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Crohn’s disease is an inflammatory bowel disease (IBD) which most often presents with patchy lesions in the terminal ileum and colon and requires complex clinical care. Recent advances in the targeting of cytokines and leukocyte migration have greatly advanced treatment options, but most patients still relapse and inevitably progress. Although single-cell approaches are transforming our ability to understand the barrier tissue biology of inflammatory disease, comprehensive single-cell RNA-sequencing (scRNA-seq) atlases of IBD to date have largely sampled pre-treated patients with established disease. This has limited our understanding of which cell types, subsets, and states at diagnosis are predictive of disease severity and response to treatment. Here, through a combined clinical, flow cytometric, and scRNA-seq study, we profile diagnostic human biopsies from the terminal ileum of treatment-naive pediatric patients with Crohn’s disease (pediCD; n=14) and from non-inflamed pediatric controls with functional gastrointestinal disorders (FGID; n=13). To fully resolve and annotate epithelial, stromal, and immune cell states among the 201,883 single-cell transcriptomes, we develop and deploy a principled and unbiased tiered clustering approach, ARBOL, yielding 138 FGID and 305 pediCD end cell clusters. Notably, through both flow cytometry and scRNA-seq, we observe that at the level of broad cell types, treatment-naive pediCD is not readily distinguishable from FGID in cellular composition. However, by integrating high-resolution scRNA-seq analysis, we identify significant differences in cell states that arise during pediCD relative to FGID. Furthermore, by closely linking our scRNA-seq analysis with clinical meta-data, we resolve a vector of lymphoid, myeloid, and epithelial cell states in treatment-naive samples which can distinguish patients with less severe disease (those not on anti-TNF therapies (NOA)), from those with more severe disease at presentation who require anti-TNF therapies. Moreover, this vector was also able to distinguish those patients that achieve a full response (FR) to anti-TNF blockade from those more treatment-resistant patients who only achieve a partial response (PR). Our study jointly leverages a treatment-naive cohort, high-resolution principled scRNA-seq data analysis, and clinical outcomes to understand which baseline cell states may predict inflammatory disease trajectory.

PURPOSE Severe (grade 3-4) acute graft-versus-host disease (AGVHD) is a major cause of death after unrelated-donor (URD) hematopoietic cell transplant (HCT), resulting in particularly high mortality after HLA-mismatched transplantation. There are no approved agents for AGVHD prevention, underscoring the critical unmet need for novel therapeutics. ABA2 was a phase II trial to rigorously assess safety, efficacy, and immunologic effects of adding T-cell costimulation blockade with abatacept to calcineurin inhibitor (CNI)/methotrexate (MTX)-based GVHD prophylaxis, to test whether abatacept could decrease AGVHD. METHODS ABA2 enrolled adults and children with hematologic malignancies under two strata: a randomized, double-blind, placebo-controlled stratum (8/8-HLA-matched URD), comparing CNI/MTX plus abatacept with CNI/MTX plus placebo, and a single-arm stratum (7/8-HLA-mismatched URD) comparing CNI/MTX plus abatacept versus CNI/MTX CIBMTR controls. The primary end point was day +100 grade 3-4 AGVHD, with day +180 severe-AGVHD-free-survival (SGFS) a key secondary end point. Sample sizes were calculated using a higher type-1 error (0.2) as recommended for phase II trials, and were based on predicting that abatacept would reduce grade 3-4 AGVHD from 20% to 10% (8/8s) and 30% to 10% (7/8s). ABA2 enrolled 142 recipients (8/8s, median follow-up = 716 days) and 43 recipients (7/8s, median follow-up = 708 days). RESULTS In 8/8s, grade 3-4 AGVHD was 6.8% (abatacept) versus 14.8% (placebo) ( P = .13, hazard ratio = 0.45). SGFS was 93.2% (CNI/MTX plus abatacept) versus 82% (CNI/MTX plus placebo, P = .05). In the smaller 7/8 cohort, grade 3-4 AGVHD was 2.3% (CNI/MTX plus abatacept, intention-to-treat population), which compared favorably with a nonrandomized matched cohort of CNI/MTX (30.2%, P < .001), and the SGFS was better (97.7% v 58.7%, P < .001). Immunologic analysis revealed control of T-cell activation in abatacept-treated patients. CONCLUSION Adding abatacept to URD HCT was safe, reduced AGVHD, and improved SGFS. These results suggest that abatacept may substantially improve AGVHD-related transplant outcomes, with a particularly beneficial impact on HLA-mismatched HCT.

Organ infiltration by donor T cells is critical to the development of acute graft-versus-host disease (aGVHD) in recipients after allogeneic hematopoietic stem cell transplant (allo-HCT). However, deconvoluting the transcriptional programs of newly recruited donor T cells from those of tissue-resident T cells in aGVHD target organs remains a challenge. Here, we combined the serial intravascular staining technique with single-cell RNA sequencing to dissect the tightly connected processes by which donor T cells initially infiltrate tissues and then establish a pathogenic tissue residency program in a rhesus macaque allo-HCT model that develops aGVHD. Our results enabled creation of a spatiotemporal map of the transcriptional programs controlling donor CD8+ T cell infiltration into the primary aGVHD target organ, the gastrointestinal (GI) tract. We identified the large and small intestines as the only two sites demonstrating allo-specific, rather than lymphodepletion-driven, T cell infiltration. GI-infiltrating donor CD8+ T cells demonstrated a highly activated, cytotoxic phenotype while simultaneously developing a canonical tissue-resident memory T cell (TRM) transcriptional signature driven by interleukin-15 (IL-15)/IL-21 signaling. We found expression of a cluster of genes directly associated with tissue invasiveness, including those encoding adhesion molecules (ITGB2), specific chemokines (CCL3 and CCL4L1) and chemokine receptors (CD74), as well as multiple cytoskeletal proteins. This tissue invasion transcriptional signature was validated by its ability to discriminate the CD8+ T cell transcriptome of patients with GI aGVHD from those of GVHD-free patients. These results provide insights into the mechanisms controlling tissue occupancy of target organs by pathogenic donor CD8+ TRM cells during aGVHD in primate transplant recipients.

One of the central challenges in the field of allo-immunity is deciphering the mechanisms driving T cells to infiltrate and subsequently occupy target organs to cause disease. The act of CD8-dominated T cell infiltration is critical to acute graft-versus-host disease (aGVHD), wherein donor T cells become activated, tissue-infiltrating and highly cytotoxic, causing wide-spread tissue damage after allogeneic hematopoietic stem cell transplant (allo-HCT). However, in human and non-human primate studies, deconvolving the transcriptional programs of newly recruited relative to resident memory T cells in the gastrointestinal (GI) tract has remained a challenge. In this study, we combined the novel technique of Serial Intravascular Staining (SIVS) with single-cell RNA-Seq (scRNA-seq) to enable detailed dissection of the tightly connected processes by which T cells first infiltrate tissues and then establish a pathogenic tissue residency program after allo-HCT in non-human primates. Our results have enabled the creation of a spatiotemporal map of the transcriptional drivers of CD8 T cell infiltration into the primary aGVHD target-organ, the GI tract. We identify the large and small intestines as the only two sites demonstrating allo-specific, rather than lymphdepletion-driven T cell infiltration. The donor CD8 T cells that infiltrate the GI tract demonstrate a highly activated, cytotoxic phenotype while simultaneously rapidly developing canonical tissue-resident memory (TRM) protein expression and transcriptional signatures, driven by IL-15/IL-21 signaling. Moreover, by combining SIVS and transcriptomic analysis, we have been able to work backwards from this pathogenic TRM programing, and, for the first time, identify a cluster of genes directly associated with tissue invasiveness, prominently including specific chemokines and adhesion molecules and their receptors, as well as a central cytoskeletal transcriptional node. The clinical relevance of this new tissue invasion signature was validated by its ability to discriminate the CD8 T cell transcriptome of patients with GI aGVHD. These results provide new insights into the mechanisms controlling tissue infiltration and pathogenic CD8 TRM transcriptional programing, uncovering critical transitions in allo-immune tissue invasion and destruction.One sentence summaryFlow cytometric and transcriptomic analysis reveals coordinated tissue-infiltration and tissue-residency programs driving gastrointestinal aGVHD.

Organ infiltration by donor T cells is critical to the development of acute graft-versus-host disease (aGVHD) in recipients after allogeneic hematopoietic stem cell transplant (allo-HCT). However, deconvoluting the transcriptional programs of newly recruited donor T cells from those of tissue-resident T cells in aGVHD target organs remains a challenge. Here, we combined the Serial Intravascular Staining technique with single-cell RNA-seq to dissect the tightly connected processes by which donor T cells initially infiltrate tissues and then establish a pathogenic tissue-residency program in a rhesus macaque allo-HCT model that develops aGVHD. Our results enabled creation of a spatiotemporal map of the transcriptional programs controlling donor CD8(+) T cell infiltration into the primary aGVHD target organ, the gastrointestinal (GI) tract. We identified the large and small intestines as the only two sites demonstrating allo-specific, rather than lymphodepletion-driven, T cell infiltration. GI-infiltrating donor CD8(+) T cells demonstrated a highly activated, cytotoxic phenotype while simultaneously developing a canonical tissue-resident memory T cell (T(RM)) transcriptional signature driven by IL-15/IL-21 signaling. We discovered expression of a cluster of genes directly associated with tissue invasiveness, including those encoding adhesion molecules (ITGB2), specific chemokines (CCL3, CCL4L1) and chemokine receptors (CD74), as well as multiple cytoskeletal proteins. This tissue invasion transcriptional signature was validated by its ability to discriminate the CD8(+) T cell transcriptome of patients with GI aGVHD from those of GVHD-free patients. These results provide insights into the mechanisms controlling tissue occupancy of target organs by pathogenic donor CD8(+) T(RM) cells during aGVHD in primate transplant recipients.

Introduction: The gut microbiome is a potentially modifiable factor in treatment related outcomes in allogeneic hematopoietic cell transplant (HCT). Prior studies have linked pre- or mid-treatment gut microbiome diversity with risk for treatment related morbidity and mortality. However, these studies have been limited by the inclusion of one or only a few institutions and the lack of longitudinal sampling with high quality metadata. These limitations complicate the interpretation of microbiome alterations over the course of HCT. Methods: To overcome these, we devised and implemented a large-scale biospecimen collection protocol in conjunction with BMT CTN 1703, a randomized, multicenter, Phase III trial of tacrolimus/methotrexate vs. post-transplant cyclophosphamide/tacrolimus/mycophenolate mofetil in reduced intensity conditioning (RIC) allogeneic HCT (NCT03959241). Patients enrolled on 1703 were optionally co-enrolled in the companion immune and microbiome profiling study, 1801 (MI-IMMUNE). This involved blood, urine and stool sampling before conditioning (PCON), then weekly starting on day 0 through day 77 (through day 84 for blood), and on days 98, 180, 270, 365 and 730. For all enrolled participants where the donor consented, residual donor cells were saved from the empty hematopoietic stem cell product bag for later analysis. Additionally, the protocol included one-time blood, urine and stool sample collection from consented matched related donors (MRDs) prior to stem cell collection. Starting with protocol version 4.0 on February 1, 2021, participation in 1801 stool collection was required for the first six out of eighteen sample collection timepoints. Participation in later stool and urine timepoint collections remained optional. Here we review the feasibility of creating a multi-institutional biobank. Additionally, we assess the success of our strategies by calculating sample collection compliance and standard deviation in compliance across centers for each timepoint. Results: On June 18, 2021, BMT CTN 1703/1801 closed to accrual with 431 patients enrolled on 1703; 323 patients from 36 centers were co-enrolled on 1801. 304 (94%) provided study samples, making this the largest prospective microbiome and immune profiling study in HCT patients to date. As of July 6, 2021, 3,683 blood, 2,668 urine, and 2,098 stool samples had been collected. Across the first 6 timepoints for all participating centers, blood, urine and stool sample collection averaged 93%, 82%, and 74% compliance, respectively. Of the 99 (30%) patients enrolled on 1801 with a MRD, 34 (34%) donors consented to sample collection. Sample collection compliance was lower for MRDs than for patients on the study with 76%, 74%, and 62% of expected blood, urine and stool samples collected, respectively, from this group. For stool collection exclusively, a median of 5 samples were collected per patient across the first 6 timepoints (median of 6 across all timepoints) with 93 (31%) of patients completing a full sample set through Day 28. 139 (46%) patients provided at least one sample after day 28; these represented 37% of the total samples collected to date. The PCON sample, which provides an important measure of pre-treatment gut microbiome diversity, had the third highest compliance with 74% of patients providing a sample. Surprisingly, Day 28 had the lowest compliance (66%) and highest standard deviation (37%) possibly because this timepoint often falls around the time of hospital discharge. Between PCON and day 28, the standard deviation between sites in the average collection compliance (24%) and number of samples collected per patient (1.1) was small indicating the successful adoption of stool collection across institutions. Table 1 summarizes sample collection statistics. Conclusion: Overall this study has resulted in a large, novel biobank of blood, urine and stool samples from patients undergoing RIC allogeneic HCT at 36 centers across the US. This will serve as a valuable resource for investigating the role of the gut microbiome in long term health outcomes following HCT. Although the results of 1801 are forthcoming given ongoing sample collection, the size and composition of the biobank to date clearly demonstrate the feasibility of implementing multi-institutional stool collection. This study represents a critical step towards the large-scale adoption of microbiome sampling as a diagnostic tool. Figure 1 Figure 1. Disclosures Chhabra: GSK: Honoraria. Clark: Kadmon: Consultancy. Horowitz: Mesoblast: Research Funding; Shire: Research Funding; Vertex: Research Funding; Stemcyte: Research Funding; Vor Biopharma: Research Funding; Janssen: Research Funding; Miltenyi Biotech: Research Funding; Kiadis: Research Funding; Sobi: Research Funding; Kite/Gilead: Research Funding; Pfizer, Inc: Research Funding; Jazz Pharmaceuticals: Research Funding; Magenta: Consultancy, Research Funding; Medac: Research Funding; Novartis: Research Funding; GlaxoSmithKline: Research Funding; Daiicho Sankyo: Research Funding; Xenikos: Research Funding; Omeros: Research Funding; Orca Biosystems: Research Funding; Pharmacyclics: Research Funding; Regeneron: Research Funding; Tscan: Research Funding; Takeda: Research Funding; CSL Behring: Research Funding; Genentech: Research Funding; Gamida Cell: Research Funding; Chimerix: Research Funding; Bristol-Myers Squibb: Research Funding; bluebird bio: Research Funding; Astellas: Research Funding; Amgen: Research Funding; Allovir: Consultancy; Actinium: Research Funding; Sanofi: Research Funding; Seattle Genetics: Research Funding. Jenq: Microbiome DX: Consultancy; Merck: Consultancy; Prolacta: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kaleido: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seres: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; LisCure: Consultancy, Membership on an entity's Board of Directors or advisory committees; MaaT Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Karius: Consultancy. Levine: Equillium Bio: Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Research Funding; Kamada: Research Funding; Biogen: Research Funding; Omeros: Membership on an entity's Board of Directors or advisory committees; Symbio: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Talaris Therapeutics: Membership on an entity's Board of Directors or advisory committees; Viracor: Patents & Royalties: GVHD biomarker patent with royalties from Viracor; Mesoblast: Consultancy, Research Funding; X4 Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Murthy: CRISPR Therapeutics: Research Funding. Riches: ATARA Biotherapeutics: Other: Payment; BioIntelect: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Other: Payment. Sung: Merck: Research Funding; Novartis: Research Funding; Enterome: Research Funding; Seres: Research Funding; AVROBIO: Consultancy; Abbott Nutrition: Honoraria; Clasado: Other: Research Product; DSM: Other: Research Product. Al Malki: Neximmune: Consultancy; Jazz Pharmaceuticals, Inc.: Consultancy; Rigel Pharma: Consultancy; Hansa Biopharma: Consultancy; CareDx: Consultancy. Rezvani: Kaleido: Other: One-time scientific advisory board; Nohla Therapeutics: Other: One-time scientific advisory board; Pharmacyclics-Abbvie: Research Funding; US Department of Justice: Consultancy. Bolaños-Meade: Incyte Corp: Consultancy. Holtan: Incyte: Consultancy, Research Funding; Generon: Consultancy. Saber: Govt. COI: Other. Hamadani: Sanofi, Genzyme, AstraZeneca, BeiGene: Speakers Bureau; Janssen, Incyte, ADC Therapeutics, Omeros, Morphosys, Kite: Consultancy; Takeda, Spectrum Pharmaceuticals and Astellas Pharma: Research Funding. Kean: Bluebird Bio: Research Funding; Bristol Myers Squibb: Patents & Royalties: From clinical trial data, Research Funding; Vertex: Consultancy; Novartis: Consultancy; Gilead: Research Funding; Regeneron: Research Funding; EMD Serono: Consultancy. Perales: Cidara: Honoraria; Servier: Honoraria; Incyte: Honoraria, Other; Equilium: Honoraria; Takeda: Honoraria; Novartis: Honoraria, Other; Nektar Therapeutics: Honoraria, Other; NexImmune: Honoraria; MorphoSys: Honoraria; Omeros: Honoraria; Karyopharm: Honoraria; Sellas Life Sciences: Honoraria; Merck: Honoraria; Miltenyi Biotec: Honoraria, Other; Medigene: Honoraria; Bristol-Myers Squibb: Honoraria; Kite/Gilead: Honoraria, Other; Celgene: Honoraria.

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.

Allogeneic hematopoietic cell transplantation (HCT) may be curative for patients with marrow and immune disorders, but graft-vs-host-disease (aGVHD) and infections cause significant morbidity and non-relapse mortality. We have conducted a multicenter, double blind, placebo-controlled phase II trial of costimulation blockade with abatacept (Aba) combined with standard GVHD prophylaxis with a calcineurin inhibitor and methotrexate (CNI + MTX) following HLA matched unrelated donor transplant (n=142). In order to assess the effects of Aba on immune reconstitution, and to assess whether this reconstitution is influenced during CMV reactivation, we longitudinally evaluated post-transplant whole blood samples with multiparameter flow cytometry using markers for CD3, CD4, CD8, CD197 and CD45RA to measure reconstitution of CD4 and CD8 T cell populations and their respective memory subsets over time. Results: We observe that post-transplant CMV reactivation induces a marked expansion of CD8 effector memory (EM) cells, which is similar in magnitude for Aba vs placebo patients. We found that development of moderate (gr 2-4) or severe (gr 3-4) GVHD was not associated with an increased frequency of CMV reactivation, but patients with moderate GVHD showed a blunted expansion of CD8 EM cells compared to those without GVHD, and CD8 EM expansion was essentially absent among CMV reactivating patients with severe aGVHD. Clinical correlates will be presented. Conclusions: Our results suggest that adding abatacept to CNI/MTX does not materially affect reconstitution of T cell immunity in the presence or absence of CMV reactivation, but aGVHD remains a major driver of compromised immune recovery after HCT. Disclosures Watkins: Bristol Myers Squibb: Research Funding. Qayed:Mesoblast: Consultancy; Novartis: Consultancy. Horan:Bristol Myers Squib: Honoraria, Research Funding. Kean:gilead: Research Funding; bluebird bio: Research Funding; fortyseven: Consultancy; magenta: Research Funding; regeneron: Research Funding; hifibio: Consultancy; kymab: Consultancy; Bristol Meyers Squibb: Research Funding; novartis: Consultancy. Langston:Kadmon Corporation: Research Funding; Bristol Myers Squib: Research Funding; Incyte: Research Funding; Chimerix: Research Funding; Takeda: Research Funding; Astellas Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Research Funding.

Allogeneic hematopoietic cell transplantation (HCT) may be curative for patients with marrow and immune disorders, but acute graft-vs-host-disease (aGVHD) and infections cause significant morbidity and non-relapse mortality. We conducted a multicenter, double blind, placebo-controlled phase II trial of costimulation blockade with abatacept (Aba) combined with standard GVHD prophylaxis with a calcineurin inhibitor and methotrexate (CNI + MTX) following HLA matched unrelated donor transplant (n=140). In order to assess whether Aba produces clinically relevant compromise of immune reconstitution, and whether this reconstitution is influenced during CMV reactivation, we longitudinally evaluated post-transplant whole blood samples with multiparameter flow cytometry using markers for CD3, CD4, CD8, CD197 and CD45RA to measure reconstitution of CD4 and CD8 T cell populations and their respective memory subsets over time. We show here that post-transplant CMV reactivation induces a marked expansion of CD8 effector memory (EM) cells, which is similar in Aba vs placebo pts. We found that development of moderate (gr 2–4) or severe (gr 3–4) GVHD does not cause more CMV reactivation in patients, but patients with moderate GVHD show a blunted expansion of CD8 EM cells compared to those without GVHD. Remarkably, CD8 EM expansion was essentially absent among CMV reactivating patients with severe aGVHD. Our results suggest that adding abatacept to CNI/MTX does not materially affect reconstitution of T cell immunity in the presence or absence of CMV reactivation, but aGVHD remains a major driver of compromised immune recovery after HCT.

We performed a Ph2 trial in adults and children to test abatacept for AGVHD prevention (‘ABA2', Clinicaltrials # NCT01743131), based on our promising preclinical and pilot patient data. ABA2 had 2 cohorts: A) HLA-mismatched (‘7/8', n = 43), a single-arm study with pre-specified CIBMTR matched analysis (vs CNI+MTX or CNI+MTX+ATG). B) HLA-matched (‘8/8', n = 142), randomized double-blind, comparing CNI+MTX+placebo vs CNI+MTX+ABA (‘ABA'). For each ABA arm, patients received 4 doses of 10mg/kg on d -1,5,14,28. ABA2 was designed as a screening Ph2 trial, with relaxed Type 1 error (0.2) and standard Type 2 error (0.2). Power analysis assumed ABA would decrease Gr 3-4 AGVHD from 30%–>10% in 7/8s and from 20%–>10% in 8/8s. Here we report top-line results for the 7/8s (median f/u = 708d, 264-1491) and 8/8s (median f/u = 369d, 180-1175). Reduced Grade 3-4 AGVHD: ABA was associated with decreased d180 Gr 3-4 AGVHD. In 7/8s, Gr 3-4 AGVHD = 2.5% in ABA (1 event/43 patients) vs 31% (CNI+MTX) and 22% (+ATG), 1 sided p = 0.001, 0.005,). In 8/8s, Gr 3-4 AGVHD = 6.85 % (5 events/73 patients) in ABA vs 14.6% (10 events) in placebo, 1 sided p = 0.068). Reduced Grade 2-4 AGVHD in 8/8s: ABA was associated with decreased d180 Gr 2-4 AGVHD. In 7/8s, Gr 2-4 AGVHD = 42% (ABA) vs 54% (CNI+MTX) and 45% (+ATG, 1 sided p = 0.098, 0.25). In 8/8s, Gr 2-4 AGVHD = 44.5% in ABA vs 62.3% in placebo (1 sided p = 0.004). Chronic GVHD: For 7/8s, 1 yr CGVHD = 38.8% (ABA) vs 43.5% (CNI+MTX) and 35.5% (+ATG, p = 0.4, 0.99). For 8/8s, follow up is still too short to adequately evaluate, and adjudication is ongoing. No Increase In Relapse: In 7/8s, relapse = 9.37% at 1 y vs 12.9% in CNI+MTX and 13.6% in +ATG (p = 0.115 and 0.085). In 8/8s, relapse = 13.8% at 1y (ABA) vs 20.5% (placebo, p = 0.7). Remarkably, in 7/8s, where median follow up=708d, there have been no further relapses reported in ABA2, with 9.37% relapse at 2 yr vs 20.63% (CNI+MTX) and 23.4% (ATG), despite matched disease risk. Statistically significant survival advantage in 7/8s: TRM: For 7/8s, 1 yr TRM = 10.5% (ABA) vs 32.7% (CNI+MTX) and 26% (+ATG, p = 0.024, 0.365). For 8/8s, TRM = 7.1% vs 14.6% at 1 yr (p = 0.5). Relapse-Free Survival (RFS): For 7/8s, RFS = 73.7% (ABA) vs 38.7% in CNI+MTX and 48.7% in +ATG (p = 0.001 and 0.027). For 8/8s, RFS = 79.1% for ABA vs 64.9% (placebo, p = 0.38). OS: For 7/8s, OS = 71% (ABA) vs 47.5% (CNI+MTX) and 58% (+ATG, p = 0.01 and 0.145). For 8/8s, OS = 83.2% (ABA) vs 76.6 (placebo, p = 0.32). Our results suggest that short-course (4 doses) ABA can safely prevent AGVHD without compromising relapse. While this was a modestly sized study (7/8s: n = 43, 8/8 n = 142), the comparative event size for 7/8s was high enough that the protective effect of ABA was highly statistically significant. For 8/8s, there was a statistically significant improvement for Gr 2-4 GVHD and a trend toward an advantage for ABA in all other parameters. These results are the first to demonstrate efficacy of in vivo T cell costimulation blockade in preventing AGVHD.

Although acute graft-versus-host-disease (AGVHD) is one of the major causes of non-relapse mortality after hematopoietic stem cell transplant (HCT), we are still unable to predict which patients will develop the most severe form of this disease, or which molecular pathways are dysregulated in the T cells that cause disease. Thus, understanding the molecular features of AGVHD is a critical unmet need. To address this, we have performed a companion mechanistic study as a part of our completed Phase 2 trial of abatacept, a CD28:CD80/86 costimulation blockade agent, for severe AGVHD prevention (Clinicaltrials.gov # NCT01743131, 'ABA2'). ABA2 has demonstrated significant improvement in AGVHD in patients prophylaxed with abatacept in addition to calcineurin inhibition (CNI) + Methotrexate (MTX) compared to controls receiving CNI/MTX alone. To begin to uncover mechanisms responsible for the control of AGVHD with abatacept, and given that CD4+ T cells have been consistently documented to be the main therapeutic target of this drug, we interrogated the transcriptome of CD4+ T cells reconstituting in patients prophylaxed with abatacept compared to CNI/MTX. To perform this analysis, we flow cytometrically sorted CD4+ T cells on Days 21-28 post-transplant from all patients on ABA2, as well as a cohort of 12 untransplanted healthy controls, and subsequently performed mRNA-sequencing on these cells. Weighted Gene Correlation Network Analysis (WGCNA) was performed on the top 6000 most variant transcripts from the resulting sequencing data. Hierarchical clustering of the WGCNA co-expression matrix enabled the identification of self-assembling modules (SAMs) that met a threshold of coexpression (Figure 1A). For the ABA2 dataset, we considered the following variables in the WGCNA model: patient cohort (7/8 patients, 8/8 patients, healthy controls), +/- prophylaxis with abatacept, CMV reactivation, EBV reactivation, Grade of GVHD (0-4), relapse, non-relapse mortality, and all-cause mortality. The WGCNA clustering analysis resulted in the identification of 4 discrete SAMs, which were highly correlated with clinical variable metamodules. This analysis revealed a strong positive correlation of a 476-gene SAM (the Turquoise module) in patients prophylaxed with CNI/MTX + placebo and anti-correlation of this module in patients prophylaxed with CNI/MTX + abatacept, as demonstrated in both the WGCNA heatmap and through Gene Set Enrichment Analysis (Figure 1 A-B). These opposing correlations suggested that interrogation of this module would reveal mechanistic correlates with standard prophylaxis that were decoupled by abatacept. Pathway analysis using the Reactome database (Figure 1C) revealed the turquoise SAM to be dominated by four types of pathways: (1) Those that define canonical cell-cycle pathways (2) Those involved in T cell metabolism (3) Those involved in apoptosis and (4) Those involved in T cell activation, consistent with upregulation of these transcripts in placebo versus abatacept patients. In addition to being highly correlated with patients receiving placebo, the expression of a subset of the transcripts in the Turquoise module were also directly correlated with the severity of AGVHD in these patients. Thus, linear regression analysis of the 476 transcripts in this module identified a subset of 93 genes for which transcript expression level was increased both in placebo compared to abatacept, and for which expression level also positively correlated with Grade of AGVHD. As with the Turquoise module as a whole, this subset of genes also formed a highly correlated network, linking transcripts involved in T cell proliferation, apoptosis, activation, metabolism as well as the T cell checkpoint (Figure 1D). This analysis represents the first comprehensive interrogation of the transcriptomic correlates of AGVHD. It identifies a novel set of transcripts which positively associate with the severity of AGVHD, and which costimulation blockade with abatacept down-regulates and de-couples from AGVHD severity. These results suggest a profound reprograming of T cell activation with abatacept that is correlated with the control of AGVHD. Disclosures Qayed: Bristol-Myers Squibb: Honoraria. Langston:Astellas Pharma: Other: Research Support; Incyte: Other: Research Support; Jazz Pharmaceuticals: Other: Research Support; Chimerix: Other: Research Support; Takeda: Other: Research Support; Kadmon Corporation: Other: Research Support; Novartis: Other: Research Support; Bristol Myers Squibb: Other: Research Support. Blazar:Fate Therapeutics, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; Abbvie Inc: Research Funding; Leukemia and Lymphoma Society: Research Funding; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-Founder; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees. Kean:HiFiBio: Consultancy; BlueBirdBio: Research Funding; Gilead: Research Funding; Regeneron: Research Funding; EMDSerono: Consultancy; FortySeven: Consultancy; Magenta: Research Funding; Kymab: Consultancy; Jazz: Research Funding; Bristol Meyers Squibb: Patents & Royalties, Research Funding. OffLabel Disclosure: Abatacept: Approved for Rheumatoid Arthritis; used in this trial for prevention of GVHD.