid growth (Fig 3C). Finally, immunofluorescent staining showed Myc protein expression in intestinal crypts after allo-BMT in a STAT1-dependent manner (Fig 3D), and Myc inhibitor treatment in vivo suppressed crypt epithelial proliferation in mice with GVHD. In summary, we found that epithelial STAT1 contributes to crypt regeneration in GVHD by transmitting T-cell-derived JAK/STAT cytokine signaling to activate Myc expression in ISCs. Clinical use of JAK inhibitors in GVHD may inhibit this regenerative response, necessitating concurrent interventions aimed at restoring it. Figure 1 Figure 1. Disclosures Hanash: Evive Biotech: Ended employment in the past 24 months.

Graft-versus-host disease (GVHD) and posttransplant immunodeficiency are frequently related complications of allogeneic hematopoietic transplantation. Alloreactive donor T cells can damage thymic epithelium, thus limiting new T-cell development. Although the thymus has a remarkable capacity to regenerate after injury, endogenous thymic regeneration is impaired in GVHD. The mechanisms leading to this regenerative failure are largely unknown. Here we demonstrate in experimental mouse models that GVHD results in depletion of intrathymic group 3 innate lymphoid cells (ILC3s) necessary for thymic regeneration. Loss of thymic ILC3s resulted in deficiency of intrathymic interleukin-22 (IL-22) compared with transplant recipients without GVHD, thereby inhibiting IL-22-mediated protection of thymic epithelial cells (TECs) and impairing recovery of thymopoiesis. Conversely, abrogating IL-21 receptor signaling in donor T cells and inhibiting the elimination of thymic ILCs improved thymopoiesis in an IL-22-dependent fashion. We found that the thymopoietic impairment in GVHD associated with loss of ILCs could be improved by restoration of IL-22 signaling. Despite uninhibited alloreactivity, exogenous IL-22 administration posttransplant resulted in increased recovery of thymopoiesis and development of new thymus-derived peripheral T cells. Our study highlights the role of innate immune function in thymic regeneration and restoration of adaptive immunity posttransplant. Manipulation of the ILC-IL-22-TEC axis may be useful for augmenting immune reconstitution after clinical hematopoietic transplantation and other settings of T-cell deficiency.

Background IL-22 has been shown to support intestinal mucosa after damage through multiple mechanisms, including promotion of epithelial survival and regeneration as well as induction of innate antimicrobial molecules such as REG3. In experimental murine models, aGVHD resulted in elimination of host-derived IL-22-producing cells, IL-22 deficiency increased GVHD mortality and GI pathology, and early initiation of treatment with exogenous IL-22 reduced GI pathology and improved survival. We thus tested if the addition of IL-22 to corticosteroids could promote healing of GI tract injury and improve treatment response in patients with lower GI aGVHD. Methods We conducted a 27-patient open-label, single-cohort, multicenter prospective phase 2 study between 05/16 and 05/19. Eligible pts were ≥ 18 yrs old and had new onset biopsy-proven grade II-IV lower GI aGVHD following an allo-HCT. Pts received F-652 once weekly at a dose of 45 μg/kg x 4 weeks in combination with standard corticosteroid treatment. Primary endpoints included PK, safety, and day 28 lower GI aGVHD response. Additional endpoints included day 56 treatment response, evaluation of changes in gut microbiota by 16S sequencing, and plasma GVHD biomarkers. The study was powered to distinguish between an unpromising response rate of 35% and a promising response rate of 60%. Results The 27 pts (median age 55 yrs, mostly PBSC recipients) had predominantly stage 2-4 lower GI GVHD (17/27, 63%), with 6/27 (22%) pts having stage 3 and 6/27 (22%) pts having stage 4 disease. All pts had detectable F-652 levels. Overall, 19/27 achieved a day 28 response (70%, 90%CI: 56-79, Fig 1A). Response to treatment based on Ann Arbor Risk, evaluable in 20 pts, was 7/12 (58%) with high, 3/4 (75%) with intermediate, and 4/4 (100%) with low risk biomarkers (Fig 1B). At day 56, 16 pts (59%) remained treatment responders. 3 pts had repeat GI biopsy after treatment and demonstrated improvement in GI epithelial injury (Fig 2). Additionally, in a subset of 17 pts with stool samples collected, microbial diversity and relative abundance of protective commensal Blautia appeared increased in pts with a clinical response to F-652 (p = 0.082 and 0.048, respectively, Fig 3A-B). Serious TEAEs were observed in 11 pts (40%) including enterocolitis (n = 1), pyrexia (n = 1), infection (2 sepsis, 1 device-related, 1 pneumonia, 1 sinusitis), musculoskeletal (n = 2), and respiratory (n = 1). One patient was diagnosed with squamous cell carcinoma 8 months after F-652 treatment. Conclusion IL-22 in combination with corticosteroids was well tolerated and the 70% lower GI aGVHD response rate met the primary efficacy endpoint. These findings support further development of this approach and provide a proof-of-concept for combining standard immunosuppression with tissue-supportive strategies to enhance recovery of damaged mucosa, promote microbial health, and improve GVHD treatment response.

Gastro-intestinal (GI) Graft-versus-Host Disease (GvHD) involves a complex interplay of conditioning-related injury and the pathologic immune response at the epithelial level. Enhanced tissue damage is a risk factor for GVHD. We demonstrated that donor T cell-derived IFNγ causes intestinal stem cell damage in GvHD. Recent studies have proposed that donor T cells directly interact with GI epithelium in GvHD in vivo, but the precise mechanisms remain incompletely understood. Here, we evaluate the influence of both cytokine- and chemotherapy-induced intestinal epithelial damage on T cell migration and direct epithelial cell-T cell interactions using GI organoids as a model system. We developed a human intestinal organoid-T cell interaction co-culture system and studied the role of T cells in intestinal damage during GVHD. We observed that polyclonally activated T cells accumulate in close proximity to intestinal organoids (Fig. 1). In this co-culture system T cell-derived IFNg- plays a key role in tissue damage. RNA-seq analyses of IFNg-treated intestinal organoids indicated significant upregulation of T cell chemotaxis associated genes including CXCL9, 10 and 11. Luminex analysis of conditioned media confirmed release of these chemokines in co-culture versus controls. In addition, stimulation of organoids with IFNγ led to upregulation of HLA-I and HLA-II at both the RNA and membrane protein expression level (Fig. 2A). These experiments place activated T cells at the cusp of intestinal damage during GVHD through the production of cytokines, the induction of T cell recruiting chemokines and mechanisms of antigen driven cytotoxicity. Next, we set out to characterize the effect of chemotherapy on epithelium by treating organoids with Busulfan (Bu), Fludarabine (Flu) or Clofarabine (Clo). Epithelial damage was confirmed by the presence of increasing levels of caspase activation (Fig. 3A), a decrease in the number of surviving organoids, a smaller organoid size and decreased proliferation. Bu, Flu, Clo treatment did not affect HLA-I-II mRNA expression, but a variable, organoid donor-dependent upregulation of membrane HLA-I upon exposure to Bu was observed (Fig. 2B). By using a migration assay, we established that medium that we obtained from chemotherapy-treated organoids exerts increased T cell chemotaxis compared to control medium (Fig. 3B). In conclusion, both (T cell derived) IFNγ- and chemotherapy induced intestinal epithelial damage can lead to increased T cell migration ex vivo, resulting in direct contact between allogeneic T cells and co-cultured human intestinal epithelium. Cytokine signaling and conditioning-related injury may contribute to GVHD pathogenesis in vivo by promoting recruitment to intestinal crypt epithelium.

Despite the importance of intestinal stem cells (ISCs) for epithelial maintenance, there is limited understanding of how immune-mediated damage affects ISCs and their niche. We found that stem cell compartment injury is a shared feature of both alloreactive and autoreactive intestinal immunopathology, reducing ISCs and impairing their recovery in T cell-mediated injury models. Although imaging revealed few T cells near the stem cell compartment in healthy mice, donor T cells infiltrating the intestinal mucosa after allogeneic bone marrow transplantation (BMT) primarily localized to the crypt region lamina propria. Further modeling with ex vivo epithelial cultures indicated ISC depletion and impaired human as well as murine organoid survival upon coculture with activated T cells, and screening of effector pathways identified interferon-γ (IFNγ) as a principal mediator of ISC compartment damage. IFNγ induced JAK1- and STAT1-dependent toxicity, initiating a proapoptotic gene expression program and stem cell death. BMT with IFNγ-deficient donor T cells, with recipients lacking the IFNγ receptor (IFNγR) specifically in the intestinal epithelium, and with pharmacologic inhibition of JAK signaling all resulted in protection of the stem cell compartment. In addition, epithelial cultures with Paneth cell-deficient organoids, IFNγR-deficient Paneth cells, IFNγR-deficient ISCs, and purified stem cell colonies all indicated direct targeting of the ISCs that was not dependent on injury to the Paneth cell niche. Dysregulated T cell activation and IFNγ production are thus potent mediators of ISC injury, and blockade of JAK/STAT signaling within target tissue stem cells can prevent this T cell-mediated pathology.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for hematological malignancies. However, graft-versus-host disease (GVHD) and relapse after allo-HSCT remain major impediments to the success of allo-HSCT. Chimeric antigen receptors (CARs) direct tumor cell recognition of adoptively transferred T cells. CD19 is an attractive CAR target, which is expressed in most B cell malignancies, as well as in healthy B cells. Clinical trials using autologous CD19-targeted T cells have shown remarkable promise in various B cell malignancies. However, the use of allogeneic CAR T cells poses a concern in that it may increase risk of the occurrence of GVHD, although this has not been reported in selected patients infused with donor-derived CD19 CAR T cells after allo-HSCT. To understand the mechanism whereby allogeneic CD19 CAR T cells may mediate anti-lymphoma activity without causing a significant increase in the incidence of GVHD, we studied donor-derived CD19 CAR T cells in allo-HSCT and lymphoma models in mice. We demonstrate that alloreactive T cells expressing CD28-costimulated CD19 CARs experience enhanced stimulation, resulting in the progressive loss of both their effector function and proliferative potential, clonal deletion, and significantly decreased occurrence of GVHD. Concurrently, the other CAR T cells that were present in bulk donor T cell populations retained their anti-lymphoma activity in accordance with the requirement that both the T cell receptor (TCR) and CAR be engaged to accelerate T cell exhaustion. In contrast, first-generation and 4-1BB-costimulated CAR T cells increased the occurrence of GVHD. These findings could explain the reduced risk of GVHD occurring with cumulative TCR and CAR signaling.

Intestinal graft-versus-host disease (GVHD) remains a significant obstacle to the success of allogeneic hematopoietic cell transplantation. The intestinal mucosa comprises the inner lining of the intestinal tract and maintains close proximity with commensal microbes that reside within the intestinal lumen. Recent advances have significantly improved our understanding of the interactions between the intestinal mucosa and the enteric microbiota. Changes in host mucosal tissue and commensals posttransplant have been actively investigated, and provocative insights into mucosal immunity and the enteric microbiota are now being translated into clinical trials of novel approaches for preventing and treating acute GVHD. In this review, we summarize recent findings related to aspects of the intestinal mucosa during acute GVHD.

The spectrum of somatic alterations in hematologic malignancies includes substitutions, insertions/deletions (indels), copy number alterations (CNAs), and a wide range of gene fusions; no current clinically available single assay captures the different types of alterations. We developed a novel next-generation sequencing-based assay to identify all classes of genomic alterations using archived formalin-fixed paraffin-embedded blood and bone marrow samples with high accuracy in a clinically relevant time frame, which is performed in our Clinical Laboratory Improvement Amendments-certified College of American Pathologists-accredited laboratory. Targeted capture of DNA/RNA and next-generation sequencing reliably identifies substitutions, indels, CNAs, and gene fusions, with similar accuracy to lower-throughput assays that focus on specific genes and types of genomic alterations. Profiling of 3696 samples identified recurrent somatic alterations that impact diagnosis, prognosis, and therapy selection. This comprehensive genomic profiling approach has proved effective in detecting all types of genomic alterations, including fusion transcripts, which increases the ability to identify clinically relevant genomic alterations with therapeutic relevance.

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

Patient outcomes for steroid-dependent or -refractory chronic graft-versus-host diesease (cGVHD) are poor, and only ibrutinib has been US Food and Drug Administration (FDA) approved for this indication. cGVHD is often driven by the germinal center (GC) reaction, in which T follicular helper cells interact with GC B cells to produce antibodies that are associated with disease pathogenesis. The transcriptional corepressor B-cell lymphoma 6 (BCL6) is a member of the Broad-complex, Tramtrack, and Bric-abrac/poxvirus and zinc finger (BTB/POZ) transcription factor family and master regulator of the immune cells in the GC reaction. We demonstrate that BCL6 expression in both donor T cells and B cells is necessary for cGVHD development, pointing to BCL6 as a therapeutic cGVHD target. A small-molecule BCL6 inhibitor reversed active cGVHD in a mouse model of multiorgan system injury with bronchiolitis obliterans associated with a robust GC reaction, but not in cGVHD mice with scleroderma as the prominent manifestation. For cGVHD patients with antibody-driven cGVHD, targeting of BCL6 represents a new approach with specificity for a master GC regulator that would extend the currently available second-line agents.

Cytokines are critical drivers of both pathology (i.e., graft-versus-host disease [GVHD]) and protection (i.e., graft-versus-leukemia [GVL]) after hematopoietic stem cell transplantation, and an understanding of their biology is important for understanding both the pathophysiology of disease, together with existing and future treatment modalities. The critical cytokines promoting pathology in GVHD are interferon-gamma, tumor necrosis factor, and interleukin (IL)-6, as these have involvement in the gastrointestinal tract, skin and lung, though paradoxically interferon-gamma signaling provides protection from lung GVHD/idiopathic pneumonia syndrome. Many other cytokines are important in the overall inflammatory cytokine storm that is characteristic of acute GVHD, including the largely “proinflammatory” molecules IL-1, IL-2, IL-12, IL-17, IL-21, IL-33, and the regulatory/protective molecules IL-10, IL-22, and TGF-β. This chapter provides an overview of cytokine signaling in GVHD and GVL and highlights the preclinical and clinical evidence for current cytokine-associated paradigms in GVHD pathophysiology.

Background Anti-thymocyte globulin-free dCBT is associated with high rates of acute GVHD (aGVHD) especially involving the GI tract. Investigation of augmented aGVHD prophylaxis is indicated. Methods We are investigating the addition of a single dose of tocilizumab 8 mg/kg (day -1) to CSA/ MMF (starting day -3) aGVHD prophylaxis in adult patients (pts) with hematologic malignancies undergoing dCBT with intermediate intensity Cy 50/ Flu 150/ Thio 10/ TBI 400 conditioning (NCT03434730). Outcomes of the first 26 patients (with survivor follow-up of at least 100 days) were analyzed & compared to 54 dCBT historic controls transplanted with identical conditioning & only CSA/ MMF. All patients received letermovir if CMV seropositive. Results 26 pts [median age 47 years (range 26-60), median weight 82 kg (range 59-125), 11 AML, 8 ALL, 2 MPAL, 3 MDS/ CML, 2 NHL, median age-adjusted HCT-CI 2.5 (range 0-6)] received dCB grafts with a median CD34+ cell dose of 1.5 × 105/kg/unit (range 0.23-5.94) & median unit-recipient 8-allele HLA-match of 5/8 (range 3-6). One patient had graft failure in the setting of low graft viability/ disseminated adenovirus & a second pt had early transplant-related mortality (TRM) with incomplete count recovery. The remaining 24 patients engrafted at a median of 24 days (range 18-40) for a cumulative incidence of 92% (95%CI:67-98). 88% (95%CI:59-97) of pts engrafted platelets at a median of 43 days (range 32-78). Of evaluable pts, 68% & 83% had 100% single-unit donor chimerism in the blood at days 30 & 100, respectively; single-unit dominance was observed in the remaining cases. Six of 26 pts (23%) developed pre-engraftment syndrome (PES) at a median of 12.5 days, & only 3 of these (12%) required systemic corticosteroids. Additionally, 16 pts (62%) had no neutropenic fever prior to engraftment. The day 100 cumulative incidence of grade II-III & grade III aGVHD was 62% (95%CI:39-78) & 4% (95%CI: Conclusions Tocilizumab-based dCBT appears safe with reduced PES & grade III-IV aGVHD. While many pts developed upper GI aGVHD, the marked reduction in lower GI aGVHD supports preferential reduction of T-cell homing to the lower gut. Investigation of this approach including aGVHD treatment responses, immune recovery & correlative biomarker/ microbiota studies are ongoing.

Nuclear factor erythroid-derived 2-like 2 (Nrf2) is a ubiquitously expressed transcription factor that is well known for its role in regulating the cellular redox pathway. Although there is mounting evidence suggesting a critical role for Nrf2 in hematopoietic stem cells and innate leukocytes, little is known about its involvement in T-cell biology. In this study, we identified a novel role for Nrf2 in regulating alloreactive T-cell function during allogeneic hematopoietic cell transplantation (allo-HCT). We observed increased expression and nuclear translocation of Nrf2 upon T-cell activation in vitro, especially in CD4+ donor T cells after allo-HCT. Allo-HCT recipients of Nrf2−/− donor T cells had significantly less acute graft-versus-host disease (GVHD)-induced mortality, morbidity, and pathology. This reduction in GVHD was associated with the persistence of Helios+ donor regulatory T cells in the allograft, as well as defective upregulation of the gut-homing receptor LPAM-1 on alloreactive CD8+ T cells. Additionally, Nrf2−/− donor CD8+ T cells demonstrated intact cytotoxicity against allogeneic target cells. Tumor-bearing allo-HCT recipients of Nrf2−/− donor T cells had overall improved survival as a result of preserved graft-versus-tumor activity and reduced GVHD activity. Our findings characterized a previously unrecognized role for Nrf2 in T-cell function, as well as revealed a novel therapeutic target to improve the outcomes of allo-HCT.

Background GI symptoms are common after CBT & GI aGVHD is frequent. The correlation between symptoms, macroscopic appearance (macro), histology, aGVHD severity & therapy response, however, is not established. Methods We analyzed 1st endoscopies done days +21-180 in engrafted adult double unit CBT recipients transplanted 10/2009-12/2017 with Cy 50/ Flu 150/ Thio 5-10/ TBI 400 & CSA/ MMF. All patients (pts) received fungal prophylaxis, early CMV pre-emptive therapy & C. difficile/ viral PCR stool assays (& blood for adenovirus) were routinely performed. Abnormal histology was defined as mild (grade 1-2) vs severe (grade 3-4). Results Of 156 pts, 88 pts (56%) [median 49 years (range 22-66), 65 leukemia, 5 MDS/ MPD, 18 NHL/ HL] were scoped. Their median donor-recipient HLA-allele match was 5/8 (range 3-7) & 36 (38%) pts also received haplo-CD34+ cells. Pts had 44 upper, 2 lower & 42 combined upper/ lower scopes at a median of 52 days (range 22-176) post-CBT. Of 86 pts who had upper scopes (Table 1), 67/86 (78%) were treated for upper GI aGVHD & only 3 had infection (Figure). The majority (51/67,76%) of aGVHD treated pts had normal macro appearance & the majority (49/67, 73%) had aGVHD compatible histology (typically apoptotic epithelial cells, mild in all but 2). Overall, the majority of treated pts (48/67, 72%) had a day 28 CR (37 budesonide, 11 high dose corticosteroids). Of 18 treated pts with normal macro & histology, 11 had CR & 7 PR. Nearly all (40/44, 91%) pts who had lower GI scopes (Table 2) were treated for aGVHD & none had infection (Figure). Most (33/40, 83%) were stage I-II ( Conclusions With pre-emptive CMV therapy, yeast prophylaxis, & stool PCR studies, the majority of CBT pts who required endoscopies had aGVHD & infection was very rare. This was especially true in pts with diarrhea. Normal mucosal appearance was frequent in aGVHD treated pts & nearly all pts with diarrhea & abnormal macro had aGVHD. Most had aGVHD compatible histology (usually mild). Notably, 9/23 treated pts with normal histology only had a PR supporting aGVHD is a clinical diagnosis. Moreover, pts with severe histologic abnormality were at high risk for PR/ refractory disease. This data supports early corticosteroid therapy if aGVHD compatible symptoms & not waiting for biopsy results. This is critical in pts with diarrhea. Correlation of this data with serum biomarkers & microbiota to improve the efficiency of diagnosis & risk stratification are needed.

Innate lymphoid cells (ILCs) are associated with protective mucosal response after experimental allogeneic BMT. However, the specific in vivo roles of ILCs and their capacity for reconstitution post-transplant remain unclear. We therefore sought to characterize the significance and tissue distribution of ILCs after allo-BMT. To study the role of ILCs after allo-BMT, we established a clinically relevant acute GVHD model based on H-Y antigen mismatch. Lethally irradiated Rag2−/− male mice (Thy1.2, B6) were transplanted with T cell depleted (TCD) bone marrow and purified T cells obtained from wild-type female mice (Thy1.1, B6). To deplete host-derived ILCs, recipient mice were treated with an anti-Thy1.2 antibody or an anti-isotype antibody prior to BMT. Pre-transplant depletion of ILCs induced significantly worse systemic signs of GVHD, as well as increased thymic injury and intestinal GVHD pathology, indicating an important role for ILCs after BMT (Fig. 1). To better understand the systemic roles of ILCs, we evaluated ILC subsets in the intestine, liver, and lungs after allo-BMT. Two weeks after MHC-mismatched (C57BL6 into BALB/c) TCD-BMT, both donor- and host-derived ILCs could be detected in all tissues. Liver was characterized by a high number of NK cells and ILC1s, while the lungs contained a high proportion of ILC2s, and small intestine contained relatively greater ILC3s. For all tissues analyzed, the majority of ILCs were host-derived. During T cell replete BMT resulting in GVHD, both donor and host-derived ILCs were significantly reduced in all tissues compared to TCD-BMT mice (Fig. 2). To better understand the loss of donor-derived ILCs caused by donor T cells, we investigated ILC precursors in recipient marrow post-BMT, and found a significant loss of CLPs, αLPs, CHILPs, and ILC2 precursors in mice with GVHD. These data suggested that bone marrow involvement with GVHD induced a loss of ILC precursors impairing their reconstitution post-BMT. Given the loss of ILCs occurring post-BMT and the increased GVHD severity in BMT recipients lacking ILCs, we hypothesized that expansion of the ILC compartment pre-transplant could help protect BMT recipients from GVHD. Aryl hydrocarbon receptor (AhR) signaling has been associated with ILC3 development and maintenance during intestinal homeostasis. We thus evaluated the effect of diet-derived AhR ligands (AhRL) on intestinal ILCs and BMT outcomes. Prior to transplantation, mice were given diets containing or lacking AhRL. Mice on an AhRL-poor diet showed reduced intestinal ILC3s, increased intestinal GVHD pathology, and increased mortality post-BMT. In conclusion, ILCs contribute to tissue protection following allo-BMT, but GVHD leads to elimination of pre-existing ILCs and abolishes their development. Dietary supplementation with AhRL-rich foods may represent an approach to enhance mucosal immunity and intestinal recovery post-BMT.

Background Adult dCBT recipients are at significant risk for aGVHD especially involving the GI tract. Investigation of augmented prophylaxis is indicated. Methods Based on adult donor allograft data (Kennedy et al, Lancet 2015; Drobyski et al Haematologica 2018), we are investigating the addition of a single dose of tocilizumab 8 mg/kg (cap 800mg) on day -1 to standard cyclosporine/ MMF starting day -3 in adult patients (pts) with hematologic malignancies undergoing intermediate intensity Cy 50/ Flu 150/ Thio 10/ TBI 400 dCBT. We report an interim analysis of the first 16 pts of a 27 pt phase II trial (NCT03434730). All pts received standard bacterial/ fungal prophylaxis & Letermovir if CMV seropositive. Results 16 pts [median 50 years (range 27-59), median 85 kg (range 59-125), 6 AML, 5 ALL, 2 MPAL, 2 MDS, 1 CML, median aaHCT-CI of 3 (range 0-6)] received double-unit CB grafts with a median CD34+ cell dose of 1.33 × 105/kg/unit (range 0.24-3.83) & median unit-recipient 8-allele HLA-match of 5/8 (range 3-6). Pt outcomes are shown (Table). All (100%) pts engrafted with a median neutrophil recovery of 26 days (range 19-40). 94% of pts engrafted platelets (median 44 days, range 32-59). All pts had single unit dominance (median day 28 blood winning unit chimerism 100% (range 58-100). Prior to engraftment, 11/16 (69%) had no neutropenic fever, 3/16 (19%) pts had fever with bacteremia, 1 pt (6%) had febrile neutropenia (duration 2 days) responding to antibiotics, & 1 pt (6%) had initial fever due to bacteremia followed by fever/ rash attributed to pre-engraftment syndrome (PES). An additional 2 pts developed rash without fever prior to engraftment attributed to PES. Thus, a total of 3/16 (19%) pts had PES (onset ranging 12-14 days). Ten pts developed grade II aGVHD (median onset 45 days, range 26-81) for a cumulative incidence of 63% (95%CI: 33-82). This was limited primarily to skin &/or the upper gut with one pt having stage I lower GI. No pt has developed grade III-IV aGVHD & no pt has had stage II-IV lower GI aGVHD to date (Figure). With a median follow-up of 147 days (range 74-214), 15 pts are alive & disease-free (1 death due to HHV-6 pneumonitis). Conclusions Preliminary data suggests tocilizumab is safe with no adverse effects upon engraftment or unit dominance. Tocilizumab mitigates PES (expected incidence 65% in dCBT controls, Bhatt et al, ASBMT 2019). Expected days of febrile neutropenia were also reduced especially given the prolonged neutrophil recovery. Notably, lower gut aGVHD appears markedly reduced with no cases of grade III-IV aGVHD or aGVHD mortality to date. Our findings appear consistent with the published adult donor data. Overall, one tocilizumab dose has improved the early post-dCBT morbidity & investigation of this promising & potentially cost-effective approach is ongoing.

Sustained hematopoiesis after double-unit cord blood transplantation (dCBT) is mediated by 1 unit in nearly all patients. To investigate the associations between nondominant unit characteristics and neutrophil engraftment, we studied 129 consecutive myeloablative dCBT recipients. Ninety-five percent (95% confidence interval, 90 to 98) of patients engrafted. Detection of the nondominant unit 21 to 28 days after dCBT was not associated with improved neutrophil engraftment. In univariate analyses, nondominant unit characteristics (infused total nucleated cell [TNC] and viable CD3(+) cell doses) were significantly associated with speed and success of neutrophil engraftment as were dominant unit characteristics (infused TNC; viable CD34(+), viable CD3(+), and viable CD3-56(+)16(+) cell doses; and post-thaw CD34(+) cell viability). In multivariate analysis, higher infused TNC dose of the nondominant unit was independently associated with improved neutrophil engraftment, even when this unit did not contribute to donor hematopoiesis. In further subgroup analysis, this association was only evident when the infused viable CD34(+) cell dose of the dominant unit was low (1.20 × 10(5)/kg). These findings suggest nondominant units mediate a dose-dependent facilitation of engraftment in myeloablative dCBT and support continued investigation of dCBT biology and the clinical practice of dCBT in adults in whom low cell dose grafts are common.

The relationship between intestinal microbiota composition and acute graft-versus-host disease (GVHD) after allogeneic blood/marrow transplantation (allo BMT) is not well understood. Intestinal bacteria have long been thought to contribute to GVHD pathophysiology, but recent animal studies in non-transplant settings have found that anti-inflammatory effects are mediated by certain subpopulations of intestinal commensals. Hypothesizing that a more nuanced relationship may exist between the intestinal bacteria and GVHD, we evaluated the fecal bacterial composition of 64 patients 12 days after BMT. We found that increased bacterial diversity was associated with reduced GVHD-related mortality. Furthermore, harboring increased amounts of bacteria belonging to the genus Blautia was associated with reduced GVHD lethality in this cohort and was confirmed in another independent cohort of 51 patients from the same institution. Blautia abundance was also associated with improved overall survival. We evaluated the abundance of Blautia with respect to clinical factors and found that loss of Blautia was associated with: 1) treatment with antibiotics that inhibit anaerobic bacteria and 2) receiving total parenteral nutrition (TPN) for longer durations. We conclude that increased abundance of commensal bacteria belonging to the Blautia genus is associated with reduced lethal GVHD and improved overall survival.

The thymus is not only extremely sensitive to damage but also has a remarkable ability to repair itself. However, the mechanisms underlying this endogenous regeneration remain poorly understood, and this capacity diminishes considerably with age. We show that thymic endothelial cells (ECs) comprise a critical pathway of regeneration via their production of bone morphogenetic protein 4 (BMP4) ECs increased their production of BMP4 after thymic damage, and abrogating BMP4 signaling or production by either pharmacologic or genetic inhibition impaired thymic repair. EC-derived BMP4 acted on thymic epithelial cells (TECs) to increase their expression of Foxn1, a key transcription factor involved in TEC development, maintenance, and regeneration, and its downstream targets such as Dll4, a key mediator of thymocyte development and regeneration. These studies demonstrate the importance of the BMP4 pathway in endogenous tissue regeneration and offer a potential clinical approach to enhance T cell immunity.

While cord blood transplantation (CBT) is an effective therapy for hematologic malignancies, acute graft-versus-host disease (aGVHD) is a leading cause of transplant-related mortality (TRM). We investigated if biomarkers could predict aGVHD and TRM after day 28 in CBT recipients. Day 28 samples from 113 CBT patients were analyzed. Suppressor of tumorigenicity 2 (ST2) was the only biomarker associated with grades II-IV and III-IV aGVHD and TRM. Day 180 grade III-IV aGVHD in patients with high ST2 levels was 30% (95% confidence interval [CI], 18-43) vs 13% (95% CI, 5-23) in patients with low levels (P = .024). The adverse effect of elevated ST2 was independent of HLA match. Moreover, high day 28 ST2 levels were associated with increased TRM with day 180 estimates of 23% (95% CI, 13-35) vs 5% (95% CI, 1-13) if levels were low (P = .001). GVHD was the most common cause of death in high ST2 patients. High concentrations of tumor necrosis factor receptor-1, interleukin-8, and regenerating islet-derived protein 3-α were also associated with TRM. Our results are consistent with those of adult donor allografts and warrant further prospective evaluation to facilitate future therapeutic intervention to ameliorate severe aGVHD and further improve survival after CBT.

The mammalian gut microbiota provides essential metabolites to the host and promotes the differentiation and accumulation of extrathymically generated regulatory T (pTreg) cells. To explore the impact of these cells on intestinal microbial communities, we assessed the composition of the microbiota in pTreg cell-deficient and -sufficient mice. pTreg cell deficiency led to heightened type 2 immune responses triggered by microbial exposure, which disrupted the niche of border-dwelling bacteria early during colonization. Moreover, impaired pTreg cell generation led to pervasive changes in metabolite profiles, altered features of the intestinal epithelium, and reduced body weight in the presence of commensal microbes. Absence of a single species of bacteria depleted in pTreg cell-deficient animals, Mucispirillum schaedleri, partially accounted for the sequelae of pTreg cell deficiency. These observations suggest that pTreg cells modulate the metabolic function of the intestinal microbiota by restraining immune defense mechanisms that may disrupt a particular bacterial niche.

A new study highlights dynamic changes in the enteric virome after hematopoietic stem cell transplantation in humans, pointing to a correlation between these changes and graft-versus-host disease.

Nfil3 is critical for normal development of innate lymphoid cell (ILC) progenitors. Nfil3-deficient mice have severely reduced lung and visceral adipose tissue ILC2s and gut-associated ILC3s, and compromised innate immunity to acute bacterial infection., The bZIP transcription factor Nfil3 (also known as E4BP4) is required for the development of natural killer (NK) cells and type 1 innate lymphoid cells (ILC1s). We find that Nfil3 plays a critical role in the development of other mucosal tissue-associated innate lymphocytes. Type 3 ILCs (ILC3s), including lymphoid tissue inducer (LTi)–like cells, are severely diminished in both numbers and function in Nfil3-deficient mice. Using mixed bone marrow chimeric mice, we demonstrate that Nfil3 is critical for normal development of gut-associated ILC3s in a cell-intrinsic manner. Furthermore, Nfil3 deficiency severely compromises intestinal innate immune defense against acute bacterial infection with Citrobacter rodentium and Clostridium difficile. Nfil3 deficiency resulted in a loss of the recently identified ILC precursor, yet conditional ablation of Nfil3 in the NKp46+ ILC3 subset did not perturb ILC3 numbers, suggesting that Nfil3 is required early during ILC3 development but not for lineage maintenance. Lastly, a marked defect in type 2 ILCs (ILC2s) was also observed in the lungs and visceral adipose tissue of Nfil3-deficient mice, revealing a general requirement for Nfil3 in the development of all ILC lineages.

A potential role for endogenous innate lymphoid cells (ILCs) in prevention of gastrointestinal (GI) GVHD has recently been described in murine and clinical models, and experimental approaches for adoptive cellular therapy with ILCs have recently been described as well. However, the specific in vivo roles of tissue-resident ILCs and their capacity for reconstitution post-transplant remain unclear. We thus sought to characterize the significance and tissue distribution of ILCs after allogeneic bone marrow transplant (allo-BMT) in mouse models and in patients. In order to specifically assess the functional role of ILCs after allo-BMT, we established a clinically relevant acute GVHD model based on sex mismatched H-Y antigens. Lethally irradiated Rag2-/-male mice (Thy1.2, B6) were transplanted with T cell-depleted (TCD) BM and purified T cells obtained from wild-type female mice (Thy1.1, B6). To specifically deplete host-derived ILCs (and not donor cells), recipient mice were treated with an anti-Thy1.2 antibody or an anti-isotype antibody prior to BMT. Pre-transplant depletion of ILCs induced significantly worse systemic signs of GVHD, as well as increased thymic injury and intestinal GVHD pathology, indicating an important role for endogenous ILCs in tissue protection after allo-BMT in this context. To better understand the roles of tissue-resident ILCs in reducing pathology post-BMT, we evaluated ILC subsets in different GVHD target organs after allo-BMT. Two weeks after MHC-mismatched (C57BL6 into BALB/c) TCD-BMT, both donor- and host-derived ILCs could be detected in the liver, lungs, and intestines. Liver was characterized by a high number of NK cells and ILC1s, while lungs contained a high proportion of ILC2s. The small intestine contained a relatively greater proportion of ILC3s. For all tissues analyzed, the majority of ILCs were host-derived. During T cell replete BMT resulting in GVHD, both donor and host-derived ILCs were significantly reduced in all three tissues compared to TCD-BMT mice, and this was true for all ILC lineages. To better understand the loss of donor-derived ILCs in GVHD, we investigated ILC precursors in recipient marrow post-BMT, hypothesizing that an impairment of ILC precursors could explain the loss of all ILC lineages in the various tissues analyzed. Indeed, we found a significant loss of CLPs, αLPs, CHILPs, and ILC2Ps in mice with GVHD, suggesting that bone marrow involvement with GVHD induced a loss of ILC precursors impairing their reconstitution. We next sought to evaluate if the loss of tissue-resident ILCs in GVHD target organs after experimental BMT was relevant for clinical transplantation. To evaluate ILC frequencies in patient tissues post-transplant, duodenal biopsies were collected from patients undergoing clinically indicated endoscopy to work up symptoms of acute upper GI (UGI) GVHD. In total, duodenal samples were collected from ten patients post-transplant and 6/10 had histologic evidence of GVHD on biopsy (Table 1). While frequencies were low, ILCs could be identified by FACS in digested lamina propria of duodenal biopsy specimens. Notably, ILC frequencies were significantly lower in patients with biopsy-proven UGI GVHD, and duodenal ILCs could be identified in only 1/6 patients with histologic evidence of GVHD (Fig 1). Among the ILCs that could be identified, ILC1s represented the greatest proportion of ILC populations within the duodenal lamina propria. Accordingly, the frequency of ILC1s was significantly reduced in transplant patients with biopsy-proven UGI GVHD. Additionally, we were able to evaluate paired peripheral blood and duodenal biopsy samples in a small subset of patients. Although the numbers were limiting, this cohort suggested that ILC2s may represent a greater proportion of ILCs in peripheral blood than in the duodenum post-transplant. Overall, clinical findings were consistent with experimental models demonstrating a reduction of ILCs in GVHD. In conclusion, tissue-resident ILCs contribute to tissue protection after allo-BMT, but GVHD leads to elimination of pre-existing ILCs and loss of ILC precursors in the marrow, impairing their reconstitution. Most duodenal ILCs in clinical biopsy samples post-transplant were ILC1s in this patient cohort, and patients with biopsy-proven UGI GVHD demonstrated significant loss of the few lamina propria ILCs that could be identified in clinical duodenal biopsies. Disclosures Hanash: Nexus Global Group LLC: Consultancy.

Crypt base intestinal stem cells (ISCs) marked by Lgr5 and Olfm4 maintain the intestinal epithelium, and Paneth cells (PCs) provide an epithelial niche for ISCs in the small bowel. ISCs are reduced during gastrointestinal (GI) GVHD, but the precise mechanisms including the role of niche injury are unknown. Additionally, the specific effects of Interferon-γ (IFNγ) on intestinal epithelium in GVHD remain ill-defined. We evaluated kinetics of ISC loss by histology after allogeneic (allo) BMT using Lgr5-LacZ reporter mice. In both MHC- and miHA-mismatched models (LP>B6 and B6>BDF1), ISC numbers quickly recovered from pretransplant TBI conditioning in recipients of T-cell-depleted (TCD) BMT by day +10, but ISCs failed to recover in recipients of allo T cells. T-cell-induced ISC reduction was functionally validated by genetic marking of stem cell progeny and by culturing intestinal organoids from crypts isolated post-BMT. Similar to the kinetics of ISC loss, ISC-dependent organoid-forming capacity was impaired in recipients of allo T cells compared with TCD BMT recipients on day +10 (p To better understand the potential for T cells to interact with the ISC compartment, we performed whole-mount 3-D microscopy to distinguish T cell localization within the intraepithelial and lamina propria compartments post-transplant. We found that donor T cells invading the small bowel after BMT were mostly in the crypt region, and infiltration within the lamina propria adjacent to the ISC compartment was much greater than invasion within the epithelium itself (Fig. 2). We next established an ex vivo co-culture system, to model interactions between intestinal epithelium and donor T cells and investigate mechanisms of T-cell-mediated ISC injury. Screening of effector pathways revealed no impact of perforin or FasL, but identified IFNγ as a principal mediator of ISC injury. Culture with allo T cells significantly reduced viable human and mouse intestinal organoid numbers, and this was inhibited by IFNγ neutralization. IFNγ receptor knockout (IFNγR-/-) organoids were resistant to T cells. IFNγ increased expression of Bak1 and decreased expression of Bcl2 in organoids, and induced ISC apoptosis defined by Annexin+Dapi-Lgr5+ phenotype. ISC killing was mediated by intraepithelial JAK/STAT signaling, as JAK1- and STAT1-deficient organoids were resistant, and it was inhibited by Ruxolitinib. Investigating the role of IFNγ in vivo, FACS analysis confirmed donor T cells to be the primary producers of IFNγ in crypt lamina propria, and BMT with IFNγ-/- donor T cells reduced crypt apoptosis, and preserved ISC frequencies. Moreover, BMT with recipients lacking IFNγR specifically in the intestinal epithelium significantly protected ISCs, reduced crypt apoptosis, and ameliorated GI GVHD pathology (Fig.3). Furthermore, ISCs were also protected by epithelial deletion of STAT1 and by Ruxolitinib treatment. As specific genetic manipulation of ISCs in vivo is not possible because genetic targeting of ISCs results in the same changes in their progeny, we utilized ex vivo models to determine if IFNγ kills ISCs by directly inducing their apoptosis or by damaging the PC niche. Manipulation of the niche by culturing wild-type ISCs with IFNγR-/- PCs was not protective to allo T cells. Using a niche-independent high-purity Lgr5+ ISC culture system based on combined GSK3β and HDAC inhibition, IFNγ directly induced cleaved-caspase-3+ ISC apoptosis and substantial ISC colony death, which were inhibited by Bak/Bax deficiency and by the pan-caspase inhibitor QVD. These results confirmed that IFNγ can directly induce ISC apoptosis independent of other cytotoxic effector molecules and independent of injury to the PC niche. In summary, T cells migrating to the GI tract primarily infiltrated the lamina propria adjacent to the ISC compartment, and T-cell-derived IFNγ directly targeted intestinal epithelium via JAK1/STAT1 signaling to induce ISC apoptosis in a PC-independent manner. ISC reduction and GI GVHD pathology were prevented by inhibiting the IFNγR/JAK1/STAT1 axis within the intestinal epithelium, indicating that in addition to their effects on T cells, JAK inhibitors may treat GVHD by inhibiting pathologic cytokine signaling within target organs and shielding them from allo T cells. Disclosures Hanash: Nexus Global Group LLC: Consultancy.

The key sites within the gastrointestinal (GI) tract where T cells mediate effector responses and the impact of these responses on intestinal stem cells (ISCs) remain unclear. Using experimental bone marrow transplantation to model immune-mediated GI damage and 3D imaging to analyze T cell localization, we found that the ISC compartment is the primary intestinal site targeted by T cells after transplantation. Recruitment to the crypt base region resulted in direct T cell engagement with the stem cell compartment and loss of crypt base columnar ISCs, which expressed both MHC classes I and II. Vasculature expressing the adhesion molecule MAdCAM-1 clustered near the crypt base, preferentially regulating crypt compartment invasion and ISC reduction without affecting T cell migration to villi. These findings indicate that allogeneic T cells rapidly access the stem cell niche after transplantation, and this targeted recruitment to the stem cell compartment results in ISC loss during immune-mediated GI damage.

Manifestations of and risk factors for graft-versus-host disease (GVHD) after double-unit cord blood transplantation (DCBT) are not firmly established. We evaluated 115 DCBT recipients (median age, 37 years) who underwent transplantation for hematologic malignancies with myeloablative or nonmyeloablative conditioning and calcineurin inhibitor/mycophenolate mofetil immunosuppression. Incidence of day 180 grades II to IV and III to IV acute GVHD (aGVHD) were 53% (95% confidence interval, 44 to 62) and 23% (95% confidence interval, 15 to 31), respectively, with a median onset of 40 days (range, 14 to 169). Eighty percent of patients with grades II to IV aGVHD had gut involvement, and 79% and 85% had day 28 treatment responses to systemic corticosteroids or budesonide, respectively. Of 89 engrafted patients cancer-free at day 100, 54% subsequently had active GVHD, with 79% of those affected having persistent or recurrent aGVHD or overlap syndrome. Late GVHD in the form of classic chronic GVHD was uncommon. Notably, grades III to IV aGVHD incidence was lower if the engrafting unit human leukocyte antigen (HLA)-A, -B, -DRB1 allele match was >4/6 to the recipient (hazard ratio, 0.385; P = .031), whereas engrafting unit infused nucleated cell dose and unit-to-unit HLA match were not significant. GVHD after DCBT was common in our study, predominantly affected the gut, and had a high therapy response, and late GVHD frequently had acute features. Our findings support the consideration of HLA- A,-B,-DRB1 allele donor–recipient (but not unit–unit) HLA match in unit selection, a practice change in the field. Moreover, new prophylaxis strategies that target the gastrointestinal tract are needed.

Current strategies to suppress graft-versus-host disease (GVHD) also compromise graft-versus-tumor (GVT) responses. Furthermore, most experimental strategies to separate GVHD and GVT responses merely spare GVT function without actually enhancing it. We have previously shown that endogenously expressed TNF-related apoptosis-inducing ligand (TRAIL) is required for optimal GVT activity against certain malignancies in recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT). In order to model a donor-derived cellular therapy, we genetically engineered T cells to overexpress TRAIL and adoptively transferred donor-type unsorted TRAIL+ T cells into mouse models of allo-HSCT. We found that murine TRAIL+ T cells induced apoptosis of alloreactive T cells, thereby reducing GVHD in a DR5-dependent manner. Furthermore, murine TRAIL+ T cells mediated enhanced in vitro and in vivo antilymphoma GVT response. Moreover, human TRAIL+ T cells mediated enhanced in vitro cytotoxicity against both human leukemia cell lines and against freshly isolated chronic lymphocytic leukemia (CLL) cells. Finally, as a model of off-the-shelf, donor-unrestricted antitumor cellular therapy, in vitro-generated TRAIL+ precursor T cells from third-party donors also mediated enhanced GVT response in the absence of GVHD. These data indicate that TRAIL-overexpressing donor T cells could potentially enhance the curative potential of allo-HSCT by increasing GVT response and suppressing GVHD.

Despite significant advances in prevention and management, graft versus host disease (GVHD) is still a leading complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Although skin, gut, liver, thymus, and lung are GVHD targets, neurological complications (NC) have also been reported following allo-HSCT. We demonstrate that the central nervous system (CNS) can be a direct target of alloreactive T cells following allo-HSCT in mice. We found significant infiltration of the CNS with donor T lymphocytes and cell death of neurons and neuroglia in allo-HSCT recipients with GVHD. We also found that allo-HSCT recipients with GVHD had deficits in spatial learning/memory and demonstrated increased anxious behavior. These findings highlight CNS sensitivity to damage caused by alloreactive donor T cells and represent the first characterization of target cell subsets and NC during GVHD. Therefore, these clinically relevant studies offer a novel and rational explanation for the well-described neurological symptoms observed after allo-HSCT.