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We previously described clinically relevant reductions in fecal microbiota diversity in patients undergoing allogeneic hematopoietic cell transplantation (allo-HCT). Recipients of high-dose chemotherapy and autologous HCT (auto-HCT) incur similar antibiotic exposures and nutritional alterations. To characterize the fecal microbiota in the auto-HCT population, we analyzed 1161 fecal samples collected from 534 adult recipients of auto-HCT for lymphoma, myeloma, and amyloidosis in an observational study conducted at 2 transplantation centers in the United States. By using 16S ribosomal gene sequencing, we assessed fecal microbiota composition and diversity, as measured by the inverse Simpson index. At both centers, the diversity of early pretransplant fecal microbiota was lower in patients than in healthy controls and decreased further during the course of transplantation. Loss of diversity and domination by specific bacterial taxa occurred during auto-HCT in patterns similar to those with allo-HCT. Above-median fecal intestinal diversity in the periengraftment period was associated with decreased risk of death or progression (progression-free survival hazard ratio, 0.46; 95% confidence interval, 0.26-0.82; P = .008), adjusting for disease and disease status. This suggests that further investigation into the health of the intestinal microbiota in auto-HCT patients and posttransplant outcomes should be undertaken.

A goal in precision medicine is to use patient-derived material to predict disease course and intervention outcomes. Here, we use mechanistic observations in a preclinical animal model to design an ex vivo platform that recreates genetic susceptibility to T-cell–mediated damage. Intestinal graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation. We found that intestinal GVHD in mice deficient in Atg16L1, an autophagy gene that is polymorphic in humans, is reversed by inhibiting necroptosis. We further show that cocultured allogeneic T cells kill Atg16L1-mutant intestinal organoids from mice, which was associated with an aberrant epithelial interferon signature. Using this information, we demonstrate that pharmacologically inhibiting necroptosis or interferon signaling protects human organoids derived from individuals harboring a common ATG16L1 variant from allogeneic T-cell attack. Our study provides a roadmap for applying findings in animal models to individualized therapy that targets affected tissues.

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.

Patients with multiple myeloma (MM) who achieve minimal residual disease (MRD) negativity after upfront treatment have superior outcomes compared with those who remain MRD+. Recently, associations have been shown between specific commensal microbes and development of plasma cell disorders. Here, we report the association between intestinal microbiota composition and treatment outcome in MM. Microbiota composition of fecal samples collected from 34 MM patients after induction therapy and at the time of flow cytometry–based bone marrow MRD testing was determined by 16S ribosomal RNA sequencing. We observed a higher relative abundance of Eubacterium hallii in the 16 MRD− patients relative to the 18 MRD+ patients. No association was observed between microbial relative abundance and autologous stem cell transplantation history or MM paraprotein isotype. No differences in microbiota α diversity were observed between MRD− and MRD+ patients. The potential association of microbiota composition with treatment response in MM patients is an important parameter for additional correlative and clinical investigation.

Studies of the relationship between the gastrointestinal microbiota and outcomes in allogeneic hematopoietic stem cell transplantation (allo-HCT) have thus far largely focused on early complications, predominantly infection and acute graft-versus-host disease (GVHD). We examined the potential relationship of the microbiome with chronic GVHD (cGVHD) by analyzing stool and plasma samples collected late after allo-HCT using a case-control study design. We found lower circulating concentrations of the microbe-derived short-chain fatty acids (SCFAs) propionate and butyrate in day 100 plasma samples from patients who developed cGVHD, compared with those who remained free of this complication, in the initial case-control cohort of transplant patients and in a further cross-sectional cohort from an independent transplant center. An additional cross-sectional patient cohort from a third transplant center was analyzed; however, serum (rather than plasma) was available, and the differences in SCFAs observed in the plasma samples were not recapitulated. In sum, our findings from the primary case-control cohort and 1 of 2 cross-sectional cohorts explored suggest that the gastrointestinal microbiome may exert immunomodulatory effects in allo-HCT patients at least in part due to control of systemic concentrations of microbe-derived SCFAs.

Background Acute graft-versus-host disease (aGVHD) remains a major limitation of allogeneic hematopoietic cell transplantation (allo-HCT) despite prophylactic immunosuppression. Not all patients who develop aGVHD respond to currently available treatment and thus there is a pressing need for novel prophylactic and therapeutic strategies. Interleukin-2-Inducible T-Cell Kinase (ITK) is a Tec-family, non-receptor tyrosine kinase expressed in T-cells, homologous with Bruton's tyrosine kinase (BTK) in B cells and is involved in proximal T-cell receptor (TCR) signaling, activating Phospholipase Cγ1 (PLCγ1) and initiating a signaling cascade that includes the NFAT, NF-κB, and MAPK pathways tuning T-cell activation, proliferation, and differentiation. Targeting ITK (ITK-/- in T cell graft and short-term exposure of donor graft to ITK inhibitor) has been shown to be associated with less GVHD in preclinical studies (Mammadli et al 2020, Kondo et al 2021). CPI-818 is a highly selective irreversible ITK inhibitor that covalently binds to cysteine 442 of ITK and abolishes kinase activity. In vitro assays demonstrate that CPI-818 inhibits phosphorylation of the ITK substrate PLC γ1 (Y783) and downstream signaling molecules ERK and S6, and blocks IL-2 secretion. CPI-818 reduced clinical and histological disease severity in a Th1-driven T cell adoptive transfer model of colitis (not published). CPI-818 is currently being evaluated in a Phase 1/1b study in patients with relapsed/refractory T-cell lymphoma (NCT03952078), and thus far appears well tolerated. Methods and Results We used 2 well established preclinical aGVHD models (MHC-disparate B6 → BALB/c and minor MHC-mismatched B6 → 129) to explore the effects of ITK inhibition by CPI-818. Recipient mice were treated with CPI-818-formulated (300 mg/kg/day) or control diet from day -7 to day 90 relative to allo-HCT. We found significantly improved survival (MHC-disparate: p Conclusion These data in two clinically relevant GVHD models demonstrate that ITK inhibition has potential as a novel targeted approach to prevent aGVHD through a) the suppression of T cell activation and proliferation, b) decreased concentrations of pro-inflammatory cytokines and increased concentration of anti-inflammatory cytokines. CPI-818 is the most potent and selective ITK inhibitor reported to date and these data highlight its promise as a novel agent for the prevention of aGVHD. Figure 1 Figure 1. Disclosures Smith: Janssen: Consultancy, Honoraria. Perales: Celgene: Honoraria; Kite/Gilead: Honoraria, Other; MorphoSys: Honoraria; Merck: Honoraria; Incyte: Honoraria, Other; Cidara: Honoraria; Bristol-Myers Squibb: Honoraria; Omeros: Honoraria; Novartis: Honoraria, Other; Medigene: Honoraria; Karyopharm: Honoraria; Nektar Therapeutics: Honoraria, Other; Equilium: Honoraria; Takeda: Honoraria; Sellas Life Sciences: Honoraria; NexImmune: Honoraria; Miltenyi Biotec: Honoraria, Other; Servier: Honoraria. Buggy: Corvus Pharmaceuticals Inc: Consultancy, Current holder of individual stocks in a privately-held company. Janc: Corvus Pharmaceuticals Inc: Current Employment, Current holder of individual stocks in a privately-held company. van den Brink: Forty-Seven, Inc.: Honoraria; WindMILTherapeutics: Honoraria; Kite Pharmaceuticals: Other; Da Volterra: Other: has consulted, received honorarium from or participated in advisory boards; Notch Therapeutics: Honoraria; Pluto Therapeutics: Current holder of stock options in a privately-held company, Other: has consulted, received honorarium from or participated in advisory boards ; GlaskoSmithKline: Other: has consulted, received honorarium from or participated in advisory boards; Synthekine (Spouse): Other: has consulted, received honorarium from or participated in advisory boards; Priothera: Research Funding; Wolters Kluwer: Patents & Royalties; Pharmacyclics: Other; Rheos: Honoraria; Nektar Therapeutics: Honoraria; Ceramedix: Other: has consulted, received honorarium from or participated in advisory boards ; Novartis (Spouse): Other: has consulted, received honorarium from or participated in advisory boards; Lygenesis: Other: has consulted, received honorarium from or participated in advisory boards ; Frazier Healthcare Partners: Honoraria; DKMS (nonprofit): Other; Juno Therapeutics: Other; MagentaTherapeutics: Honoraria; Merck & Co, Inc: Honoraria; Amgen: Honoraria; Therakos: Honoraria; Jazz Pharmaceuticals: Honoraria; Seres: Other: Honorarium, Intellectual Property Rights, Research Fundingand Stock Options.

Background and Scientific Rationale: Multiple myeloma (MM) is often preceded by the premalignant conditions monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM). Obesity, low adiponectin levels, and diets high in inflammatory, insulinemic foods or lacking plant-based foods are known risk factors for the development of MGUS/SMM, as well as for progression to MM. Therefore, there is an opportunity to study a dietary intervention in cancer progression among patients with MGUS/SMM, for which the standard of care is observation even though some patients will eventually progress to MM. This is a pilot nutrition-based intervention study of a whole food, plant-based diet (WFPBD) in overweight and obese MGUS and SMM patients to enable weight loss, assess associated changes in disease biomarkers, epigenetics, and the gut microbiome. We expect that the findings will enable larger lifestyle-based studies of prevention and survivorship in plasma cell disorders. Study Design and Methods: This is a single-arm, single-center pilot study of a WFPBD for 12 weeks and nutrition counseling for 24 weeks which will enroll 20 patients (Figure). Clinical trial registry number: NCT04920084, actively recruiting. Study Population and Inclusion Criteria i) SMM or MGUS ii) Body mass index ≥25 iii) Monoclonal protein spike ≥0.2 g/dL or abnormal free light chain ratio with increased level of the appropriate involved light chain iv) ECOG performance status 0-3 v) Willingness to comply with study-related procedures Statistical Methods: The average weight loss from baseline at 12 weeks will be reported as sample mean along with 95% confidence interval. Adherence will be estimated by sample proportion, with confidence intervals based on exact binomial distribution. Patients who have completed evaluation at 12 weeks will be evaluable for the weight loss outcome. All patients who have received at least one WFPBD intervention will be evaluable for adherence assessment. We will consider this intervention promising if 1) we detect weight loss at 12 weeks and 2) estimated adherence to the intervention is ≥70%. Study Treatment: For 12 weeks, patients will receive two premade meals per day, for lunch and dinner for 6 days per week, prepared and shipped by Plantable. The meals will have a low glycemic index and contain vegetables, whole grains, and plant-based fats that have undergone minimal processing. Detailed recommendations for snacks and breakfasts meeting the standard of a WFPBD will also be given to supplement their daily calorie needs with access to an online portal or phone application from Plantable which contains education materials and access to a coach daily for 24 weeks. Patients will receive dietary education and counseling from a research dietitian every 2 weeks for the 12-week intervention period. Endpoints: Primary: - To determine the feasibility of a WFPBD, as measured by weight loss and adherence at 12 weeks. Secondary: - To determine the feasibility of a WFPBD, as measured by safety, and quality of life. - To assess weight loss at 24, and 52 weeks. - To assess alterations in metabolic, and myeloma markers. Exploratory: - To assess alterations in T cell and plasma cell epigenetic markers - To assess alterations in the fecal microbiome - To assess changes in body composition as determined by PET imaging and correlate with weight change as well as disease markers. Figure 1 Figure 1. Disclosures Shah: Janssen: Research Funding; Celgene/BMS: Research Funding. Adintori: Vidafuel Inc.: Current holder of stock options in a privately-held company. Mailankody: Jansen Oncology: Research Funding; Physician Education Resource: Honoraria; Bristol Myers Squibb/Juno: Research Funding; Plexus Communications: Honoraria; Fate Therapeutics: Research Funding; Takeda Oncology: Research Funding; Allogene Therapeutics: Research Funding; Legend Biotech: Consultancy; Evicore: Consultancy. Korde: Medimmune: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding. Hultcrantz: Intellisphere LLC: Consultancy; Amgen: Research Funding; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi Sankyo: Research Funding; Curio Science LLC: Consultancy. Hassoun: Celgene, Takeda, Janssen: Research Funding. D'Souza: Sanofi, Takeda, Teneobio, CAELUM, Prothena: Research Funding; Imbrium, Pfizer, BMS: Membership on an entity's Board of Directors or advisory committees; Janssen, Prothena: Consultancy. Giralt: AMGEN: Membership on an entity's Board of Directors or advisory committees; PFIZER: Membership on an entity's Board of Directors or advisory committees; JAZZ: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; SANOFI: Membership on an entity's Board of Directors or advisory committees; Actinnum: Membership on an entity's Board of Directors or advisory committees; JENSENN: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; CELGENE: Membership on an entity's Board of Directors or advisory committees. Iyengar: Novartis: Consultancy; Seattle Genetics: Consultancy; Novartis: Research Funding. Landgren: Janssen: Other: IDMC; Janssen: Honoraria; Amgen: Honoraria; Celgene: Research Funding; Janssen: Research Funding; Amgen: Research Funding; Takeda: Other: IDMC; GSK: Honoraria. van den Brink: Pluto Therapeutics: Current holder of stock options in a privately-held company, Other: has consulted, received honorarium from or participated in advisory boards ; Novartis (Spouse): Other: has consulted, received honorarium from or participated in advisory boards; Forty-Seven, Inc.: Honoraria; MagentaTherapeutics: Honoraria; Da Volterra: Other: has consulted, received honorarium from or participated in advisory boards; Wolters Kluwer: Patents & Royalties; Ceramedix: Other: has consulted, received honorarium from or participated in advisory boards ; Notch Therapeutics: Honoraria; DKMS (nonprofit): Other; Pharmacyclics: Other; Kite Pharmaceuticals: Other; GlaskoSmithKline: Other: has consulted, received honorarium from or participated in advisory boards; Lygenesis: Other: has consulted, received honorarium from or participated in advisory boards ; Nektar Therapeutics: Honoraria; Rheos: Honoraria; Seres: Other: Honorarium, Intellectual Property Rights, Research Fundingand Stock Options; Amgen: Honoraria; Therakos: Honoraria; WindMILTherapeutics: Honoraria; Juno Therapeutics: Other; Merck & Co, Inc: Honoraria; Frazier Healthcare Partners: Honoraria; Priothera: Research Funding; Synthekine (Spouse): Other: has consulted, received honorarium from or participated in advisory boards; Jazz Pharmaceuticals: Honoraria. Lesokhin: pfizer: Consultancy, Research Funding; Genetech: Research Funding; Trillium Therapeutics: Consultancy; Behringer Ingelheim: Honoraria; Serametrix, Inc: Patents & Royalties; bristol myers squibb: Research Funding; Janssen: Honoraria, Research Funding; Iteos: Consultancy.

Microbial diversity is associated with improved outcome in patients receiving allogeneic hematopoietic cell transplantation (allo-HCT), however, the mechanism underlying this observation is unclear. Unconventional T cells recognize specific metabolites of bacterial biosynthesis and their role in the post-HCT immunity has not yet been fully clarified. Here we have performed an observational study (n = 174 patients) using 16S rRNA sequencing of early post-HCT patient stool samples (day 7-21 after HCT) paired with multiparameter flow cytometry (performed at day 30 and day 100 after HCT) to explore the relationship between the intestinal microbiome early after HCT and the unconventional T cell populations in circulation. Our data extend findings of other groups suggesting that mucosal-associated invariant T (MAIT) cells are dependent on a diverse microbiome and are also associated with favorable allo-HCT outcome. In addition, we report for the first time that the Vδ2 subset of γδ T cells is positively correlated with MAIT cells as well as independent predictors of favorable transplant outcome. We first focused on MAIT cells as these cells respond to metabolites of the bacterial riboflavin biosynthesis pathway and should therefore be responsive to changes in the gut microbiome. MAIT cell frequency on day 30 after HCT was significantly higher in peripheral blood stem cell (PBSC) graft recipients who had higher peri-engraftment stool diversity (day 7-21; p=0.014, n=118, α-diversity measured using Simpson's reciprocal index, Figure A). Patients with higher-than-median MAIT cell frequency had improved 2-year overall survival (p=0.047, n=118 PBSC recipients, Figure B) and lower non-relapse mortality (p=0.031) compared with patients with lower-than-median frequency. High dimensional flow cytometry analysis using clustering algorithms Uniform Manifold Approximation and Projection (UMAP) and Self Organizing Map (FlowSOM) identified the Vδ2 subset of γδ T cells as the only differentially abundant population associated with higher MAIT cell frequency, and furthermore, frequencies of these two cell types were highly correlated (R=0.38, p=2.8e-05, Figure C). Vδ2 cells are activated by the bacterial metabolite, 4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), an intermediate of the microbial isoprenoid biosynthetic pathway. Vδ2 cell frequencies were higher in patients with higher stool α-diversity (p=0.0026, n=118 PBSC recipients, Figure D) and interestingly, appeared protective with regard to acute graft versus host disease (aGVHD), as we observed a higher frequency of Vδ2 cells among patients who had no or grade 1 disease compared with patients who experienced grade 2-4 aGVHD (p=0.013). We next used Linear Discriminant effect Size (LefSE) analysis to identify statistically significant differences between bacterial taxa in our groups of interest (threshold p>0.01 and effect size>4) and observed that higher MAIT and Vδ2 cell numbers are associated with higher abundance of bacteria belonging to the phylum Bacteroidetes and lower MAIT and Vδ2 cell numbers are associated with higher abundance of the members of the phylum Firmicutes. Furthermore, using the PICRUSt2 algorithm, which uses 16S amplicon abundance data to predict the abundance of functional pathways, we observed a significantly increased predicted abundance of HMBPP, the phosphoantigen ligand for Vδ2 cells, in the patients with higher circulating Vδ2 cell frequencies (p=0.00054). Our findings confirm our hypothesis that a diverse microbiota supports the reconstitution of protective unconventional T cell populations, namely MAIT and Vδ2 cells, which are in turn associated with a favorable HCT outcome. Although further studies are needed to dissect the functional contribution of these cells to the post-transplantation immune milieu, our work offers a valuable insight into the interplay between the intestinal microbiome and reconstitution of immune subsets after allo-HCT and may aid in the design of microbiota-targeted interventions. Figure 1 Figure 1. Disclosures Gomes: Xbiome: Current Employment. Zappasodi: iTeos Therapeutics: Consultancy; Astra Zeneca: Research Funding; Bristol Myers Squibb: Research Funding. Ponce: CareDx: Consultancy, Honoraria; Takeda Pharmaceuticals: Research Funding; Generon Pharmaceuticals: Consultancy; Kadmon pharmaceuticals: Consultancy, Honoraria; Ceramedix: Consultancy, Honoraria; Seres Therapeutics: Consultancy, Research Funding. Giralt: JENSENN: Membership on an entity's Board of Directors or advisory committees; SANOFI: Membership on an entity's Board of Directors or advisory committees; AMGEN: Membership on an entity's Board of Directors or advisory committees; PFIZER: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; JAZZ: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; CELGENE: Membership on an entity's Board of Directors or advisory committees; Actinnum: Membership on an entity's Board of Directors or advisory committees. Peled: Seres: Other: Intellectual Property Rights, Research Funding and Travelfees; DaVolterra: Consultancy; Other: Other: Jonathan U. Peled had filed intellectual property applications related to the microbiome (referencenumbers #62/843,849, #62/977,908, and #15/756,845); MaaT Pharma: Consultancy. Perales: Cidara: Honoraria; Celgene: Honoraria; Merck: Honoraria; MorphoSys: Honoraria; Nektar Therapeutics: Honoraria, Other; Equilium: Honoraria; Karyopharm: Honoraria; Takeda: Honoraria; Bristol-Myers Squibb: Honoraria; Kite/Gilead: Honoraria, Other; Medigene: Honoraria; Novartis: Honoraria, Other; Sellas Life Sciences: Honoraria; Servier: Honoraria; Miltenyi Biotec: Honoraria, Other; Omeros: Honoraria; Incyte: Honoraria, Other; NexImmune: Honoraria. van den Brink: Rheos: Honoraria; Merck & Co, Inc: Honoraria; Therakos: Honoraria; Amgen: Honoraria; GlaskoSmithKline: Other: has consulted, received honorarium from or participated in advisory boards; Synthekine (Spouse): Other: has consulted, received honorarium from or participated in advisory boards; Kite Pharmaceuticals: Other; Notch Therapeutics: Honoraria; Nektar Therapeutics: Honoraria; Priothera: Research Funding; Wolters Kluwer: Patents & Royalties; Juno Therapeutics: Other; Frazier Healthcare Partners: Honoraria; DKMS (nonprofit): Other; Seres: Other: Honorarium, Intellectual Property Rights, Research Fundingand Stock Options; Forty-Seven, Inc.: Honoraria; Lygenesis: Other: has consulted, received honorarium from or participated in advisory boards ; WindMILTherapeutics: Honoraria; Pharmacyclics: Other; Jazz Pharmaceuticals: Honoraria; Novartis (Spouse): Other: has consulted, received honorarium from or participated in advisory boards; Da Volterra: Other: has consulted, received honorarium from or participated in advisory boards; Ceramedix: Other: has consulted, received honorarium from or participated in advisory boards ; MagentaTherapeutics: Honoraria; Pluto Therapeutics: Current holder of stock options in a privately-held company, Other: has consulted, received honorarium from or participated in advisory boards .

Introduction: Cellular immunotherapy with CD19-targeted chimeric antigen receptor (CAR) T cells has provided new therapeutic options for patients with high-risk hematologic malignancies. Following this therapy, patients may experience disease relapse or CAR-mediated toxicity due to cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS). Recent studies have confirmed that the intestinal microbiome can modulate the anti-tumor immune response to chemotherapy, immune checkpoint blockade, graft-versus-host disease after allogeneic hematopoietic cell transplantation, and adoptive cellular therapy. The contribution of the intestinal microbiome on the function of CAR T cells in vivo both with respect to their anti-tumor function and their propensity to induce toxicities is not known. Hence, in a multi-center study we analyzed the association between clinical outcomes and (1) antibiotic exposure prior to CAR T cell infusion and (2) the composition and diversity of the fecal microbiome. Methods and Results: We retrospectively collected clinical data and antibiotic exposures from patients with acute lymphoblastic leukemia (ALL, n=91) and non-Hodgkin lymphoma (NHL, n=137) treated with investigational or commercial CD19 CAR T cells at Memorial Sloan Kettering Cancer Center (MSK) and the University of Pennsylvania (Penn). We considered any antibiotic exposure between day -30 and the day of CAR T cell infusion. We focused our analysis on anaerobe-targeting antibiotics used in the setting of neutropenic fever: piperacillin-tazobactam, imipenem-cilastatin, and meropenem (here referred to as "P-I-M"). We found that forty-seven (20.6%) of 228 patients were exposed to P-I-M in the four weeks before CAR T cell infusion. Patient characteristics at the time of CAR T cell infusion were similar between the P-I-M-exposed and not-exposed groups, although a worse performance status was observed in patients with NHL treated with P-I-M. We found that overall survival (OS) was significantly decreased following CAR T cell infusion in patients exposed to P-I-M (Fig 1A; OS HR, 2.58; 95% CI, 1.68 - 3.98; p= We also prospectively collected baseline fecal samples prior to cell infusion from CD19 CAR T cells recipients (n=48) at MSK and Penn. Samples were submitted for 16S RNA sequencing of the V4-V5 region on the Illumina MiSeq platform and the amplicon sequence variants (ASVs) were annotated according to the NCBI 16S database using BLAST. In comparison to healthy controls (n=30), we found that alpha-diversity was significantly lower in fecal samples from CAR T cell patients (p= 0.0023, Fig 1B) and the composition of fecal samples was significantly different (p= Conclusion: Our results suggest that exposure to antibiotics, in particular P-I-M, in the four weeks before therapy was associated with worse survival. Profiling of the baseline fecal microbiome samples by 16S revealed that CD19 CAR T cell patients presented with evidence of an altered fecal microbiome as measured by lower alpha-diversity and a composition that is distinct from that of healthy controls. Finally, we identified bacterial taxa that were associated with Day 100 CR and CAR-mediated toxicity. Our findings indicate that the intestinal microbiome can affect the efficacy of CD19 CAR T cell therapy and provides a rationale to target the intestinal microbiome to improve clinical outcomes of patients treated with cellular therapies. Figure 1 Figure 1. Disclosures Smith: Janssen: Consultancy, Honoraria. Gomes: Xbiome: Current Employment. Schluter: Postbiotics Plus LLC: Other: cofounder. Park: Kura Oncology: Consultancy; BMS: Consultancy; Servier: Consultancy; Autolus: Consultancy; Curocel: Consultancy; Artiva: Consultancy; Kite Pharma: Consultancy; Amgen: Consultancy; Novartis: Consultancy; Affyimmune: Consultancy; Intellia: Consultancy; Innate Pharma: Consultancy; Minerva: Consultancy; PrecisionBio: Consultancy. Palomba: Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Kite Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Jain: Targeted Healthcare Communications: Consultancy; Bristol Myers Squibb: Other: for advisory board participation; CareDx: Other: for advisory board participation; CTI Biopharma: Research Funding; Syneos Health: Research Funding. Pennisi: Gilead Sciences: Consultancy. Perales: Miltenyi Biotec: Honoraria, Other; Novartis: Honoraria, Other; Omeros: Honoraria; NexImmune: Honoraria; Bristol-Myers Squibb: Honoraria; Merck: Honoraria; Celgene: Honoraria; Takeda: Honoraria; Kite/Gilead: Honoraria, Other; Medigene: Honoraria; Nektar Therapeutics: Honoraria, Other; Cidara: Honoraria; Servier: Honoraria; Sellas Life Sciences: Honoraria; Karyopharm: Honoraria; MorphoSys: Honoraria; Equilium: Honoraria; Incyte: Honoraria, Other. Garfall: Amgen: Honoraria; CRISPR Therapeutics: Research Funding; GlaxoSmithKline: Honoraria; Janssen: Honoraria, Research Funding; Novartis: Research Funding; Tmunity: Research Funding. Landsburg: Triphase: Research Funding; Morphosys: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Other: DSMB member; Incyte: Membership on an entity's Board of Directors or advisory committees; ADCT: Membership on an entity's Board of Directors or advisory committees; Curis: Research Funding; Takeda: Research Funding. Gerson: Kite: Consultancy; Pharmacyclics: Consultancy; Abbvie: Consultancy; TG Therapeutics: Consultancy. Svoboda: Imbrium: Consultancy; Genmab: Consultancy; Astra Zeneca: Consultancy, Research Funding; Atara: Consultancy; BMS: Consultancy, Research Funding; Adaptive: Consultancy, Research Funding; Incyte: Research Funding; Merck: Research Funding; Pharmacyclics: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding; TG: Research Funding. Giralt: AMGEN: Membership on an entity's Board of Directors or advisory committees; PFIZER: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; SANOFI: Membership on an entity's Board of Directors or advisory committees; CELGENE: Membership on an entity's Board of Directors or advisory committees; JAZZ: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; JENSENN: Membership on an entity's Board of Directors or advisory committees; Actinnum: Membership on an entity's Board of Directors or advisory committees. Gill: Interius Biotherapeutics: Current holder of stock options in a privately-held company, Research Funding; Novartis: Other: licensed intellectual property, Research Funding; Carisma Therapeutics: Current holder of stock options in a privately-held company, Research Funding. Rivière: FloDesign Sonics: Other: Provision of Services; Centre for Commercialization of Cancer Immunotherapy: Other: Provision of Services; Fate Therapeutics: Other: Provision of Services, Patents & Royalties; The Georgia Tech Research Corporation (GTRC): Other: Provision of Services (uncompensated); Juno Therapeutics: Patents & Royalties. Porter: Kite/Gilead: Membership on an entity's Board of Directors or advisory committees; Wiley and Sons Publishing: Honoraria; Tmunity: Patents & Royalties; Novartis: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; ASH: Membership on an entity's Board of Directors or advisory committees; DeCart: Membership on an entity's Board of Directors or advisory committees; Genentech: Current equity holder in publicly-traded company, Ended employment in the past 24 months; American Society for Transplantation and Cellular Therapy: Honoraria; National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees. Schuster: Abbvie: Consultancy, Research Funding; Acerta Pharma: Consultancy; AstraZeneca: Consultancy; Adaptive Biotechnologies: Research Funding; BeiGene: Consultancy; Celgene: Consultancy, Honoraria, Research Funding; DTRM: Research Funding; Genetech: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Incyte: Research Funding; Juno Theraputics: Consultancy, Research Funding; Loxo Oncology: Consultancy; Merck: Research Funding; Nordic Nanovector: Consultancy; Novartis: Consultancy, Honoraria, Patents & Royalties, Research Funding; Pharmaclcyclics: Research Funding; Tessa Theraputics: Consultancy; TG Theraputics: Research Funding. Sadelain: NHLBI Gene Therapy Resource Program: Other: Provision of Services (uncompensated); Fate Therapeutics: Other: Provision of Services (uncompensated), Patents & Royalties; Atara Biotherapeutics: Patents & Royalties; Ceramedix: Patents & Royalties; Mnemo Therapeutics: Patents & Royalties; Takeda Pharmaceuticals: Other: Provision of Services, Patents & Royalties; St. Jude Children's Research Hospital: Other: Provision of Services; Juno Therapeutics: Patents & Royalties; Minerva Biotechnologies: Patents & Royalties. Frey: Novartis: Research Funding; Kite Pharma: Consultancy; Sana Biotechnology: Consultancy; Syndax Pharmaceuticals: Consultancy. Brentjens: Gracell Biotechnologies, Inc: Consultancy, Ended employment in the past 24 months; BMS: Consultancy, Patents & Royalties, Research Funding; sanofi: Patents & Royalties; Caribou: Patents & Royalties. June: AC Immune, DeCART, BluesphereBio, Carisma, Cellares, Celldex, Cabaletta, Poseida, Verismo, Ziopharm: Consultancy; Novartis: Patents & Royalties; Tmunity, DeCART, BluesphereBio, Carisma, Cellares, Celldex, Cabaletta, Poseida, Verismo, Ziopharm: Current equity holder in publicly-traded company. Pamer: Diversigen: Other: Advisory board; Bristol Myers Squibb, Celgene, Seres Therapeutics, MedImmune, Novartis and Ferring Pharmaceuticals: Honoraria. Peled: DaVolterra: Consultancy; MaaT Pharma: Consultancy; CSL Behring: Consultancy; Seres Therapeutics: Research Funding. Ruella: BMS, BAYER, GSK: Consultancy; Novartis: Patents & Royalties; AbClon: Consultancy, Research Funding; Tmunity: Patents & Royalties; viTToria biotherapeutics: Research Funding. van den Brink: WindMILTherapeutics: Honoraria; Pluto Therapeutics: Current holder of stock options in a privately-held company, Other: has consulted, received honorarium from or participated in advisory boards ; Priothera: Research Funding; Forty-Seven, Inc.: Honoraria; MagentaTherapeutics: Honoraria; GlaskoSmithKline: Other: has consulted, received honorarium from or participated in advisory boards; Ceramedix: Other: has consulted, received honorarium from or participated in advisory boards ; Merck & Co, Inc: Honoraria; Synthekine (Spouse): Other: has consulted, received honorarium from or participated in advisory boards; Kite Pharmaceuticals: Other; Amgen: Honoraria; Frazier Healthcare Partners: Honoraria; Seres: Other: Honorarium, Intellectual Property Rights, Research Fundingand Stock Options; Rheos: Honoraria; Therakos: Honoraria; Jazz Pharmaceuticals: Honoraria; Notch Therapeutics: Honoraria; Nektar Therapeutics: Honoraria; Wolters Kluwer: Patents & Royalties; Juno Therapeutics: Other; DKMS (nonprofit): Other; Pharmacyclics: Other; Da Volterra: Other: has consulted, received honorarium from or participated in advisory boards; Novartis (Spouse): Other: has consulted, received honorarium from or participated in advisory boards; Lygenesis: Other: has consulted, received honorarium from or participated in advisory boards .

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.

Hematopoietic cell transplantation (HCT) is a critical treatment of patients with high-risk hematopoietic malignancies, hematological deficiencies, and other immune diseases. In allogeneic HCT (allo-HCT), donor-derived T cells recognize host tissues as foreign, causing graft-versus-host disease (GVHD) which is a main contributor to morbidity and mortality. The intestine is one of the organs most severely affected by GVHD and research has recently highlighted the importance of bacteria, particularly the gut microbiota, in HCT outcome and in GVHD development. Loss of intestinal bacterial diversity is common during the course of HCT and is associated with GVHD development and treatment with broad-spectrum antibiotics. Loss of intestinal diversity and outgrowth of opportunistic pathogens belonging to the phylum Proteobacteria and Enterococcus genus have also been linked to increased treatment-related mortality including GVHD, infections, and organ failure after allo-HCT. Experimental studies in allo-HCT animal models have shown some promising results for prebiotic and probiotic strategies as prophylaxis or treatment of GVHD. Continuous research will be important to define the relation of cause and effect for these associations between microbiota features and HCT outcomes. Importantly, studies focused on geographic and cultural differences in intestinal microbiota are necessary to define applicability of new strategies targeting the intestinal microbiota.

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.

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.

Members of the B7 family have been shown to be important for regulating immune responses by providing either positive or negative costimulatory signals. The function of B7-H3 has been controversial. We show that B7-H3 is upregulated in graft-versus-host disease (GVHD) target organs, including the colon, liver, and lung. Infusion of allogeneic donor T cells into B7-H3(-/-) vs wild-type (WT) recipients resulted in increased GVHD lethality associated with increased T-cell proliferation, colonic inflammatory cytokines, and destruction of epithelial barriers. Allogeneic B7-H3(-/-) vs WT donor T cells also had increased T-cell proliferation and GVHD lethality associated with increased proliferation and cytokine secretion in the spleen, intraepithelial lymphocyte inflammatory cytokines, and intestinal permeability. Both resting and activated regulatory T cells (Tregs) lack B7-H3 messenger RNA. Consistent with these data, GVHD was augmented in recipients of B7-H3(-/-) Treg-depleted grafts. In two delayed lymphocyte infusion (DLI) models, T cells lacking B7-H3 are capable of providing graft-versus-leukemia (GVL) effects. We conclude that B7-H3 is responsible for providing a negative costimulatory signal. Our studies provide support for developing and testing new therapies directed toward the B7-H3 pathway, including approaches to augment host B7-H3 early after bone marrow transplantation to prevent GVHD and to develop potent antagonistic antibodies later after transplant to facilitate DLI-mediated GVL without GVHD complications.

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.

TO THE EDITOR: Bacterial infection remains one of the major causes of morbidity and mortality early after allogeneic hematopoietic cell transplantation (allo-HCT) because of a patient’s immunocompromised state requiring use of antibiotic treatments as prophylactic regimens or to treat manifest

Introduction: Dysbiosis of the gut microbiome during hematopoietic stem cell transplantation (HCT) is associated with adverse post-transplant outcomes such as graft-versus-host disease, bloodstream infections, and mortality. In order to learn more about the role of the microbiome in HCT in adverse clinical outcomes, researchers collect stool samples from patients at various time points throughout HCT. However, unlike blood samples or skin swabs, stool collection requires active subject participation, particularly in the outpatient setting, and may be limited by patient aversion to handling stool. By providing study participants with compensation for their stool samples, we hypothesize that we can significantly increase stool collection rates. Methods: We performed a prospective cohort study on the impact of financial incentives on stool collection rates for microbiome studies. The intervention group consisted of allogeneic (allo)-HCT patients from 05/2017-05/2018 who were compensated with a $10 gas gift card for each stool sample. The intervention group was compared to a historical control group consisting of allo-HCT patients from 11/2016-05/2017 who provided stool samples before the incentive was implemented. To control for potential changes in collections over time, we also compared a contemporaneous control group of autologous (auto)-HCT patients from 05/2017-05/2018 with a historical control group of auto-HCT patients from 11/2016-05/2017; neither auto-HCT groups were compensated. Allo-HCT patients were required to give samples at pre-HCT, day 0 (the day of HCT), and days 7, 14, 21, 30, 60, and 90 post-HCT. Auto-HCT patients were required to give samples at pre-HCT and days 7, 14, and 90 post-HCT. Collection rates were defined as the number of samples provided divided by the number of time points for which we attempted to obtain samples. Patient characteristics were summarized by proportions for categorical variables and median with interquartile ranges for continuous variables. Chi-square tests or Fisher's exact tests were used to compare categorical variables, as appropriate, and Wilcoxon Rank Sum tests or t-tests were used to compare continuous variables, as appropriate. This study was approved by the Duke Institutional Review Board, and informed consent was obtained from all patients. Results: There were 35 allo-HCT patients in the intervention group, 19 allo-HCT patients in the historical control group, 142 auto-HCT patients in the contemporaneous control group, and 75 auto-HCT patients in the historical control group. Groups were similar with regard to baseline demographics such as age, race, and gender. While allo-HCT patients were more likely to have leukemia and auto-HCT patients were more likely to have lymphoma and multiple myeloma, there were no differences in disease rates across the study periods. Allo-HCT patients in the intervention group had significantly higher average overall collection rates when compared to the historical control group allo-HCT patients (80% vs 37%, p Discussion: Our results demonstrate that even a modest incentive can significantly increase collection rates. Use of a contemporaneous control group to account for potential differences in stool collection rates over time strengthens our finding that financial incentives increase stool collection rates. Furthermore, the significant increase in collection rates in the outpatient setting highlights the role of the incentive when patient participation is needed, as opposed to the inpatient setting in which the nurse assists with collection. While this study uses a specialized HCT patient population, these results may be generalizable to future studies and aid other researchers in obtaining stool samples needed for future microbiome studies. Disclosures Peled: Seres Therapeutics: Other: IP licensing fees, Research Funding. van den Brink:Acute Leukemia Forum (ALF): Consultancy, Honoraria; Juno Therapeutics: Other: Licensing; Merck & Co, Inc.: Consultancy, Honoraria; Seres Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Therakos: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Flagship Ventures: Consultancy, Honoraria; Evelo: Consultancy, Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Magenta and DKMS Medical Council: Membership on an entity's Board of Directors or advisory committees. Sung:Novartis: Research Funding; Merck: Research Funding; Seres: Research Funding.

The intestinal microbiota undergoes major perturbations during allogeneic hematopoietic stem cell transplantation (allo-HCT), and low microbiota diversity during this period is associated with an increased risk of graft-versus-host disease and mortality. Identifying the environmental variables that might impact intestinal microbiota could inform strategies to maintain and restore a healthy microbiota state. However, understanding microbial dynamics is challenging due to the high-dimensional nature of microbiota data. Here, we simplified complex microbiota communities into clusters and investigated the dynamics under different conditions in terms of transition probabilities in a large dataset of allo-HCT fecal specimens (Fig. a). The bacterial compositions of 7,930 samples from 1,076 allo-HCT patients were determined by 16S rRNA deep-sequencing. Samples were then clustered into 10 distinct states by k-means clustering of a Bray-Curtis β-diversity matrix (Fig. b). These clusters captured variations in diversity and microbiota compositions (Fig. c-d). Cluster 1 represented a high-diversity state, and Lachnospiraceae and other Clostridiales were the most commonly observed taxa in this cluster. The low-diversity clusters 9 and 10 consisted mostly of Streptococcus-dominated and Enterococcus-dominated samples, respectively. We utilized a regression-based predictive approach to model cluster transition probabilities in terms of a weight for remaining in the same cluster over time (self-weight) and a weight for attracting transitions from other clusters over time (attractor-weight). Controlling for the effect of time, the weights measured the contribution of different environmental exposures to intestinal microbial behaviors. A negative parameter coefficient indicates cluster destabilization or decreased cluster transition likelihood in the case of self-weights and attractor-weights, respectively. We evaluated the impact of the 3 most commonly used non-prophylactic antibacterial drugs using 2359 daily samples from 385 allo-HCT patients collected between day -14 to 7 relative to transplant. High-diversity cluster 1 was significantly destabilized by piperacillin-tazobactam (pip-tazo) exposure (β = -0.87, P < 0.05). Meanwhile, exposure to cefepime and meropenem did not have a significant effect on cluster 1 stability (Fig. e). Exposure to pip-tazo also increased the transition probability to the Streptococcus-dominated cluster 9 (β = 1.83, P < 0.001), while cefepime (β = 2.69, P < 0.05) and meropenem (β = 1.96, P < 0.01) exposure favored transitions to the Enterococcus-dominant cluster 10. These results suggest that antibiotic exposures are associated with different composition outcomes depending on patient microbiota states during transplant period. In a small subset of 242 daily samples from 46 allo-HCT patients with detailed daily dietary information, we observed that an increase in total protein intake (range = 0-137.4g; median = 36g) was associated with low self-maintenance of cluster 1 (β = -1.29, P < 0.05), while an increase in total fat intake (range = 0-183.3g; median = 34.5g) improved cluster 1 stability (β = 1.44, P < 0.05). Overall, dietary intakes could also modulate transition probabilities between microbial communities in allo-HCT patients. While prior studies have assessed specific bacterial taxa or diversity indices as biomarkers of clinical outcomes, here we considered the entire intestinal communities and demonstrated that various environmental exposures were associated with changes in microbiota composition during allo-HCT. Using a regression-based approach that predicts cluster transitions in response to environmental conditions, we found that pip-tazo exposure was associated with destabilization of a high-diversity state and increased transitions to a Streptococcus-dominated state, while cefepime and meropenem exposure did not disrupt high-diversity microbial community. Furthermore, increased protein intake was also associated with disruption to the high-diversity cluster, while increased fat intake strengthened the maintenance of a diverse and healthy microbial community. Ultimately, this computation framework aims to inform strategies to optimize treatment plans for allo-HCT patients to maximize a healthy gut microbiota state and clinical outcomes. Disclosures Gomes: Seres Therapeutics: Other: Part of Salary. Peled:Seres Therapeutics: Research Funding. Slingerland:Seres Therapeutics: Other: Salary supported by Seres funding. Clurman:Seres Therapeutics: Research Funding. Giralt:Celgene: Consultancy, Research Funding; Takeda: Consultancy; Sanofi: Consultancy, Research Funding; Amgen: Consultancy, Research Funding. Perales:Bristol-Meyers Squibb: 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; Bellicum: Honoraria, Membership on an entity's Board of Directors or advisory committees; NexImmune: Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Nektar Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: 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; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy, Honoraria; Medigene: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; Kyte/Gilead: Research Funding; Miltenyi: Research Funding; MolMed: Membership on an entity's Board of Directors or advisory committees. Pamer:MedImmune: Honoraria; Seres Therapeutics: Honoraria, Patents & Royalties; Bristol Myers Squibb: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Ferring Pharmaceuticals: Honoraria. van den Brink:Acute Leukemia Forum (ALF): Consultancy, Honoraria; Seres Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Flagship Ventures: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Evelo: Consultancy, Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria; Therakos: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Merck & Co, Inc.: Consultancy, Honoraria; Juno Therapeutics: Other: Licensing; Magenta and DKMS Medical Council: Membership on an entity's Board of Directors or advisory committees.

Background Fecal microbiota composition is associated with important outcomes after allo-HCT including survival, relapse, GVHD, and infections. We previously demonstrated in a multicenter observational study that HCT patients present with fecal microbiota configurations that have lower diversity and are distinct from those of healthy individuals, and that pre-HCT microbiota injury predicts poor overall survival. Here, we hypothesized that pre-HCT fecal microbiota features predict development of critical illness post-HCT. Methods We analyzed 828 adults who received a first allo-HCT from 2009 to 2017 at a single institution who had an evaluable fecal sample in our biobank collected within the 10 days prior to cell infusion. The patients were heterogeneous with respect to transplant indication, conditioning intensity, graft source (cord blood, peripheral blood, marrow) and graft manipulation (CD34-selection). The V4-V5 regions of 16S rRNA genes of DNA extracted from fecal samples were amplified and annotated taxonomically. The outcome of interest was time to ICU admission, which was assessed using survival-analysis methods. The reason for admission to the ICU was evaluated for each subject. Results Seventy-five (9%) patients were admitted to the intensive care unit (ICU) between the day of cell infusion and day +50; the peak incidence of ICU admission occurred on day +10. The most common indications for ICU admission were respiratory failure (65%) and infection (27%). Patients were stratified based on fecal microbiota diversity, as assessed by 16S sequencing of stool samples collected prior to transplantation, into high (inverse Simpson index ≥4) and low ( Conclusion Pre-transplant fecal microbial diversity is an independent predictor of intensive-care-requiring critical illness in the post-HCT period. These observations highlight the pre-HCT period as a window of opportunity to (a) assess microbiota injury in conjunction with comorbidity evaluation, (b) inform selection of antibiotic prophylaxis, gut-decontamination, GVHD-prophylaxis, or conditioning regimens, and (c) intervene with microbiota injury-remediation or prevention strategies. Figure Disclosures Brereton: Seres Therapeutics: Other: Salary Support. Clurman:Seres Therapeutics: Research Funding. Slingerland:Seres Therapeutics: Other: Salary supported by Seres funding. Shah:Janssen Pharmaceutica: Research Funding; Amgen: Research Funding. Scordo:McKinsey & Company: Consultancy; Angiocrine Bioscience, Inc.: Consultancy. Politikos:Angiocrine Bioscience Inc: Research Funding. Gyurkocza:Actinium Pharmaceuticals: Research Funding. Barker:Angiocrine Bioscience Inc: Research Funding; Gamida Cell: Research Funding; Merck: Research Funding. Perales:Kyte/Gilead: Research Funding; Miltenyi: Research Funding; MolMed: Membership on an entity's Board of Directors or advisory committees; NexImmune: Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bellicum: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Nektar Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: 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; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy, Honoraria; Medigene: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees. Giralt:Amgen: Consultancy, Research Funding; Spectrum Pharmaceuticals: Consultancy; Actinium: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Kite: Consultancy; Johnson & Johnson: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy; Miltenyi: Research Funding; Takeda: Consultancy, Research Funding; Novartis: Consultancy. van den Brink:Merck & Co, Inc.: Consultancy, Honoraria; Acute Leukemia Forum (ALF): Consultancy, Honoraria; Magenta and DKMS Medical Council: Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Other: Licensing; Amgen: Consultancy, Honoraria; Therakos: Consultancy, Honoraria; Seres Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Flagship Ventures: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Evelo: Consultancy, Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria. Pamer:Bristol Myers Squibb: Honoraria; Celgene: Honoraria; Seres Therapeutics: Honoraria, Patents & Royalties; MedImmune: Honoraria; Novartis: Honoraria; Ferring Pharmaceuticals: Honoraria. Peled:Seres Therapeutics: Research Funding.

Introduction Translocation of intestinal bacteria across impaired mucosal barriers has long been believed to occur following exposure to chemotherapy. Consistent with this, we and others have previously reported that expansions of potentially pathogenic bacteria within the gastrointestinal microbiome precedes bloodstream infection. While the blood microbiome represents a rich area for investigation, detailed unbiased characterization of the blood microbiome has not previously been possible. Here, we sought to investigate the relationship between the blood and stool microbiome in patients undergoing allogeneic hematopoietic stem cell transplantation (HCT) who are at high risk for gut barrier dysfunction, severe infection, and the immunological complications of transplantation including graft-versus-host disease (GVHD). We show preliminary data suggesting that the blood microbiome may hold biomarkers for gut integrity. Subsequently, it may offer microbiological data supporting causative organisms in "culture-negative" fevers, or in itself, perform an immunomodulatory role acting as a damage/pathogen associated molecular pattern (DAMP/PAMP). Methods We sequenced serially-collected plasma and stool samples (n = 61 unique samples of each type) from a cohort of 19 patients who underwent allogeneic HCT at our center. Microbial cell-free DNA (mcfDNA) was extracted from plasma and sequenced using a next generation sequencing assay (Karius, Inc, Redwood City, CA). After sequences are processed, mcfDNA abundances are reported in molecules per microliter. Stool samples underwent were profiled using 16S-targeted sequencing (V4-V5 region) on the Illumina MISEQ platform and analyzed using the DADA2 pipeline. Association of blood microbial burden with clinical factors was assessed using a Wilcoxon rank sum test. Results We confirmed a high sequence homology between the DNA found in plasma and stool samples, thus demonstrating that circulating mcfDNA is gut-derived in these patients. By comparing microbial sequences from paired plasma and stool samples collected equivalent time points during the neutropenic nadir after HCT, we observed a correlation between the abundance of bacterial mcfDNA in plasma and DNA from the same microbes in stool. Of note, this occurred independently of conditioning regimen intensity, and was observed in patients who had received myeloablative, non-myeloablative and reduced intensity therapy. We assessed the association of bacterial mcfDNA burden (only considering sequences common to stool sequences) with various clinical factors. Translocation was significantly higher in patients who experienced pre-engraftment mucositis (any observed grade) compared with those who did not (p = 0.02, n = 5 in the mucositis group, any grade, and 12 in the mucositis-free group), but there was no relationship between the degree of translocation and HCT-CI, conditioning intensity, donor type, age or pre-engraftment fevers. We next asked whether translocation events were associated with acute GVHD (aGVHD) by assessing a subgroup of patients who received unmodified grafts (peripheral blood stem cell or bone marrow; n = 10) and tracked the degree of translocation from the gut to the blood stream pre-transplant, during the neutropenic nadir, and following engraftment. As shown in Figure 1, we observed low pre-transplant translocation (as quantified by the total bacterial DNA abundance in plasma where sequences were also shared in stool samples), and a marked increase during neutropenic nadir. Remarkably, even in this small cohort, we observe a higher burden of translocation in the post-engraftment period in patients who subsequently develop aGVHD, while it decreased to baseline levels in those who do not (n = 7 in the aGVHD group; n = 3 aGVHD-free; p = 0.05). Conclusions Here we demonstrate the first use of a culture-free molecular assay to track the blood microbiome and identify features of the circulating mcfDNA that correlate with the microbial DNA in stool samples. Despite the small sample size, these data suggest that the maintenance of a high mcfDNA-burden beyond the neutropenic nadir is associated with subsequent GVHD development, and provides some evidence for early gut barrier dysfunction that permits translocation in these patients. Disclosures Blair: Karius, Inc: Employment. Peled:Seres Therapeutics: Other: IP licensing fees, Research Funding. Giardina:Seres Therapeutics: Other: Salary funding. Slingerland:Seres Therapeutics: Other: Salary supported by Seres funding. Hollemon:Karius, Inc: Employment. Ho:Karius, Inc: Employment. Bercovici:Karius, Inc: Employment. Ahmed:Karius, Inc: Employment. Hong:Karius, Inc: Employment. Giralt:Celgene: Consultancy, Research Funding; Takeda: Consultancy; Sanofi: Consultancy, Research Funding; Amgen: Consultancy, Research Funding. van den Brink:Therakos: Consultancy, Honoraria; Merck & Co, Inc.: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Acute Leukemia Forum (ALF): Consultancy, Honoraria; Magenta and DKMS Medical Council: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria; Seres Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Flagship Ventures: Consultancy, Honoraria; Juno Therapeutics: Other: Licensing; Evelo: Consultancy, Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria.

Introduction. Clinical graft-versus-host disease (GVHD) phenotypes can selectively involve the upper or lower tract and extraintestinal tissues and organs. High gut microbiota diversity and the abundance of certain bacterial commensals within the Clostridiales order are associated with decreased risk of GVHD-related mortality after allogeneic stem cell transplantation (allo-HCT). The microbiota-derived short-chain fatty acid butyrate has been found beneficial in reducing murine GI aGVHD. Conversely, microbiota disruption is associated with expansions of potentially pathogenic bacteria, including facultative anaerobes, and can worsen allo-HCT outcomes. Since the vast majority of the intestinal microbiota biomass resides within the colon, we hypothesized that certain features of the colonic microbiota confer local protection to the lower GI tract. Methods. We evaluated 216 patients (median age 55 years) who underwent unmodified allo-HCT and had stool samples collected under our institutional fecal microbiome biobank between 01/2011 and 02/2017. Patients were classified in three groups according to the organ involved in aGVHD: Upper GI only (UGI, n = 56), lower GI with or without upper GI involvement (LGI, n =61 ), no GI tract involvement (non-GI, n = 29). A fourth group of 70 control patients with no GVHD whatsoever were selected on the basis of the study inclusion and exclusion criteria and having evaluable samples in our biobank in a temporal distribution that matched onset of GVHD. Microbial diversity was computed using Simpson's reciprocal index. Results. A total of 902 stool specimens were analyzed with an average of 4.2 samples/patient. Samples were grouped into pre-onset (day -20 to day 0) and post-onset (day 1 to day 20) aGVHD. Trends in intestinal microbiota features relative to the day of aGVHD onset showed distinct dynamics among the groups. Prior to aGVHD onset, all groups showed similar microbial diversity, and similar abundance of anaerobic commensals, including members of genus Blautia (Fig 1A-C). After aGVHD onset, microbial diversity and commensal anaerobe abundance in LGI cases were significantly lower than non-GI (p = 0.01 and 0.008, respectively) and UGI (p = 0.03 and 0.03, respectively) groups (Fig. 1A, 1B). The LGI group also had significantly lower abundance of the genus Blautia (p = 0.02, Fig 1C). Conversely, the LGI group had increased prevalence of facultative anaerobes (LGI vs. non-GI, p = 0.03, Fig. 1D). Prior to aGVHD onset, the LGI cases had lower abundance of predicted butyrate-producing bacteria (LGI vs. no-GVHD, p = 0.03), which was maintained in all three GVHD groups post-onset aGVHD (Fig 1E). Conclusions. Patients without GVHD, non-GI and UGI aGVHD phenotypes had higher microbial diversity, abundance of anaerobe commensals including genus Blautia, and predicted butyrate-producing bacteria after aGVHD onset than patients with lower GI tract involvement by aGVHD. In contrast, the LGI cases had features consistent with microbiota dysbiosis including prevalence of facultative anaerobes, and lower abundance of butyrate-producing bacteria before and after aGVHD. These findings are consistent with a model in which a diverse microbiota abundant in potential beneficial commensals exerts a local protective effect in the lower GI tract and have potential practical implications in future prophylactic and therapeutic interventions in aGVHD. Figure 1 Disclosures Gomes: Seres Therapeutics: Other: Part of Salary. Slingerland:Seres Therapeutics: Other: Salary supported by Seres funding. Pamer:Bristol Myers Squibb: Honoraria; Celgene: Honoraria; Seres Therapeutics: Honoraria, Patents & Royalties; MedImmune: Honoraria; Novartis: Honoraria; Ferring Pharmaceuticals: Honoraria. Peled:Seres therapeutics: Consultancy, Honoraria. van den Brink:Acute Leukemia Forum (ALF): Consultancy, Honoraria; Merck & Co, Inc.: Consultancy, Honoraria; Magenta and DKMS Medical Council: Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Other: Licensing; Seres Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria; Therakos: Consultancy, Honoraria; Flagship Ventures: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Evelo: Consultancy, Honoraria.

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.

GVHD and tumor relapse are fundamental problems in allogeneic HSCT. Recent research has linked neovascularization to GVHD, tumor growth, and graft-versus-tumor (GVT) activity. Damage of the endothelium by the conditioning regimen provides the initiation stimulus for recruitment of donor-derived endothelial cells and their progenitors. During the early inflammatory phase of GVHD there is considerable neovascularization facilitating migration of inflammatory cells to target organs. In the course of GVHD, however, the vasculature itself becomes a target of alloreactive donor T cells. As a consequence, later stages of GVHD are characterized by fibrosis and rarefaction of blood vessels. Importantly, the inhibition of tumor-neovascularization by activated donor T cells that release antiangiogenic substances contributes to GVT and may be enhanced by pharmacologic inhibition of neovascularization. Furthermore, the therapeutic inhibition of neovascularization may improve immunotherapy for cancer by enhancing leukocyte infiltration in tumor tissue because of normalization of tumor vessels and stimulation of leukocyte–vessel wall interactions. These insights identify important mechanisms underlining the importance of neovascularization for allogeneic immune responses and move therapeutic approaches targeting neovascularization into the spotlight. This perspective covers current knowledge of the role of neovascularization during GVHD as well as GVT and its implications for HSCT.

Background : While dCBT is associated with high rates of sustained donor engraftment, delayed neutrophil recovery in adults is frequent and can contribute to extended hospitalization and early transplant-related mortality. Methods : We investigated engraftment after myeloablative dCBT supplemented with CD34+ selected haploidentical PBSC (haploCD34+) in patients (pts) with high risk hematologic malignancies or aplastic anemia in a phase II clinical trial. The aim was to abrogate neutropenia (ANC >/= 500 within 14 days) with a haplo myeloid bridge prior to CB engraftment. Pts did not receive ATG due to the increased mortality risk reported in adult CBT. Double unit CB grafts allowed comparison to dCBT controls without haploidentical graft supplementation. Results : 78 adult pts [median age 48.5 years (range 21-68), median weight 82 kgs (range 48-138), 44 (56%) CMV+, 3 with prior allografts] underwent haplo-dCBT between 9/2012-12/2017. Diagnoses included 54 (69%) acute leukemias, 10 (13%) MDS/ MPN (all ≤ 10% blasts at work-up), 10 (17%) NHL/ HD and 1 aplastic anemia. Conditioning was myeloablative (1 Cy 120/ Flu 75/ TBI 1375, 77 intermediate intensity Cy 50/ Flu 150/ Thio 5-10/ TBI 400) with CSA/ MMF. CB units had a median infused TNC of 2.3 (range 1-5.7) x 107/kg/unit & median infused viable CD34+ cell dose of 1.1 (range 0.1-3.1) x 105/kg/unit with a median 5/8 (range 2-7) unit-recipient HLA-allele match. Haplo CD34+ grafts [procured from children (46%), siblings (31%), parents (13%) or extended family (10%)] had a median infused CD34+ dose of 5.2 x 106/kg (range 1.1-16.8) and a median infused CD3+ dose of 1.6 x 103/kg (range 0.3-13.7). Sixty-one (78%) haplos were 4/8 and 17 (22%) were 5-7/8 HLA-matched to the pt. In 77 evaluable pts (1 pt died on day 14), 4 engraftment patterns were observed (Table 1). All but 2 pts had sustained CB engraftment with either an optimal haplo-bridge (Gp. 1, 34/77, 44%), a transient bridge with a second nadir preceding CB engraftment (Gp. 2, 20/77, 26%), or no bridge (Gp. 3, 21/77, 27%). The 2 remaining pts had CB/ haplo graft failure (Gp. 4, 2/77, 3%); both were successfully re-transplanted with single CB units. While there was no difference in the day 100 TRM in the 34 optimal bridge pts vs pts with transient or no bridge [9% (95%CI 2-21) vs 15% (95%CI 6-27), p = 0.388], optimal bridge pts had faster platelet recovery [19 (range 14-41) vs 44.5 days (range 14-67)] and earlier hospital discharge [28.5 (range 20-60) vs 36 days (range 28-98)]. Similar to dCBT alone, chimerism analysis revealed sustained engraftment in haplo-dCBT is mediated by a "winning" CB unit. This was heralded by winning unit-derived T-cells seen as early as day +28. Although universal, the speed of haplo rejection varied, and a high haplo chimerism percentage early post-transplant did not guarantee successful bridging. Analysis of factors potentially predicting an optimal bridge is shown in Table 2. The median winning CB unit-haplo 8-allele HLA-match was 3/8 (range 1-7/8). In univariate analysis, higher haplo CD34+ dose/kg, > 4/8 haplo-recipient HLA-match and ≥ 3/8 winning unit-haplo HLA-match were associated with a higher likelihood of bridging. Haplo CD34+ dose and winning unit-haplo HLA-match remained significant in multivariate analysis. Conclusions: While haplo-dCBT can be associated with enhanced neutrophil recovery, this platform does not guarantee a successful myeloid bridge due to early haplo rejection by the winning CB unit. This universal haplo rejection highlights the importance of the CB graft dose and quality with this ATG-free strategy as the CB will mediate sustained engraftment. Our findings have significance for strategies that combine unmanipulated CB with any third-party or ex vivo expanded T-cell depleted product given higher product CD34+ cell dose and better HLA-match to the unmanipulated CB unit could improve the likelihood of successful myeloid bridging. The data also support alternative approaches to improve myeloid recovery after CBT such as optimized unit selection and vivo expansion. Disclosures O'Reilly: Atara Biotherapeutics: Consultancy, Patents & Royalties, Research Funding. Perales:Takeda: Other: Personal fees; Novartis: Other: Personal fees; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees and Clinical trial support; Merck: Other: Personal fees; Abbvie: Other: Personal fees. Sauter:Juno Therapeutics: Consultancy, Research Funding; Sanofi-Genzyme: Consultancy, Research Funding; Spectrum Pharmaceuticals: Consultancy; Novartis: Consultancy; Precision Biosciences: Consultancy; Kite: Consultancy. Shah:Janssen: Research Funding; Amgen: Research Funding.

Introduction Cellular therapy with chimeric antigen receptor (CAR) T cells has fundamentally changed the treatment of many cancers. Unfortunately, not all patients who receive this therapy have a favorable response. Additionally, some patients may develop toxicity due to cytokine release syndrome (CRS) or neurotoxicity. Recent studies have found a relationship between the intestinal microbiome and the response to immunotherapy with checkpoint blockade. We propose the intestinal microbiota as a factor that influences the efficacy and toxicity of CAR T cells. We hypothesize that the differences in outcomes of patients who receive CAR T cells are related to the composition of their intestinal microbiota at baseline. We report a single-center analysis of pre-CAR T cell infusion microbiota composition. Methods We collected stool samples from recipients of CAR T cells at Memorial Sloan Kettering Cancer Center (MSKCC). A baseline sample was collected prior to CAR T cell infusion. Samples were submitted for 16S RNA sequencing of the V4-V5 region on the Illumina MiSeq platform and the operational taxonomic units (OTUs) were classified using the NCBI Reference Sequence Database. Clinical response to assess efficacy was classified as either complete response (CR) or no complete response. Given the sample size, toxicity was pooled to encompass Grade 1 to 4 CRS and Grade 1 to 4 neurotoxicity. Linear discriminant analysis effect size (LEfSe) was used to identify microbial biomarkers for efficacy and toxicity between groups using relative abundances with a linear discriminant analysis score threshold >2.5. Results We analyzed 24 baseline samples from 24 patients treated at MSKCC. The patients were all adult recipients of cellular therapy with CAR T cells. The patients varied in conditioning regimen, CAR construct and underlying diagnosis, which included solid tumors and hematologic malignancies. First, we assessed the 16S relative abundance of the intestinal microbiota of the patients at baseline. We found that the composition of the microbiota prior to CAR T cell infusion was diverse, as defined by an Inverse Simpson >4 in all of the patients, although the level of diversity amongst the patient samples varied (Fig A). An assessment of the efficacy of CAR T cells with LEfSe analysis found increased abundance in several families of the Clostridiales order (Firmicutes phylum), including Oscillospiraceae, Ruminococcacaeae, and Lachnospiraceae, in those patients who achieved a CR. For the patients who did not achieve a CR, we found an increased abundance of a family in the Clostridiales order (Firmicutes phylum), Peptostreptococcaceae. Patients who experienced toxicity, either CRS or neurotoxicity, had an increased abundance of families within the Clostridiales or Lactobacillales order (Firmicutes phylum), which included Lachnospiraceae and Lactobacillaceae. Finally, patients who did not experience toxicity also had an increased abundance of a family in the Clostridiales order (Firmicutes phylum), Peptostreptococcaceae. Conclusion We demonstrate that our subset of patients had diverse microbial composition prior to receiving CAR T cell therapy despite the fact that many of them were heavily pre-treated. Additionally, we observe the abundance of the family Lachnospiraceae in the patients who achieved a CR and those who experienced toxicity. Many Lachnospiraceae are butyrate producers, whose presence has been found to be protective against Clostridium difficile infection in recipients of allogeneic hematopoietic cell transplant but whose abundance is lower in colon cancer. Conversely, we observe an abundance of the family Peptostreptococcaceae in patients who did not achieve a CR or who did experience toxicity. Peptostreptococcaceae has been found to be more abundant in the intestines of patients with colon cancer. Of note, the intestinal micriobiota that we identify are not congruent with the specific bacteria that have been found to promote anti-tumor immunity to checkpoint blockade. Our data suggests a role for the intestinal microbiota in mediating the response to CAR T cells and proposes that the baseline microbial composition may correlate with efficacy and toxicity. Further studies will investigate biochemical mechanisms to understand the interplay of the intestinal microbiota and the immune system to improve patient outcomes following CAR T cell therapy. Disclosures Park: Adaptive Biotechnologies: Consultancy; Juno Therapeutics: Consultancy, Research Funding; Novartis: Consultancy; AstraZeneca: Consultancy; Kite Pharma: Consultancy; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy; Shire: Consultancy. O'Cearbhaill:Juno: Research Funding. Mailankody:Juno: Research Funding; Janssen: Research Funding; Takeda: Research Funding; Physician Education Resource: Honoraria. Smith:Celgene: Consultancy, Patents & Royalties: CAR T cell therapies for MM, Research Funding. Palomba:Pharmacyclics: Consultancy; Celgene: Consultancy. Riviere:Fate Therapeutics Inc.: Research Funding; Juno Therapeutics, a Celgene Company: Membership on an entity's Board of Directors or advisory committees, Research Funding. Brentjens:Juno Therapeutics, a Celgene Company: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Introduction: Increasing evidence suggests that the intestinal microbiota is involved in the development of acute graft-vs.-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT). We previously reported in single center studies that Enterococcus a) is associated with GVHD (Holler et al., BBMT 2014) and b) can dominate the post-transplant gut microbiota in up to 50% of allo-HCT patients resulting in a 9-fold increased risk of bacteremia (Taur et al, CID 2012). To further investigate the hypothesis that Enterococcus can trigger the development of GVHD, we studied both allo-HCT patients and pre-clinical mouse transplant models. Methods and Results: Stool samples from 1240 allo-HCT patients at 4 different transplant centers in the U.S., Germany and Japan were collected approximately weekly during inpatient hospitalization. The V4-V5 region of the bacterial 16S rRNA genes from 6718 samples was sequenced at one central site on the Illumina platform. We observed Enterococcus mono-domination (relative abundance > 30%) in post-transplant samples ranging from 20 to 60% of patients at different centers (Fig. A, left). This mono-domination was primarily attributable to E. faecium, and was associated with a significantly increased risk for grade 2-4 acute GVHD (Fig. A, right). In three different mouse models we found a transient bloom of E. faecalis around 7 days after transplant in allo-HCT recipients with GVHD (Fig. B; C57BL/6 -> 129SV model). This bloom did not occur in allo-HCT recipients of a T cell depleted allograft without GVHD. To further investigate this Enterococcus bloom, we treated mice with an experimental E. faecalis-strain on days 4 to 6 after transplant and found significantly increased lethal GVHD. Colonizing germ-free mice with a minimal gut flora also lead to increased lethal GVHD when enterococci were added to the gnotobiotic flora (Fig. C). The allo-HCT recipients with Enterococcus-containing flora had also increased serum IFNg levels. Short chain fatty acids (SCFA) can be protective against GVHD and gut inflammation through maintenance of epithelial homeostasis and increases in anti-inflammatory regulatory T cells in the gut. In BMT mouse models, we found that Enterococcus-dominated allo-HCT recipients with GVHD have significantly less cecal butyrate, a major SCFA. Similarly, Enterococcus domination after allo-HCT also leads to a decrease in fecal SCFAs in patients (Fig. D). Next, we hypothesized that intestinal IgA might have a protective role against this pathogen in mice. 16S sequencing of flow sorted IgA-coated vs. non-coated bacteria from fecal samples of allo-HCT patients and transplanted mice revealed no specific IgA-coating pattern of enterococci both before or after transplant rather excluding the hypothesis that IgA might have a protective role against Enterococci. E. faecalis and E. faecium use the disaccharide lactose as a major carbohydrate source for growth and expansion as observed by analyses of the Enterococcus genome and in vitro growth experiments. In mice, we observed that a lactose-free diet significantly decreases the Enterococcus bloom after transplant in allo-T cell recipients and in first survival experiments attenuates lethal GVHD (Fig. E). Conclusion: Our studies in mouse and man demonstrate that the abundance of Enterococcus in the intestinal flora plays a role in the development of GVHD and the prevention of Enterococcus growth with a lactose-free diet can ameliorate GVHD. Disclosures Peled: Seres Therapeutics: Research Funding.

Introduction: Optimal CB unit selection guidelines recommend consideration of CD34+ cell dose & 8-allele donor-recipient HLA-match. How graft characteristics for these parameters vary by patient (pt) race/ ethnicity, however, is not known. Methods: We analyzed the infused graft & back-up unit cryopreserved total nucleated cell (TNC) x 107 & CD34+ x 105 cell content, the cell dose (incorporating pt weight), & 4-6/6 & 8-allele HLA-match by pt ancestry in CB transplant (CBT) recipients transplanted 1/2014-6/2018. Units were chosen based on banking practices (e.g. RBC depleted, standard cryo volumes), TNC & CD34+ dose & 4-6/6 & 8-allele HLA-match with dose usually taking priority over match given pt size at our center. The analysis included transplanted units (considered the best choice) & the next best high resolution typed back-up units (reserved but not shipped). Pt racial/ ethnic origins were prospectively obtained by detailed family history & grouped as previously described (Barker J. et al. BBMT 2010). Results: The characteristics of 513 units chosen for 136 CBT recipients by pt ancestry are shown (Table 1). Pts had highly diverse origins including 70 (51%) non-Europeans. The 513 units included 270 units infused as the graft (134 doubles & 2 singles) & 243 back-up units (109 pts had 2 back-ups, 25 pts had one & 2 had none). Thus, 4 best units were analyzed in 109 pts (all double unit recipients), 3 best in 25 pts (all doubles), & 1 unit in 2 pts (both singles). The median weight of the 136 pts was 81 kg. Asian pts (median 68 kg) had a lower weight than other groups. The median TNC content of units for the 66 European pts was higher than that for the 70 Non-Europeans (218 vs 196, p = 0.004). Units chosen for Northwestern (NW) Europeans had the highest median TNC content (235) with lower TNC content in units for Southern Europeans (202), Asian (193), African (191) & White Hispanic (189) pts. Units chosen for European pts also had a higher median CD34+ cell content (162) than Non-Europeans (138), p = 0.004. NW Europeans had units with a higher median CD34+ content (198) & the lowest CD34+ content were those for African (124) & Middle Eastern pts (124). When patient weight was considered, median TNC/kg dose per unit was similar in European and Non-European pts (2.7 vs 2.6, p = NS). Units for NW Europeans had the highest median TNC dose (3.0) whereas those for African pts had the lowest TNC dose (2.4). Units for Europeans had a higher median CD34+ dose (2.0) than Non-Europeans (1.7) although this difference was not significant (p = 0.15). Additionally, similar to TNC dose, median CD34+ dose was highest in units for NW European pts (2.2) & lowest in units chosen for African pts (1.5). 89% of chosen units were 4/6 HLA-matched with no differences between Europeans & non-Europeans. Furthermore, the median 8 allele HLA-match was 5/8 (range 2-8/8) with no overall differences between units for Europeans and Non-Europeans (p = NS). When only transplanted units were analyzed (Table 2), the median TNC & CD34+ contents were significantly lower in non-Europeans than Europeans (238 vs 216, p = 0.01 & 184 vs 160, p = 0.016). Overall, however, units received by Europeans vs non-European pts had similar TNC & CD34+ doses (p = NS). However, differences in the CD34+ content combined with differences in pt weights resulted in disparities in CD34+ doses by ancestry sub-group. NW Europeans (high weight, high CD34+ content) received the best CD34+ doses; lower CD34+ content in Asian pts was compensated for by their lower weight. African pts (high weight, low CD34+ content) received the lowest CD34+ doses. The median 8 allele HLA-match for all was 5/8 (range 3-8/8) with the exception of African pts [median 4/8 (range 3-7/8)]. Moreover, while 108 (40%) of transplanted units were 3-4/8 HLA-matched overall, there were marked differences between pt sub-groups with only 23% of units for NW Europeans being 3-4/8 vs 42% for southern Europeans, 46% for white Hispanics & 53% for Africans. Conclusions: While CB significantly extends transplant access to racial & ethnic minorities, differences in cellular content translates to many minority pts receiving lower dosed units. There are also marked racial/ ethnic differences in HLA-match grade with African pts the most likely to receive highly mismatched units. This data supports ongoing funding of public CB banks to further increase the inventory of high dosed & better matched units for all but especially racial & ethnic minority pts. Disclosures Shah: Janssen: Research Funding; Amgen: Research Funding. Kernan:National Cancer Institute: Research Funding.

Background : While incorporation of HLA-allele matching has highlighted high degrees of CB unit-recipient HLA-disparity, the less stringent HLA-match requirements extends allograft access to many patients without matched adult donors. The extent to which HLA-mismatch can adversely affect CBT outcomes, & the maximum permitted degree of HLA-mismatch, however, are not established. Methods : We analyzed TRM, relapse & PFS after intermediate intensity myeloablative cyclophosphamide 50 mg/kg, fludarabine 150 mg/m2, thiotepa 5-10 mg/kg, 400 cGy TBI double unit CBT in adults transplanted for hematologic malignancies. Eligible patients (pts) were first allograft recipients ≤ 65 years transplanted for acute leukemia/ MDS/ MPD (≤ 10% blasts pre-CBT), B-cell or T-cell lymphomas between 1/2014 - 12/2017. TNC & CD34+ dose were typically given priority over HLA-match in unit selection. Results : 102 pts [51 (50%) non-European, 59 (58%) CMV seropositive, median age 50 years (range 21-65), median weight 80 kg (range 36-137)] were transplanted. Diagnoses included 71 acute leukemias, 17 CML/ MPD/ MDS, 14 B- or T-cell NHLs. All received double unit grafts comprised of 2 (1%) 6/6 units, 21 (10%) 5/6, 181 (89%) 4/6 HLA-match, median unit-recipient 8-allele HLA-match 5/8 (range 3-7), & median infused viable CD34+ dose 1.3 x 105/kg/unit (range 0.2-8.6). Forty-eight (47%) CB grafts were supplemented with haplo-identical CD34+ cells as a myeloid bridge. The cumulative incidence of sustained CB engraftment was 97% (95%CI 90-99) with 2 pts having graft failure. A dominant (engrafting) unit was identified in all pts but 1 who died on day +14. Day 180 incidences of grades II-IV & III-IV aGVHD were 89% (95%CI 81-94) & 23% (95%CI 15-31), respectively. 1-yr cGVHD was 4% (95%CI 1-9). Cumulative incidence of TRM was 9% (95%CI 4-15) at day 180 & 14% (95%CI 8-22) at 2 yrs. 11% (95%CI 6-19) of pts relapsed by 2 yrs. With a median survivor follow-up of 2 yrs 3 months (range 6 - 51), the 2-yr overall survival is 82% (95%CI 75-90) & PFS is 74% (95%CI 66-84). Causes of death were transplant-related in 16 pts [6 aGVHD, 4 infection (2 viral, 1 bacterial, 1 fungal), 4 organ failure, 1 graft failure, 1 unknown] & due to relapse in 4 pts. By 2-yrs post-CBT, engrafting unit-recipient HLA-allele match had no association with TRM or PFS (Figure). Univariate analysis of the association between recipient variables [age, age adjusted hematopoietic cell transplant-comorbidity index (aaHCT-CI), revised disease risk index (rDRI)] & graft variables [engrafting unit-recipient HLA-allele match & infused CD34+ cell dose] & TRM, relapse & PFS are shown (Table). Age & aaHCT-CI were associated with 2-yr TRM with the 50 younger pts (< 50 years), & the 66 pts with aaHCT-CI 0-3, having especially low 2-yr TRM of 5% & 10%, respectively. Neither engrafting unit-recipient HLA-match nor CD34+ dose were associated with TRM. No pt or graft variable was associated with relapse. The only significant variable associated with 2-yr PFS was aaHCT-CI. The 66 pts with a lower aaHCT-CI score of 0-3 had a higher 2-year PFS [82% (95%CI 73-92)] than the 36 high score 4-9 pts [60% (95%CI 46-79)]. rDRI, engrafting unit-recipient 8-allele HLA-match & CD34+ cell dose had no effect. Multivariate analysis of TRM was precluded by too few events. Multivariate analysis of PFS including aaHCT-CI, rDRI & engrafting unit-recipient HLA-match showed high aaHCT-CI was associated with worse PFS [HR 2.82 (1.28-6.25) if score 4-9 vs 0-3, p = 0.01]; neither rDRI [HR 1.21 (0.54-2.69) if high-very high vs low-intermediate, p = 0.64] nor engrafting unit-recipient HLA-match [HR 2.06 (0.89-4.74) if 5-7/8 vs 3-4/8 matched (p = 0.09] were significant. Conclusions : Our finding that a high level of HLA-allele mismatch of the engrafting unit was not significantly associated with TRM or PFS in adult dCBT for hematologic malignancies has multiple implications. It supports the use of units with a relatively high degree of HLA-mismatch in the dCBT setting if needed in these pts. This facilitates extension of allograft access to the majority of pts including minorities. Moreover, the high PFS supports our centers unit selection strategy that gives unit quality & cell dose a priority in adults with malignancies to optimize engraftment. Finally, this data support CBT in many high-risk or urgent pts even if a well-matched CB graft cannot be identified. This is especially true in younger pts or those with a low 0-3 aaHCT-CI who had high 2-yr PFS. Disclosures Perales: Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees and Clinical trial support; Novartis: Other: Personal fees; Abbvie: Other: Personal fees; Merck: Other: Personal fees; Takeda: Other: Personal fees. Sauter:Juno Therapeutics: Consultancy, Research Funding; Sanofi-Genzyme: Consultancy, Research Funding; Spectrum Pharmaceuticals: Consultancy; Novartis: Consultancy; Precision Biosciences: Consultancy; Kite: Consultancy. Shah:Janssen: Research Funding; Amgen: Research Funding.

Intestinal microbiota composition is associated with important outcomes after allo-HCT including survival, relapse, GVHD, and infections. These observations have been made almost exclusively by characterizing the microbiota in the first weeks after transplantation, and in single-center studies. We previously reported that intestinal diversity measured peri-neutrophil engraftment is predictive of overall survival in a multicenter cohort. Here, we hypothesized that pre-HCT microbiota configuration may also be an important determinant of post-transplantation outcomes. We report a multicenter analysis conducted at 4 independent international institutions to test this hypothesis. We collected 1922 stool samples ~weekly from 991 unique allo-HCT patients at four international transplant centers: Cohorts 1 and 2 in the US, cohort 3 in Europe, and cohort 4 in Japan. The patients-all adult recipients of allo-HCT-varied in underlying diagnosis, donor-graft sources, conditioning intensity, and GVHD prophylaxis. Samples from all 4 centers were sequenced and analyzed at a central laboratory using the V4-V5 region of 16S rRNA. For patients with multiple samples within the pre-HCT sampling period, which we defined as day -20 to 0, median values were used. On average, patients from all 4 transplantation centers had reduced microbiota diversity pre-HCT, as measured by median α-diversity (inverse Simpson) values that were 1.7-to-2.5-fold lower than those of healthy volunteers. This comparison was made both in volunteers whose samples we sequenced ourselves and in a publicly available dataset (Fig A, p We next asked how similar these pre-HCT communities are across geography. We found that Bray-Curtis distances between institutions were reproducibly much smaller in magnitude than the changes observed over time during transplantation (Fig C, p In order to characterize these clinically relevant low-diversity phenotypes, we defined domination as a microbiota injury in which any operational taxonomic unit comprises >30% of bacterial abundance. The dominating taxa belonged to multiple genera, the most common being Enterococcus, Streptococcus, Lactobacillus, Escherichia, and Klebsiella (Fig E) as annotated by Greengenes and NCBI databases. At all four institutions, the cumulative incidence of intestinal domination by any organism was >50% by day 0 and was >87% by day +28 (Fig F). We performed additional analyses in the largest cohort and found that the low-diversity state is associated with exposure to broad-spectrum antibiotics, conditioning intensity, and low calorie intake. We demonstrate that HCT patients at 4 institutions on 3 continents presented with microbiota configurations that were similar to one another and distinct from those of healthy individuals. Severe microbiota injury as revealed by domination is a common event whose development begins before allograft infusion, and pre-HCT microbiota injury predicts poor overall survival. These observations suggest the pre-HCT period as a window of opportunity to (a) assess microbiota injury as part of comorbidity evaluation, (b) inform selection of antibiotic prophylaxis, gut-decontamination, GVHD-prophylaxis, or conditioning regimens, and (c) intervene with microbiota injury-remediation or prevention strategies. Figure. Figure. Disclosures Peled: Seres Therapeutics: Research Funding. Perales:Merck: Other: Personal fees; Takeda: Other: Personal fees; Abbvie: Other: Personal fees; Novartis: Other: Personal fees; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees and Clinical trial support. Jenq:MicrobiomeDx: Consultancy; Ziopharm Oncology: Consultancy; Seres Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees; Seres Therapeutics, Inc.: Patents & Royalties.

Introduction: We have previously reported that clinically relevant, dramatic reductions occur in intestinal bacterial diversity during allogeneic hematopoietic stem cell transplant (allo-HSCT). These are likely attributable to antibiotic exposure, nutritional alterations, and intestinal mucosa injury from high-dose chemotherapy. Patients undergoing autologous hematopoietic stem cell transplantation (AHCT) also receive antibiotics and experience nutritional alterations due to mucositis and other gastrointestinal toxicities. We hypothesized that the pattern of dysbiosis seen in AHCT patients would reflect the changes in allo-HSCT patients. Here, we present a novel analysis of microbiota diversity in AHCT patients from two independent institutions. Methods: We retrospectively identified a cohort of 365 patients (median age 60 years) who underwent AHCT for treatment of hematologic malignancy between May 2009 to February 2018 at two large-volume transplant centers in the US. The population was diverse in terms of histology, conditioning regimens and remission status prior to transplant, with 179 (49%) patients diagnosed with multiple myeloma, 153 (42%) patients diagnosed with lymphoma, and 33 (9%) patients with other diseases. Stool samples from the selected patients were collected approximately weekly during inpatient hospitalization. Sequencing of the V4-V5 region of the bacterial 16S rRNA genes from all samples was performed on the Illumina platform at a central site. Microbial diversity was measured by the Simpson reciprocal a-diversity index (S). We defined the pre-AHCT period as days -10 to 0, and computed median values for patients with multiple samples within that period. We additionally defined monodomination of the microbiota as a single operational taxonomic unit comprising >30% of bacterial abundance. For comparison, we sequenced samples from 17 healthy volunteers and used a public dataset of sequences from 313 healthy volunteers from the NIH Human Microbiome Project (HMP). Median pre-transplant microbial diversity in the healthy patient and AHCT groups was compared by a pairwise Wilcox test to a retrospective cohort of allo-HSCT patients. Results: We evaluated 857 samples from 365 adult patients undergoing AHCT, with 316 patients from Memorial Sloan Kettering Cancer Center (MSKCC) and 49 patients from Duke University Medical Center (DUMC). Median pre-transplant diversity in AHCT patients from both centers was significantly lower than in normal controls (Fig 1A) (HMP vs MSKCC AHCT, S=12.05 vs. 9.19, p50% by day 0 and was >75% by day +14. Conclusion: Microbial diversity is reduced prior to transplant in both AHCT and allo-HSCT patients. Loss of diversity after AHCT occurs across centers and the degree of injury is comparable to the dysbiosis in allo-HSCT patients. Preliminary analysis suggests that lower diversity may correlate with worse progression-free survival (PFS) in myeloma patients in our diverse AHCT cohort. Given the known associations of alterations in microbiota composition with toxicities and overall survival in allo-HSCT patients, further evaluation of microbiota injury and its associations with toxicities, PFS, and overall survival (OS) in AHCT patients is warranted. Figure 1: A: The median Simpson reciprocal a-diversity index (S) of pre-transplant (days -10 to 0) samples of AHCT and allo-HSCT patients from two centers, as well as two cohorts of healthy volunteers, was plotted and a pairwise Wilcox test was performed, with p-values as indicated. B: (S) was plotted against time relative to allo-HSCT (on L) and AHCT (on R), for samples collected from day -10 to day +30. Larger values indicate greater diversity. C: Microbiota composition and changes in bacterial monodominance after transplant (days -14 to +28); the most common genus post-transplant is Streptococcus. Figure 1. Figure 1. Disclosures Peled: Seres Therapeutics: Research Funding. Sauter:Juno Therapeutics: Consultancy, Research Funding; Sanofi-Genzyme: Consultancy, Research Funding; Spectrum Pharmaceuticals: Consultancy; Novartis: Consultancy; Precision Biosciences: Consultancy; Kite: Consultancy. Shah:Amgen: Research Funding; Janssen: Research Funding. Perales:Novartis: Other: Personal fees; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees and Clinical trial support; Merck: Other: Personal fees; Takeda: Other: Personal fees; Abbvie: Other: Personal fees. Jenq:Ziopharm Oncology: Consultancy; Seres Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees; MicrobiomeDx: Consultancy; Seres Therapeutics, Inc.: Patents & Royalties.

Background: Multiple myeloma (MM) patients who achieve minimal residual disease (MRD) negative status after upfront treatment have prolonged progression-free and overall survival compared with those who remain MRD(+) (Landgren CO, Devlin SM et al. Bone Marrow Transplant. 2016;51(12):1565-8). Commensal intestinal microbial composition has been associated with treatment outcomes in cancer patients. We sought to evaluate whether the composition of the intestinal microbiota is associated with MRD status in patients with MM. Methods: Stool samples were collected prospectively from 34 patients after completion of upfront therapy for MM at the time of MRD testing. MRD was assessed with next-generation flow cytometry of bone marrow aspirates (sensitivity 10-5). Microbial analysis was performed via sequencing of 16S rRNA V4-V5 regions using the Illumina MiSeq platform and sequence data was analyzed using UPARSE (Edgar RC, Nature Methods 2013;10(10):996-8). The linear discriminant effect size method (LEfSe) (Segata N et al. Genome Biol. 2011;12(6):R60.) was used to compare detected clades among all groups and evaluate for associations with outcomes, using MRD as class and autologous stem cell transplant (ASCT) as subclass. Alpha diversity was calculated by the Inverse Simpson index and differential relative abundance were calculated using the phyloseq package and compared using the Wilcoxon rank sum test on the R statistical computing platform. Results: Among 34 patients evaluable for microbiota composition and MRD status, the median age was 62.5 years and 16 (47.1%) were MRD(-) at time of stool collection. 24 (70.6%) were treated with carfilzomib, lenalidomide, and dexamethasone as induction therapy (MRD(-): 14 (87.5%), MRD(+):10 (55.5%). 4 (28.5%) MRD(-) patients had autologous stem cell transplant(ASCT), compared with 10 (55.5%) who were MRD(+). In the cohort's samples, we observed 19 phyla, 315 genera, 654 species, and 1549 operational taxonomic units (OTUs). There was no significant difference in alpha diversity between MRD(-) (median 12.24, IQR = 8.76-13.98) and MRD(+) patients (median 12.44, IQR = 8.36 -16.23), p=0.6 by Wilcoxon rank sum test. A positive association with MRD negativity was noted with two butyrate-producing organisms, Eubacterium hallii (p=0.001) and Faecalibacterium prausnitzii (p= 0.006). To further evaluate these relationships, we performed a differential abundance analysis of these selected taxa in MRD(+) and MRD(-) patients at the genus and species level. The relative abundance of the genera Eubacterium and Faecalibacterium were higher in fecal samples from MRD(-) patients than MRD(+) patients (Eubacterium MRD(-): median 4.51% (IQR = 2.83 - 7.32%) vs. MRD(+): median 3.07% (IQR = 1.35 - 3.87%), p=0.0326; Faecalibacterium MRD(-): median 1.68% (IQR = 0.69 - 7.48%) vs. MRD(+): median 0.003% (IQR = 0 - 3.19%), p=0.022. The relative abundance of both species of interest were higher in MRD(-) patients than in MRD(+) patients: E. hallii MRD(-): median 2.67% (IQR = 2.11 - 3.98%) vs. MRD(+): median 1.01% (IQR = 0 - 2.16%), p=0.001; F. prausnitzii MRD(-): median 1.43% (IQR = 0.53 - 7.28%) vs. MRD(+): median 0.3%, (IQR = 0 - 2.54%), p=0.022. Other species of Eubacterium and Faecalibacterium were not significantly differentially abundant between the two groups. Conclusions: Intestinal microbiota containing several butyrate-producing anaerobes appear to be associated with MRD-negativity in patients with myeloma, with higher relative abundance of Eubacterium hallii and Faecalibacterium prausnitzii in MRD(-) patients compared with MRD(+) patients. Butyrate and other short-chain fatty acids are biologically active metabolites formed during microbial fermentation of dietary or host-derived carbohydrates, which supply the host with energy and also modulate immunity, including exerting anti-inflammatory functions. Microbes of the genus Eubacterium have been associated with reduced risk of relapse in several hematologic cancers after allogeneic hematopoietic cell transplantation, including MM (Peled JU, Devlin SM et al. J Clin Oncol 2017;35(15):1650-9). This is first study to our knowledge to suggest an association between gut microbiota and MRD status in patients with myeloma and supports further investigation of a potential role for intestinal microbiota in the natural history and treatment of myeloma. Disclosures Peled: Seres Therapeutics: Research Funding. Landgren:Karyopharm: Consultancy; Merck: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Lesokhin:Squibb: Consultancy, Honoraria; Genentech: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Serametrix, inc.: Patents & Royalties: Royalties; Takeda: Consultancy, Honoraria; Janssen: Research Funding.

We have developed a dual bioluminescent reporter system allowing noninvasive, concomitant imaging of T-cell trafficking, expansion, and activation of nuclear factor of activated T cells (NFAT) in vivo. NFAT activation plays an important role in T-cell activation and T-cell development. Therefore we used this system to determine spatial-temporal activation patterns of (1) proliferating T lymphocytes during graft-versus-host disease (GVHD) and (2) T-cell precursors during T-cell development after allogeneic hematopoietic stem cell transplantation (HSCT). In the first days after HSCT, donor T cells migrated to the peripheral lymph nodes and the intestines, whereas the NFAT activation was dominant in the intestines, suggesting an important role for the intestines in the early stages of alloactivation during development of GVHD. After adoptive transfer of in vitro-derived T-cell receptor (TCR) H-Y transgenic T-cell precursors into B6 (H-2b) hosts of both sexes, NFAT signaling and development into CD4+ or CD8+ single-positive cells could only be detected in the thymus of female recipients indicating either absence of positive selection or prompt depletion of double-positive thymocytes in the male recipients. Because NFAT plays an important role in a wide range of cell types, our system could provide new insights into a variety of biologic processes.

Acute graft-versus-host disease (GVHD) remains the major obstacle to a more favorable therapeutic outcome of allogeneic hematopoietic stem cell transplantation (HSCT). GVHD is characterized by tissue damage in gut, liver, and skin, caused by donor T cells that are critical for antitumor and antimicrobial immunity after HSCT. One obstacle in combating GVHD used to be the lack of understanding the molecular mechanisms that are involved in the initiation phase of this syndrome. Recent research has demonstrated that interactions between microbial-associated molecules (pathogen-associated molecular patterns [PAMPs]) and innate immune receptors (pathogen recognition receptors [PRRs]), such as NOD-like receptors (NLRs) and Toll-like receptors (TLRs), control adaptive immune responses in inflammatory disorders. Polymorphisms of the genes encoding NOD2 and TLR4 are associated with a higher incidence of GVHD in HSC transplant recipients. Interestingly, NOD2 regulates GVHD through its inhibitory effect on antigen-presenting cell (APC) function. These insights identify important mechanisms regarding the induction of GVHD through the interplay of microbial molecules and innate immunity, thus opening a new area for future therapeutic approaches. This review covers current knowledge of the role of PAMPs and PRRs in the control of adaptive immune responses during inflammatory diseases, particularly GVHD.

Introduction The composition of the intestinal microbiota is associated with important outcomes after allo-HSCT including survival, relapse, GVHD, and infections. The microbial composition changes drastically after allo-HSCT, with precipitous drops in α-diversity and domination by single organisms. Low diversity is particularly associated with poor overall survival from transplant-related mortality, including death after GVHD. These observations were all made in single-center cohorts. Here we compare-for the first time-the kinetics of microbiota diversity in allo-HSCT patients from three independent international institutions. We also explore the role of nutrition as well as antibiotics in a single-center pilot study and the recovery from microbiota injury after discharge. Methods We collected stool samples approximately weekly from adult allo-HSCT inpatients and approximately monthly post-discharge. Samples from all three centers were sequenced at MSKCC using the V4-V5 region of 16S rRNA. In the nutrition cohort, patients self-documented oral intake at each meal; data were confirmed in focused patient interviews by a registered dietician three times weekly and nutritional compositions were obtained from the hospital kitchen database. Oral swish samples were collected prior to allo-HSCT and during post-transplant neutropenia. Results 5,823 samples from 1,118 patients were compared: 5,508 samples from 947 MSKCC patients, 108 samples from 40 Duke patients, and 152 samples from 109 Regensburg, Germany patients. The patients-all adult recipients of allo-HSCT -varied in underlying diagnosis, donor-graft sources, conditioning intensity, and GVHD prophylaxis. We mapped the microbiota composition of each sample in 2-D space using t-Distributed Stochastic Neighbor Embedding (t-SNE). Samples from all three centers spread throughout t-SNE space, revealing no transplant-center-specific effect (Fig 1A). Enterococcus domination, a low-diversity state we previously associated with subsequent enterococcal bacteremia was observed in all three centers (Fig 1A). Diversity decreased comparably after allo-HSCT transplant at all centers (Fig 1B), validating our prior findings in this larger, international multicenter cohort. In a separate cohort of MSKCC patients, we explored the relative contributions of antibiotics and nutritional intake using a multivariate linear regression model of α-diversity dynamics. Exposure to piperacillin-tazobactam (pip-tazo), a drug with broad-spectrum antibacterial activity, and calorie intake were significant predictors (p Conclusion We determine that striking microbiota injuries after allo-HSCT occur generally across geographic regions. Nutritional perturbations and antibiotic exposure are both associated with microbiota injury, and recovery from the injured state occurs over several months post-discharge. Importantly, diversity at the time of neutrophil engraftment is associated reproducibly with overall survival. Disclosures Peled: Seres: Research Funding. Sung: Merck: Research Funding; Novartis: Research Funding; Cellective: Research Funding. Jenq: Seres: Research Funding. van den Brink: Jazz Pharmaceuticals: Consultancy; Seres: Research Funding; PureTech Health: Consultancy; Therakos Institute: Other: Speaking engagement.

Endogenous thymic regeneration is a crucial function that allows for renewal of immune competence following immunodepletion caused by common cancer therapies such as cytoreductive chemotherapy or radiation; however, the mechanisms governing this regeneration remain poorly understood. Moreover, despite this capacity, prolonged T cell deficiency is a major clinical hurdle in recipients of hematopoietic stem cell transplantation (HSCT) and can precipitate high morbidity and mortality from opportunistic infections, and may even facilitate malignant relapse. We have recently described a central role for group 3 innate lymphoid cells (ILC) in a complex cellular and molecular network that drives endogenous thymic regeneration (Dudakov 2012 Science 336:91). Although IL-22 contributes considerably towards thymic regeneration and mice deficient for IL-22 lag behind WT controls in their recovery of thymic function, there is still some tissue regeneration in these mice, suggesting that other regeneration pathways also contribute to thymic repair. Unbiased transcriptome analysis on the damage-resistant non-hematopoietic compartemtn of the thymus revealed significant upregulation of Bmp4 and its downstream signalling targets (Fig. 1a). Further interrogation revealed that while thymic expression of BMP4 was restricted to fibroblasts and endothelial cells (ECs), only ECs increase their expression of Bmp4 after damage; and specific and inducible deletion of BMP4 in ECs led to significantly worse regeneration (Fig. 1b). Thymopoiesis is dependent on the close interaction between developing thymocytes and the non-hematopoietic stromal microenvironment, which includes highly specialized thymic epithelial cells (TECs) and ECs. While the role of TECs has been well studied, the contribution of ECs to thymopoiesis and thymic regeneration has thus far remained largely unclear. Careful interrogation of ECs after damage revealed that, much like ILCs, ECs are extremely resistant to multiple clinically relevant models of acute tissue injury including corticosteroids, chemotherapy and TBI. However, whole organ imaging analysis using light sheet field microscopy suggested that even though the number of ECs remain unchanged after damage, there is considerable structural changes to the vasculature including shortening of the vessels and reduced branching. Although BMP4 receptors are widely expressed in the thymus, there was enriched expression for BMP4 receptor subunits on TECs, which is consistent with the role of BMP4 in thymus ontogeny by promoting TEC development, at least partially due to its ability to induce expression of Foxn1 (Fig. 1c), a key transcription factor for the development and maintenance of TECs. Consistent with these findings, after thymic damage we observed a significant increase in the expression of Foxn1 after damage as well as GSEA enrichment for downstream FOXN1 target genes (Fig. 1d); including Dll4, the Notch ligand critical for T cell development and whose concentration we have previously shown can directly regulate thymic size (Velardi 2014 J Exp Med 211:2341). Finally, using a technique whereby ECs are transduced with the adenoviral gene E4ORF1 - ECs could be expanded ex vivo (exEC) and, when administered to mice after SL-TBI, significantly boost recovery of thymic function; but only when the exEC were derived from the thymus but not from heart or kidney (Fig. 1e). Consistent with endogenous regeneration, in vivo administration of exEC(Thy) induced the expression by TECs of Foxn1 and Dll4 . Here we demonstrate that rather than just being passive conduits that deliver oxygen and nutrients, ECs are active participants in organ function producing distinct paracrine factors that orchestrate thymic renewal. These studies thus not only detail a novel pathway promoting endogenous thymic regeneration, but also offer an innovative clinical approach to enhance T cell immunity in recipients of allo-HSCT and for individuals with T cell deficiencies due to aging, infectious disease, and common cancer treatments such as chemo- and radiation-therapy. Figure 1 Figure 1. Disclosures van den Brink: PureTech Health: Consultancy; Therakos Institute: Other: Speaking engagement; Seres: Research Funding; Jazz Pharmaceuticals: Consultancy.

The thymus is extremely sensitive to exogenous insults but has a remarkable capacity to regenerate which is lost with age. Reactive oxygen species (ROS) accumulate early after tissue damage and despite their toxic potential, ROS and their byproducts (such as lipid peroxidation products-LPPs) can act as regeneration signals by activating membrane or intracellular sensors and subsequent stress-response signalling pathways. Using Sublethal Total Body Irradiation (SL-TBI) as a model of acute thymic injury, we found a rapid accumulation of thymic ROS as well as lipid peroxidation products on cell membranes after SLTBI (Figure 1A&B). The damage-sensing ion channel Transient Receptor Potential cation channel family A member 1 (TRPA1) represents one of the major damage sensing receptors that can mediate cellular responses to oxidative stress mediators, such as LPPs. Using immunofluorescence (IF) microscopy we found that TRPA1 is enriched in the thymic medulla. Interestingly, although TRPA1 has been classically identified in nociceptive fibers, the major TRPA1 expressing structures in the thymus were not nerve fiber terminals, but primarily thymic endothelial cells (Figure1C), fibroblasts and subsets of epithelial cells. We have recently demonstrated that thymic endothelial cells can regulate regeneration through secretion of BMP-4, which can enhance Foxn1 expression and proliferation of thymic epithelial cells. In order to assess the functional role of TRPA1 in thymic regeneration after injury, we utilized TRPA1 knockout (TRPA1-/-) mice and quantified thymic reconstitution after SL-TBI. TRPA1-/- mice had significantly lower thymic cellularity compared to their age- and sex-matched WT controls, suggesting an association between TRPA1 deficiency and delayed endogenous thymic recovery (Figure 1D). The major deficit in thymocyte counts primarily affected double negative-4 (DN4), double positive (DP) and CD4+ single positive (SP-CD4+) thymocyte numbers. The thymic stroma of TRPA1-/- mice had lower endothelial cell and fibroblast counts (Figure 1D). In accordance with these findings drinking water administration of the TRPA1 agonist Allyl-Isothiocyanate (AITC), resulted in enhanced thymic regeneration after radiation exposure. Besides its positive effects on thymocyte counts, AITC significantly augmented endothelial cell counts after irradiation (Figure 1E). In conclusion these results suggest that TRPA1 plays a non-redundant role in thymic regeneration and that exogenous TRPA1 stimulation can enhance immune recovery after damage. Disclosures van den Brink: Seres: Research Funding; Jazz Pharmaceuticals: Consultancy; PureTech Health: Consultancy; Therakos Institute: Other: Speaking engagement.

Strong inter-individual variation in clinical response to checkpoint inhibitors such as anti-CTLA-4 remains a major challenge. In a retrospective analysis, expression of viral defense genes in human melanomas including the cytosolic RNA receptor RIG-I (DDx58) has been associated with clinical benefit to CTLA-4 blockade. However, possible interconnections of these two distinct pathways remain unknown. Using CRISPR/Cas9 technology to generate B16 melanoma cells lines that lack nucleic acid receptors or downstream signaling molecules (RIG-I, STING, IRF3/7) together with available genetically deficient mouse models, we addressed the importance of nucleic acid receptor signaling in both tumor and host cells for the efficacy of anti-CTLA-4 immunotherapy. We here provide experimental proof that anti-CTLA-4 immunotherapy relies on tumor cell-intrinsic RIG-I but not STING signaling. Following anti-CTLA-4 treatment, tumor-intrinsic RIG-I signaling critically impacts on cross-presentation of tumor-associated antigen by CD103+ dendritic cells, the expansion of tumor antigen-specific CD8+ T cells and ultimately the accumulation of CD8+ T cells within tumor tissue. Consistently, therapeutic targeting of RIG-I with 5'-phosphorylated-RNA in both tumor and non-malignant cells potently augmented the efficacy of CTLA-4 checkpoint blockade. These processes were additionally dependent on host STING, MAVS and type I interferon signaling and were closely linked to RIG-I-mediated tumor cell death. In summary, our study identifies both tumor- and host-intrinsic RIG-I signaling as fundamental requirements for anti-CTLA-4-mediated antitumor immunity. Our data predict that targeting RIG-I may serve as a basis for the development of new combination strategies to increase the response rate of checkpoint inhibitor-based immunotherapy of malignancy including lymphoma, particularly in individuals that do not have a sufficient spontaneous antitumor T-cell immune response. Disclosures van den Brink: Jazz Pharmaceuticals: Consultancy; Seres: Research Funding; PureTech Health: Consultancy; Therakos Institute: Other: Speaking engagement.

Alloreactive donor cytolytic T lymphocytes play a critical role in pathophysiology of acute graft-versus-host disease (GVHD). As GVHD progression involves tumor necrosis factor superfamily receptor activation, and as apoptotic signaling for some tumor necrosis factor superfamily receptors might involve acid sphingomyelinase (ASMase)–mediated ceramide generation, we hypothesized that ASMase deletion would ameliorate GVHD. Using clinically relevant mouse models of acute GVHD in which allogeneic bone marrow and T cells were transplanted into asmase+/+ and asmase−/− hosts, we identify host ASMase as critical for full-blown GVHD. Lack of host ASMase reduced the acute inflammatory phase of GVHD, attenuating cytokine storm, CD8+ T-cell proliferation/activation, and apoptosis of relevant graft-versus-host target cells (hepatocytes, intestinal, and skin cells). Organ injury was diminished in asmase−/− hosts, and morbidity and mortality improved at 90 days after transplantation. Resistance to cytolytic T lymphocyte–induced apoptosis was found at the target cell membrane if hepatocytes lack ASMase, as hepatocyte apoptosis required target cell ceramide generation for formation of ceramide-rich macrodomains, sites concentrating proapoptotic Fas. These studies indicate a requirement for target cell ASMase in evolution of GVHD in liver, small intestines, and skin and provide potential new targets for disease management.

Keratinocyte growth factor (KGF), which is given exogenously to allogeneic bone marrow transplantation (allo-BMT) recipients, supports thymic epithelial cells and increases thymic output of naive T cells. Here, we demonstrate that this improved T-cell reconstitution leads to enhanced responses to DNA plasmid tumor vaccination. Tumor-bearing mice treated with KGF and DNA vaccination have improved long-term survival and decreased tumor burden after allo-BMT. When assayed before vaccination, KGF-treated allo-BMT recipients have increased numbers of peripheral T cells, including CD8+ T cells with vaccine-recognition potential. In response to vaccination, KGF-treated allo-BMT recipients, compared with control subjects, generate increased numbers of tumor-specific CD8+ cells, as well as increased numbers of CD8+ cells producing interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). We also found unanticipated benefits to antitumor immunity with the administration of KGF. KGF-treated allo-BMT recipients have an improved ratio of T effector cells to regulatory T cells, a larger fraction of effector cells that display a central memory phenotype, and effector cells that are derived from a broader T-cell–receptor repertoire. In conclusion, our data suggest that KGF can function as a potent vaccine adjuvant after allo-BMT through its effects on posttransplantation T-cell reconstitution.

CD4+ interleukin-17 (IL-17)+ T cells (Th17 cells) have been implicated in allograft rejection of solid organs and several autoimmune diseases. However, the functional role of Th17 cells in the development of acute graft-versus-host disease (GVHD) has not been well-characterized. We detected significant numbers of alloreactive CD4+ donor T cells expressing IL-17, IL-17F, or IL-22 in the lymphoid organs of recipients of an allogeneic bone marrow transplant. We found no differences in GVHD mortality or graft-versus-tumor (GVT) activity between wild type (WT) and IL-17−/− T-cell recipients. However, upon transfer of murine IL-17−/− CD4+ T cells in an allogeneic BMT model, GVHD development was significantly delayed behind recipients of WT CD4+ T cells, yet overall GVHD mortality was unaffected. Moreover, recipients of IL-17−/− CD4+ T cells had significantly fewer Th1 cells during the early stages of GVHD. Furthermore, we observed a decrease in the number of IFN-γ–secreting macrophages and granulocytes and decreased production of proinflammatory cytokines (interferon [IFN]-γ, IL-4, and IL-6) in recipients of IL-17−/− CD4+ T cells. We conclude that IL-17 is dispensable for GVHD and GVT activity by whole T cells, but contributes to the early development of CD4-mediated GVHD by promoting production of proinflammatory cytokines.

Delayed T-cell recovery is an important complication of allogeneic bone marrow transplantation (BMT). We demonstrate in murine models that donor BM-derived T cells display increased apoptosis in recipients of allogeneic BMT with or without GVHD. Although this apoptosis was associated with a loss of Bcl-2 and Bcl-XL expression, allogeneic recipients of donor BM deficient in Fas-, tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)- or Bax-, or BM-overexpressing Bcl-2 or Akt showed no decrease in apoptosis of peripheral donor-derived T cells. CD44 expression was associated with an increased percentage of BM-derived apoptotic CD4+ and CD8+ T cells. Transplantation of RAG-2-eGFP–transgenic BM revealed that proliferating eGFPloCD44hi donor BM-derived mature T cells were more likely to undergo to apoptosis than nondivided eGFPhiCD44lo recent thymic emigrants in the periphery. Finally, experiments using carboxyfluorescein succinimidyl ester–labeled T cells adoptively transferred into irradiated syngeneic hosts revealed that rapid spontaneous proliferation (as opposed to slow homeostatic proliferation) and acquisition of a CD44hi phenotype was associated with increased apoptosis in T cells. We conclude that apoptosis of newly generated donor-derived peripheral T cells after an allogeneic BMT contributes to delayed T-cell reconstitution and is associated with CD44 expression and rapid spontaneous proliferation by donor BM-derived T cells.

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor family that mediates epithelial cell proliferation and differentiation in a variety of tissues, including the thymus. We studied the role of KGF in T-cell development with KGF-/- mice and demonstrated that thymic cellularity and the distribution of thymocyte subsets among KGF-/-, wildtype (WT), and KGF+/- mice were similar. However, KGF-/- mice are more vulnerable to sublethal irradiation (450 cGy), and a significant decrease was found in thymic cellularity after irradiation. Defective thymopoiesis and peripheral T-cell reconstitution were found in KGF-/- recipients of syngeneic or allogeneic bone marrow transplant, but using KGF-/- mice as a donor did not affect T-cell development after transplantation. Despite causing an early developmental block in the thymus, administration of KGF to young and old mice enhanced thymopoiesis. Exogenous KGF also accelerated thymic recovery after irradiation, cyclophosphamide, and dexamethasone treatment. Finally, we found that administering KGF before bone marrow transplantation (BMT) resulted in enhanced thymopoiesis and peripheral T-cell numbers in middle-aged recipients of an allogeneic BM transplant. We conclude that KGF plays a critical role in postnatal thymic regeneration and may be useful in treating immune deficiency conditions. (Blood. 2006;107:2453-2460)

Inducible costimulator (ICOS) is expressed on activated and memory T cells and is involved in the regulation of cytokine production. We studied the role of ICOS on alloreactive T cells in graft versus host disease (GVHD) and determined that ICOS expression was up-regulated on alloreactive T cells in recipients of an allogeneic hematopoietic stem cell transplantation (allo-HSCT) with GVHD. We compared ICOS-/- T cells with wild-type (WT) T cells in 2 GVHD models. In both models, recipients of ICOS-/- T cells demonstrated significantly less GVHD morbidity and mortality, which was associated with less intestinal and hepatic GVHD but increased cutaneous GVHD. In addition, recipients of ICOS-/- donor T cells displayed a slight decrease in graft versus leukemia (GVL) activity. Further analysis of alloreactive ICOS-/- T cells showed no defect in activation, proliferation, cytotoxicity, and target organ infiltration. Recipients of ICOS-/- T cells had decreased serum levels of interferon-γ (IFN-γ), while interleukin-4 (IL-4) and IL-10 levels were increased, suggesting that alloreactive ICOS-/- T cells are skewed toward T helper-2 (Th2) differentiation. These data suggest a novel role for ICOS in the regulation of Th1/Th2 development of activated T cells. In conclusion, alloreactive ICOS-/- donor T cells induce less GVHD due to a Th2 immune deviation while GVL activity is slightly diminished.

Lymphocyte Peyer patch adhesion molecule (LPAM) or alpha(4)beta(7) integrin is expressed on lymphocytes and is responsible for T-cell homing into gut-associated lymphoid tissues through its binding to mucosal addressin cell adhesion molecule (MAdCAM), which is present on high endothelial venules of mucosal lymphoid organs. We found in murine allogeneic bone marrow transplantation (BMT) models that recipients of alpha(4)beta(7)(-) donor T cells had significantly less graft-versus-host disease (GVHD) morbidity and mortality compared with recipients of alpha(4)beta(7)(+) donor T cells. A kinetic posttransplantation analysis of lymphocytes in the intestines and mesenteric lymph nodes demonstrated a delayed invasion of lower numbers of alpha(4)beta(7)(+) T cells in recipients of alpha(4)beta(7)(-) T cells compared with recipients of alpha(4)beta(7)(+) T cells. Histopathologic analysis of GVHD target organs revealed that recipients of alpha(4)beta(7)(-) T cells developed less GVHD of the intestines and liver, whereas there was no difference in cutaneous and thymic GVHD between recipients of alpha(4)beta(7)(-) or alpha(4)beta(7)(+) T cells. Finally, we found that in vivo GVT activity of alpha(4)beta(7)(-) donor T cells was preserved. We conclude that the alpha(4)beta(7) integrin is important for the invasion of alloreactive donor T cells into the gut and the subsequent development of intestinal GVHD and overall GVHD morbidity and mortality.

Successful allogeneic hematopoietic stem cell transplantation (HSCT) requires reconstitution of normal T-cell immunity. Recipient thymic activity, biologic features of the allograft, and preparative regimens all contribute to immune reconstitution. We evaluated circulating T-cell phenotypes and T-cell receptor rearrangement excision circles (TRECs) in 331 blood samples from 158 patients who had undergone allogeneic HSCTs. All patients had received myeloablative conditioning regimens and were full donor chimeras in remission. Younger patients exhibited more rapid recovery and higher TRECs (P = .02). Recipients of T-cell–depleted allografts initially had lower TRECs than unmodified allograft recipients (P

Highly diverse bacterial populations inhabit the gastrointestinal tract and modulate host inflammation and promote immune tolerance. In allogeneic hematopoietic stem cell transplantation (allo-HSCT), the gastrointestinal mucosa is damaged, and colonizing bacteria are impacted, leading to an impaired intestinal microbiota with reduced diversity. We examined the impact of intestinal diversity on subsequent mortality outcomes following transplantation. Fecal specimens were collected from 80 recipients of allo-HSCT at the time of stem cell engraftment. Bacterial 16S rRNA gene sequences were characterized, and microbial diversity was estimated using the inverse Simpson index. Subjects were classified into high, intermediate, and low diversity groups and assessed for differences in outcomes. Mortality outcomes were significantly worse in patients with lower intestinal diversity; overall survival at 3 years was 36%, 60%, and 67% for low, intermediate, and high diversity groups, respectively (P = .019, log-rank test). Low diversity showed a strong effect on mortality after multivariate adjustment for other clinical predictors (transplant related mortality: adjusted hazard ratio, 5.25; P = .014). In conclusion, the diversity of the intestinal microbiota at engraftment is an independent predictor of mortality in allo-HSCT recipients. These results indicate that the intestinal microbiota may be an important factor in the success or failure in allo-HSCT.

Prolonged immunodeficiency after allogeneic bone marrow transplantation (BMT) causes significant morbidity and mortality from infection. This study examined in murine models the effects of interleukin-7 (IL-7) given to young and middle-aged (9-month-old) recipients of major histocompatibility complex (MHC)–matched or –mismatched allogeneic BMT. Although administration of IL-7 from day 0 to 14 after syngeneic BMT promoted lymphoid reconstitution, this regimen was ineffective after allogeneic BMT. However, IL-7 administration from day 14 (or 21) to 27 after allogeneic BMT accelerated restoration of the major lymphoid cell populations even in middle-aged recipients. This regimen significantly expanded donor-derived thymocytes and peripheral T cells, B-lineage cells in bone marrow and spleen, splenic natural killer (NK) cells, NK T cells, and monocytes and macrophages. Interestingly, although recipients treated with IL-7 had significant increases in CD4+ and CD8+ memory T-cell populations, increases in naive T cells were less profound. Most notable, however, were the observations that IL-7 treatment did not exacerbate graft-versus-host disease (GVHD) in recipients of an MHC-matched BMT, and would ameliorate GVHD in recipients of a MHC-mismatched BMT. Nonetheless, graft-versus-leukemia (GVL) activity (measured against 32Dp210 leukemia) remained intact. Although activated and memory CD4+ and CD8+ T cells normally express high levels of IL-7 receptor (IL-7R, CD127), activated and memory alloreactive donor-derived T cells from recipients of allogeneic BMT expressed little IL-7R. This might explain the failure of IL-7 administration to exacerbate GVHD. In conclusion, posttransplant IL-7 administration to recipients of an allogeneic BMT enhances lymphoid reconstitution without aggravating GVHD while preserving GVL.