Abstract: 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 & lt; 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 & lt; 0.001), while cefepime (β = 2.69, P & lt; 0.05) and meropenem (β = 1.96, P & lt; 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 & lt; 0.05), while an increase in total fat intake (range = 0-183.3g; median = 34.5g) improved cluster 1 stability (β = 1.44, P & lt; 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.

Abstract: 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 & gt;0.01 and effect size & gt;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 .

Abstract: 7015 Background: Relationships between microbiota composition and clinical outcomes following allogeneic hematopoietic cell transplantation (allo-HCT) have been described in single-center studies. Geographic variations in human microbial communities and differences in clinical practices across institutions raise the question of whether these associations are generalizable. We report the first multi-center study of the intestinal microbiota in allo-HCT. Methods: Intestinal communities in 8,768 fecal samples from 1,362 allo-HCT patients at 4 centers on 3 continents were profiled by 16S sequencing. Associations between microbiota composition and clinical outcomes were analyzed with proportional-hazards analysis in an observational study. Results: We observed reproducible patterns of microbiota injury characterized by loss of diversity and domination by single taxa. Low diversity in the neutrophil engraftment period was reproducibly associated with increased risk of death (multivariate HR 0.48 [0.30-0.77] p = 0.002 in the largest cohort). These reductions in OS were in part due to an increased risk of transplant-related mortality and graft-vs-host disease. Baseline pre-HCT samples already bore evidence of microbiome disruption; low diversity prior to transplantation was associated with poor survival. A bacterial-composition risk score that was trained in one cohort predicted mortality in the other three cohorts (multivariate HR 1.42 [1.04-1.93] p = 0.03), indicating that not only a diversity metric but also a signature of specific bacterial abundances is informative about post-HCT mortality risk across independent institutions. Conclusions: We demonstrate a relationship between microbiota and survival after allo-HCT that is independent of transplant center and geographic location. The diversity of clinical practices across institutions imposed significant heterogeneity in the study, yet we observed reproducible microbiota injury patterns and associations with outcomes. This concordance suggests that approaches to manipulate the intestinal microbiota in allo-HCT may be generalizable.

Abstract: Background Intestinal microbiome disruption is a risk factor for poor outcomes after allogeneic hematopoietic cell transplantation (allo-HCT), but the factors that contribute to microbiome injury are not well understood. We hypothesized that nutrition contributes to microbiome composition during allo-HCT. Methods Along with 16S profiling of collected fecal samples, we monitored daily inpatient nutritional intake using a customized real-time survey instrument. Data were quality-controlled by a dietitian and matched to the Food and Nutrient Database for Dietary Studies (FNDDS). Results 97 adult patients received conditioning regimens that were 68% ablative, 22% reduced, and 10% nonmyeloablative; 35% patients had acute myeloid leukemia and 35% had myelodysplastic/myeloproliferative neoplasms, while 10% had non-Hodgkin's lymphoma. Grafts were T-cell depleted in 49% and cord blood in 7%. The remaining had unmodified bone marrow or peripheral blood stem cell. 5 patients had enteral nutrition during the treatment. 22,614 food entries from 5,230 meals were collected during inpatient admissions. Among 800 sequenced stool samples, 329 were collected following exposure to an empiric antibiotic. The hierarchical organization of the FNDDS vocabulary facilitated application of alpha and beta diversities to diet data, as well as analysis of food items (e.g., chicken), which have been reported to more closely associate with microbiome composition than macronutrients (e.g., fat). Nutritional diversity declined from admission until day 3 (A). Clusters of dietary patterns were revealed by ordination with unweighted UniFrac distance applied, in which highly diverse diets clustered together (B). We observed a correlation between total calories consumed and fecal alpha diversity (r = 0.23, P & lt; 0.001) and the relative fecal abundance of the genus Blautia (r = 0.31, P & lt; 0.001), which we have previously associated with protection from lethal graft versus host disease (GVHD). In contrast, calorie intake was inversely associated with the fecal relative abundance of genus Enterococcus (r = -0.15, P & lt; 0.001), a genus we have reported exacerbates GVHD. Similar associations with microbiome features were observed for fiber. To gain insight into which types of foods are associated with microbiome injury, we constructed a Bayesian multilevel model to evaluate relationships between microbiota diversity and the amount consumed of different food groups in the two days preceding each stool sample. This model controlled for conditioning intensity, exposure to empiric antibiotics, enteral nutrition, and total parenteral nutrition. Empiric antibiotics refer to the ones for neutropenic fever such as piperacillin/tazobactam, carbapenems, cefepime, linezolid and for C. difficile such as oral vancomycin, and metronidazole. A random intercept term per patient to accommodate repeated measurements from the same patient and a random intercept term for each week relative to HCT were incorporated in the model. Intake of sugars, sweets and beverages was associated with low fecal microbiota alpha diversity (C). The model predicts that, on average, consumption of 100g sugars and sweets over two days would result in a biologically meaningful decline of diversity by 1.13-fold in inverse Simpson units. Interestingly, fruits, a food type enriched in simple sugars, trended toward associations with lower diversity as well. Conclusion Consumption of sugars and sweets is associated with lower fecal microbiota alpha diversity in allo-HCT. We hypothesize that initial insults to diverse microbial communities are exacerbated by simple sugars, which can be exploited by the remaining organisms as readily available nutrients. These results highlight the importance of developing evidence-based nutritional recommendations in allo-HCT. Figure 1 Figure 1. Disclosures Adintori: Vidafuel Inc.: Current holder of stock options in a privately-held company. Buchan: Savor Health: Current Employment. Gomes: Xbiome: Current Employment. Johnson: Diversigen: Consultancy. Knights: Diversigen: Consultancy. Jenq: Microbiome DX: Consultancy; Merck: Consultancy; Prolacta: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kaleido: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seres: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; LisCure: Consultancy, Membership on an entity's Board of Directors or advisory committees; MaaT Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Karius: Consultancy. Giralt: GSK: Membership on an entity's Board of Directors or advisory committees; PFIZER: Membership on an entity's Board of Directors or advisory committees; SANOFI: Membership on an entity's Board of Directors or advisory committees; JAZZ: Membership on an entity's Board of Directors or advisory committees; AMGEN: 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; Actinnum: Membership on an entity's Board of Directors or advisory committees. Perales: NexImmune: Honoraria; Servier: Honoraria; MorphoSys: Honoraria; Novartis: Honoraria, Other; Nektar Therapeutics: Honoraria, Other; Miltenyi Biotec: Honoraria, Other; Merck: Honoraria; Medigene: Honoraria; Takeda: Honoraria; Sellas Life Sciences: Honoraria; Kite/Gilead: Honoraria, Other; Karyopharm: Honoraria; Incyte: Honoraria, Other; Equilium: Honoraria; Cidara: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Omeros: Honoraria. van den Brink: Priothera: Research Funding; Da Volterra: Other: has consulted, received honorarium from or participated in advisory boards; Jazz Pharmaceuticals: Honoraria; Pharmacyclics: Other; Seres: Other: Honorarium, Intellectual Property Rights, Research Fundingand Stock Options; Wolters Kluwer: Patents & Royalties; Pluto Therapeutics: Current holder of stock options in a privately-held company, Other: has consulted, received honorarium from or participated in advisory boards ; Amgen: Honoraria; Frazier Healthcare Partners: Honoraria; Forty-Seven, Inc.: Honoraria; Notch Therapeutics: Honoraria; Nektar Therapeutics: Honoraria; GlaskoSmithKline: Other: has consulted, received honorarium from or participated in advisory boards; Kite Pharmaceuticals: Other; Novartis (Spouse): Other: has consulted, received honorarium from or participated in advisory boards; Juno Therapeutics: Other; MagentaTherapeutics: Honoraria; Ceramedix: Other: has consulted, received honorarium from or participated in advisory boards ; Rheos: Honoraria; DKMS (nonprofit): Other; Therakos: Honoraria; Merck & Co, Inc: Honoraria; Synthekine (Spouse): Other: has consulted, received honorarium from or participated in advisory boards; Lygenesis: Other: has consulted, received honorarium from or participated in advisory boards ; WindMILTherapeutics: Honoraria. Schluter: Postbiotics Plus LLC: Other: cofounder. Peled: MaaT Pharma: Consultancy; CSL Behring: Consultancy; DaVolterra: Consultancy; Seres Therapeutics: Research Funding.

Abstract: 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= & lt;0.001). A subgroup analysis of the patients with NHL also demonstrated decreased OS associated with P-I-M exposure whether the costimulatory domain was CD28 or 4-1BB (data not shown). Next, we queried whether patients exposed to P-I-M had more aggressive disease. We evaluated potential confounders for the findings in uni- and multi-variable models. Importantly, exposure to P-I-M remained a strong predictor of decreased OS (HR, 2.58; 95% CI, 1.55 - 4.3; p= & lt;0.001) (Table 1). Exposure to P-I-M was also associated with increased ICANS (p= 0.023) but not CRS (p= 0.058) in patients in the combined NHL and ALL cohort as well as in patients with NHL (CRS: p= 0.154, ICANS: p= 0.002) (data not shown). 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= & lt;0.001, Fig 1C). Finally, linear discriminant analysis effect size (LEfSe) identified an increased abundance of Lachnospiraceae, Ruminococcaceae, and Bacteroidaceae in patients who achieved a Day 100 complete response (CR) and those who experienced CAR-mediated toxicity (data not shown). 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 .

Abstract: The comprehension of protein and DNA binding in vivo is essential to understand gene regulation. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) provides a global map of the regulatory binding network. Most ChIP-seq analysis tools focus on identifying binding regions from coverage enrichment. However, less work has been performed to infer the physical and regulatory details inside the enriched regions. This research extends a previous blind-deconvolution approach to develop a post-peak–calling algorithm that improves binding site resolution and predicts cooperative interactions. At the core of our new method is a physically motivated model that characterizes the binding signal as an extreme value distribution. This model suggests a mathematical framework to study physical properties of DNA shearing from the ChIP-seq coverage. The model explains the ChIP-seq coverage with two signals: The first considers DNA fragments with only a single binding event, whereas the second considers fragments with two binding events (a double-binding signal). The model incorporates motif discovery and is able to detect multiple sites in an enriched region with single-nucleotide resolution, high sensitivity, and high specificity. Our method improves peak caller sensitivity, from less than 45% up to 94%, at a false positive rate 〈 11% for a set of 47 experimentally validated prokaryotic sites. It also improves resolution of highly enriched regions of large-scale eukaryotic data sets. The double-binding signal provides a novel application in ChIP-seq analysis: the identification of cooperative interaction. Predictions of known cooperative binding sites show a 0.85 area under an ROC curve.

Abstract: Ionizing-radiation causes acute radiation syndrome leading to hematopoietic, gastrointestinal and cerebrovascular injuries. Substantial federal efforts have been made to develop therapeutic radioprotectants for routine clinical use, however it remains a long-standing and unresolved problem. We investigated a population of mice that recovered from high-dose radiation to live normal lifespans. These elite-survivors harbored distinct gut microbiota that developed post-radiation and protected against radiation-induced damage and death in germ-free and conventionally housed recipients. Elevated abundances of members of the bacterial taxa Lachnospiraceae and Enterococcaceae were associated with post-radiation restoration of hematopoiesis and gastrointestinal repair. These bacteria were also found to be more abundant in leukemia patients undergoing radiotherapy who also displayed milder gastrointestinal dysfunction. Metabolomics revealed increased fecal concentrations of microbially derived propionate and tryptophan metabolites in elite-survivors. The concentrations of these metabolites correlated with long-term radioprotection, mitigation of hematopoietic and gastrointestinal syndromes, and a reduction in pro-inflammatory responses. The novelty of our work lies in the first identification of gut microbiota and downstream metabolites in providing protection against lethal radiation. Furthermore, our work provides a comprehensive omics dataset at the bacteria and metabolite levels that is of broad interest and serves as a powerful resource and example for the identification of actionable therapeutic targets derived from microbiome studies.