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While allogeneic hematopoietic stem cell transplantations have a curative potential, infections and graft-versus-host disease remain significant problems. The intestinal microbiota can influence responses to cancer chemotherapy and the role of the microbiota in affecting allogeneic hematopoietic stem cell transplantation outcomes is increasingly appreciated. The following paper discusses the most recent developments in this area.

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.

Summary Mechanisms underlying age-related defects within lymphoid-lineages remain poorly understood. We previously reported that sex steroid ablation (SSA) induced lymphoid rejuvenation and enhanced recovery from hematopoietic stem cell (HSC) transplantation (HSCT). We herein show that, mechanistically, SSA induces hematopoietic and lymphoid recovery by functionally enhancing both HSC self-renewal and propensity for lymphoid differentiation through intrinsic molecular changes. Our transcriptome analysis revealed further hematopoietic support through rejuvenation of the bone marrow (BM) microenvironment, with upregulation of key hematopoietic factors and master regulatory factors associated with aging such as Foxo1. These studies provide important cellular and molecular insights into understanding how SSA-induced regeneration of the hematopoietic compartment can underpin recovery of the immune system following damaging cytoablative treatments. These findings support a short-term strategy for clinical use of SSA to enhance the production of lymphoid cells and HSC engraftment, leading to improved outcomes in adult patients undergoing HSCT and immune depletion in general., Graphical Abstract, Highlights • Sex steroid ablation (SSA) increases number of hematopoietic stem cells (HSCs) • SSA enhances reconstitution potential and self-renewal of HSCs • SSA reverses the age-associated decline in Foxo1 expression by hematopoietic niche • There is an increase in niche expression of hematopoiesis-associated factors after SSA, Age-related declines in immune function underlie a wide range of diseases and impaired capacity to recover adaptive immunity following therapeutic immune depletion. Increasing evidence implicates a functional decline in hematopoietic stem cells (HSCs). Here, Chidgey, Dudakov, and colleagues demonstrate that reversal of HSC aging by blocking the immunosuppressive actions of sex steroids involves mechanisms both intrinsic and extrinsic to HSCs. These findings will enable progress in understanding bone marrow and HSC aging and in developing strategies for immune regeneration.

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

Antibiotic treatment can deplete the commensal bacteria of a patient’s gut microbiota and, paradoxically, increase their risk of subsequent infections. In allogeneic hematopoietic stem cell transplantation (allo-HSCT), antibiotic administration is essential for optimal clinical outcomes but significantly disrupts intestinal microbiota diversity, leading to loss of many beneficial microbes. Although gut microbiota diversity loss during allo-HSCT is associated with increased mortality, approaches to reestablish depleted commensal bacteria have yet to be developed. We have initiated a randomized, controlled clinical trial of autologous fecal microbiota transplantation (auto-FMT) versus no intervention and have analyzed the intestinal microbiota profiles of 25 allo-HSCT patients (14 who received auto-FMT treatment and 11 control patients who did not). Changes in gut microbiota diversity and composition revealed that the auto-FMT intervention boosted microbial diversity and reestablished the intestinal microbiota composition that the patient had before antibiotic treatment and allo-HSCT. These results demonstrate the potential for fecal sample banking and posttreatment remediation of a patient’s gut microbiota after microbiota-depleting antibiotic treatment during allo-HSCT.

The role of Notch signaling in human innate lymphoid cell (ILC) differentiation is unclear, although IL-7 and IL-15 promote differentiation of natural cytotoxicity receptor (NCR) NKp44+ group 3 ILCs (NCR+ILC3s) and conventional NK (cNK) cells from CD34+ hematopoietic progenitor cells (HPCs) ex vivo. In this study, we analyzed the functions of Notch in the differentiation of NCR+ILC3s and cNK cells from human HPC subpopulations circulating in peripheral blood by limiting dilution and clonal assays using high-throughput flow cytometry. We demonstrated that Notch signaling in combination with IL-7 induced NCR+ILC3 differentiation, but conversely suppressed IL-15–dependent cNK cell generation in CD45RA+Flt-3−c-Kitlow, a novel innate lymphocyte-committed HPC subpopulation. In contrast, Notch signaling induced CD45RA−Flt-3+c-Kithigh multipotent HPCs to generate CD34+CD7+CD62Lhigh, the earliest thymic progenitor–like cells, which preserved high cNK/T cell potential, but lost NCR+ILC3 potential. These findings implicate the countervailing functions of Notch signaling in the fate decision between NCR+ILC3 and cNK cell lineages at different maturational stages of human HPCs. Inhibition of Notch functions by Abs specific for either the Notch1 or Notch2 negative regulatory region suggested that both Notch1 and Notch2 signals were involved in the fate decision of innate lymphocyte-committed HPCs and in the generation of earliest thymic progenitor–like cells from multipotent HPCs. Furthermore, the synergistic interaction between Notch and IL-7 in NCR+ILC3 commitment was primarily explicable by the induction of IL-7 receptor expression in the innate lymphocyte–committed HPCs by Notch stimulation, suggesting the pivotal role of Notch in the transcriptional control required for human NCR+ILC3 commitment.

Matsuzawa-Ishimoto et al. show that autophagy gene ATG16L1, which is associated with inflammatory diseases of the gastrointestinal tract, is essential for preventing necroptotic cell death and loss of Paneth cells in the intestinal epithelium., A variant of the autophagy gene ATG16L1 is associated with Crohn’s disease, an inflammatory bowel disease (IBD), and poor survival in allogeneic hematopoietic stem cell transplant recipients. We demonstrate that ATG16L1 in the intestinal epithelium is essential for preventing loss of Paneth cells and exaggerated cell death in animal models of virally triggered IBD and allogeneic hematopoietic stem cell transplantation. Intestinal organoids lacking ATG16L1 reproduced this loss in Paneth cells and displayed TNFα-mediated necroptosis, a form of programmed necrosis. This cytoprotective function of ATG16L1 was associated with the role of autophagy in promoting mitochondrial homeostasis. Finally, therapeutic blockade of necroptosis through TNFα or RIPK1 inhibition ameliorated disease in the virally triggered IBD model. These findings indicate that, in contrast to tumor cells in which autophagy promotes caspase-independent cell death, ATG16L1 maintains the intestinal barrier by inhibiting necroptosis in the epithelium.

Background Even though hematopoietic stem cell transplantation can be curative in patients with severe combined immunodeficiency, there is a need for additional strategies boosting T cell immunity in individuals suffering from genetic disorders of lymphoid development. Here we show that image-guided intrathymic injection of hematopoietic stem and progenitor cells in NOD-scid IL2rγnull mice is feasible and facilitates the generation of functional T cells conferring protective immunity. Methods Hematopoietic stem and progenitor cells were isolated from the bone marrow of healthy C57BL/6 mice (wild-type, Luciferase+, CD45.1+) and injected intravenously or intrathymically into both male and female, young or aged NOD-scid IL2rγnull recipients. The in vivo fate of injected cells was analyzed by bioluminescence imaging and flow cytometry of thymus- and spleen-derived T cell populations. In addition to T cell reconstitution, we evaluated mice for evidence of immune dysregulation based on diabetes development and graft-versus-host disease. T cell immunity following intrathymic injection of hematopoietic stem and progenitor cells in NOD-scid IL2rγnull mice was assessed in a B cell lymphoma model. Results Despite the small size of the thymic remnant in NOD-scid IL2rγnull mice, we were able to accomplish precise intrathymic delivery of hematopoietic stem and progenitor cells by ultrasound-guided injection. Thymic reconstitution following intrathymic injection of healthy allogeneic hematopoietic cells was most effective in young male recipients, indicating that even in the setting of severe immunodeficiency, sex and age are important variables for thymic function. Allogeneic T cells generated in intrathymically injected NOD-scid IL2rγnull mice displayed anti-lymphoma activity in vivo, but we found no evidence for severe auto/alloreactivity in T cell-producing NOD-scid IL2rγnull mice, suggesting that immune dysregulation is not a major concern. Conclusions Our findings suggest that intrathymic injection of donor hematopoietic stem and progenitor cells is a safe and effective strategy to establish protective T cell immunity in a mouse model of severe combined immunodeficiency. Electronic supplementary material The online version of this article (doi:10.1186/s13045-017-0478-z) contains supplementary material, which is available to authorized users.

Infection, relapse, and GVHD can complicate allogeneic hematopoietic stem cell transplantation (allo-HSCT). Although the effect of poor immune recovery on infection risk is well-established, there are limited data on the effect of immune reconstitution on relapse and survival, especially following T-cell depletion (TCD). To characterize the pattern of immune reconstitution in the first year after transplant and its effects on survival and relapse, we performed a retrospective study in 375 recipients of a myeloablative TCD allo-HSCT for hematologic malignancies. We noted that different subsets recover sequentially, CD8 + T cells first, followed by total CD4 + and naïve CD4 + T cells, indicating thymic recovery during the first year after HSCT. In the multivariate model, a fully HLA-matched donor and recovery of T-cell function, assessed by PHA response at 6 months, were the only factors independently associated with OS and EFS. In conclusion, T-cell recovery is an important predictor of outcome after TCD allo-HSCT.

The molecular pathways that regulate the tissue repair function of type I interferon (IFN-I) during acute tissue damage are poorly understood. We describe a protective role for IFN-I and the RIG-I/MAVS signaling pathway during acute tissue damage in mice. Mice lacking mitochondrial antiviral-signaling protein (MAVS) were more sensitive to total body irradiation- and chemotherapy-induced intestinal barrier damage. These mice developed worse graft-versus-host disease (GVHD) in a preclinical model of allogeneic hematopoietic stem cell transplantation (allo-HSCT) than did wild-type mice. This phenotype was not associated with changes in the intestinal microbiota but was associated with reduced gut epithelial integrity. Conversely, targeted activation of the RIG-I pathway during tissue injury promoted gut barrier integrity and reduced GVHD. Recombinant IFN-I or IFN-I expression induced by RIG-I promoted growth of intestinal organoids in vitro and production of the antimicrobial peptide regenerating islet-derived protein 3 g (RegIIIg). Our findings were not confined to RIG-I/MAVS signaling because targeted engagement of the STING (stimulator of interferon genes) pathway also protected gut barrier function and reduced GVHD. Consistent with this, STING-deficient mice suffered worse GVHD after allo-HSCT than did wild-type mice. Overall, our data suggest that activation of either RIG-I/MAVS or STING pathways during acute intestinal tissue injury in mice resulted in IFN-I signaling that maintained gut epithelial barrier integrity and reduced GVHD severity. Targeting these pathways may help to prevent acute intestinal injury and GVHD during allogeneic transplantation. 2017

The gastrointestinal tracts of mammals are colonized by hundreds of microbial species that contribute to health, including colonization resistance against intestinal pathogens. Many antibiotics destroy intestinal microbial communities and increase susceptibility to intestinal pathogens. Among these, Clostridium difficile, a major cause of antibiotic-induced diarrhoea, greatly increases morbidity and mortality in hospitalized patients. Which intestinal bacteria provide resistance to C. difficile infection and their in vivo inhibitory mechanisms remain unclear. Here we correlate loss of specific bacterial taxa with development of infection, by treating mice with different antibiotics that result in distinct microbiota changes and lead to varied susceptibility to C. difficile. Mathematical modelling augmented by analyses of the microbiota of hospitalized patients identifies resistance-associated bacteria common to mice and humans. Using these platforms, we determine that Clostridium scindens, a bile acid 7α-dehydroxylating intestinal bacterium, is associated with resistance to C. difficile infection and, upon administration, enhances resistance to infection in a secondary bile acid dependent fashion. Using a workflow involving mouse models, clinical studies, metagenomic analyses, and mathematical modelling, we identify a probiotic candidate that corrects a clinically relevant microbiome deficiency. These findings have implications for the rational design of targeted antimicrobials as well as microbiome-based diagnostics and therapeutics for individuals at risk of C. difficile infection.

SummaryAtg16L1 mediates the cellular degradative process of autophagy and is considered a critical regulator of inflammation based on its genetic association with inflammatory bowel disease. Here we find that Atg16L1 deficiency leads to an exacerbated graft-versus-host disease (GVHD) in a mouse model of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Atg16L1-deficient allo-HSCT recipients with GVHD displayed increased T cell proliferation due to increased dendritic cell (DC) numbers and costimulatory molecule expression. Reduced autophagy within DCs was associated with lysosomal abnormalities and decreased amounts of A20, a negative regulator of DC activation. These results broaden the function of Atg16L1 and the autophagy pathway to include a role in limiting a DC-mediated response during inflammatory disease, such as GVHD.

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

The relationships between commitments of dendritic cells (DCs) and T cells in human hematopoietic stem cells are not well understood. In this study, we enumerate and characterize conventional DC and plasmacytoid DC precursors in association with T cell and thymus-derived types of NK cell precursors among CD34+ hematopoietic progenitor cells (HPCs) circulating in human peripheral blood. By limiting-dilution analyses using coculture with stroma cells expressing Notch1 ligand, the precursor frequencies (PFs) of DCs in HPCs were found to significantly correlate with T cell PFs, but not with NK cell PFs, among healthy donors. Clonal analyses showed that the majority of T/NK dual- and T single-lineage precursors—but only a minority of NK single-lineage precursors—were associated with the generation of DC progenies. All clones producing both DC and T cell progenies were found with monocyte and/or granulocyte progenies, suggesting DC differentiation via myeloid DC pathways. Analyses of peripheral blood HPC subpopulations revealed that the lineage split between DC and T/NK cell progenitor occurs at the stage prior to bifurcation into T and NK cell lineages. The findings suggest a strong linkage between DC and T cell commitments, which may be imprinted in circulating lymphoid-primed multipotent progenitors or in more upstream HPCs.

Preventing unfavorable GVHD without inducing broad suppression of the immune system presents a major challenge of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We developed a novel strategy to ameliorate GVHD while preserving graft-versus-tumor (GVT) activity by small molecule–based inhibition of the NF-κB family member c-Rel. Underlying mechanisms included reduced alloactivation, defective gut homing, and impaired negative feedback on interleukin (IL)-2 production, resulting in optimal IL-2 levels, which, in the absence of competition by effector T cells, translated into expansion of regulatory T cells. c-Rel activity was dispensable for antigen-specific T-cell receptor (TCR) activation, allowing c-Rel–deficient T cells to display normal GVT activity. In addition, inhibition of c-Rel activity reduced alloactivation without compromising antigen-specific cytotoxicity of human T cells. Finally, we were able to demonstrate the feasibility and efficacy of systemic c-Rel inhibitor administration. Our findings validate c-Rel as a promising target for immunomodulatory therapy and demonstrate the feasibility and efficacy of pharmaceutical inhibition of c-Rel activity. Significance: Chemical inhibition of c-Rel diminishes alloactivation while preserving antigen-specific TCR activation, revealing the redundancy of c-Rel in T cell–mediated antitumor activity of both mouse and human T cells. Our study provides a highly innovative immunomodulatory approach that has true potential for drug development and clinical application with broad therapeutic implications, including allo-tolerance induction after allo-HSCT, as well as antitumor therapies. Cancer Discov; 4(5); 578–91. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 495

Velardi et al. show that sex steroids regulate thymopoiesis by directly modulating Notch signaling, and provide a novel clinical strategy to boost immune regeneration., Paradoxical to its importance for generating a diverse T cell repertoire, thymic function progressively declines throughout life. This process has been at least partially attributed to the effects of sex steroids, and their removal promotes enhanced thymopoiesis and recovery from immune injury. We show that one mechanism by which sex steroids influence thymopoiesis is through direct inhibition in cortical thymic epithelial cells (cTECs) of Delta-like 4 (Dll4), a Notch ligand crucial for the commitment and differentiation of T cell progenitors in a dose-dependent manner. Consistent with this, sex steroid ablation (SSA) led to increased expression of Dll4 and its downstream targets. Importantly, SSA induced by luteinizing hormone-releasing hormone (LHRH) receptor antagonism bypassed the surge in sex steroids caused by LHRH agonists, the gold standard for clinical ablation of sex steroids, thereby facilitating increased Dll4 expression and more rapid promotion of thymopoiesis. Collectively, these findings not only reveal a novel mechanism underlying improved thymic regeneration upon SSA but also offer an improved clinical strategy for successfully boosting immune function.

Ex vivo CD34+–selected T cell depletion (TCD) has been developed as a strategy to reduce the incidence of graft-versus-host disease (GVHD) after allogeneic (allo) hematopoietic stem cell transplantation (HSCT). Clinical characteristics, treatment responses, and outcomes of patients developing acute (aGVHD) and chronic GVHD (cGVHD) after TCD allo-HSCT have not been well established. We evaluated 241 consecutive patients (median age, 57 years) with acute leukemia (n = 191, 79%) or myelodysplastic syndrome (MDS) (n = 50, 21%) undergoing CD34+–selected TCD allo-HSCT without post-HCST immunosuppression in a single institution. Cumulative incidences of grades II-IV and III-IV aGVHD at 180 days were 16% (95% confidence interval [CI], 12 to 21) and 5% (95% CI, 3 to 9), respectively. The skin was the most frequent organ involved, followed by the gastrointestinal tract. Patients were treated with topical corticosteroids, poorly absorbed corticosteroids (budesonide), and/or systemic corticosteroids. The overall day 28 treatment response was high at 82%. The cumulative incidence of any cGVHD at 3 years was 5% (95% CI, 3 to 9), with a median time of onset of 256 days (range, 95 to 1645). The 3-year transplant-related mortality, relapse, overall survival, and disease-free survival were 24% (95% CI, 18 to 30), 22% (95% CI, 17 to 27), 57% (95% CI, 50 to 64), and 54% (95% CI, 47 to 61), respectively. The 1-year and 3-year probabilities of cGVHD-free/relapse-free survival were 65% (95% CI, 59 to 71) and 52% (95% CI, 45 to 59), respectively. Our findings support the use of ex vivo CD34+–selected TCD allograft as a calcineurin inhibitor–free intervention for the prevention of GVHD in patients with acute leukemia and MDS.

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.

SUMMARY In cancer biology, the functional interpretation of genomic alterations is critical to achieve the promise of genomic profiling in the clinic. For chronic lymphocytic leukemia (CLL), a heterogeneous disease of B-lymphocytes maturing under constitutive B cell receptor (BCR) stimulation, the functional role of diverse clonal mutations remains largely unknown. Here, we demonstrate that alterations in BCR signaling dynamics underlie the progression of B cells toward malignancy. We reveal emergent dynamic features—bimodality, hypersensitivity, and hysteresis—in the BCR signaling pathway of primary CLL B cells. These signaling abnormalities in CLL quantitatively derive from BCR clustering and constitutive signaling with positive feedback reinforcement, as demonstrated through single-cell analysis of phospho-responses, computational modeling, and super-resolution imaging. Such dysregulated signaling segregates CLL patients by disease severity and clinical presentation. These findings provide a quantitative framework and methodology to assess complex and heterogeneous leukemia pathology and to inform therapeutic strategies in parallel with genomic profiling., Graphical Abstract, In Brief Using phospho-flow cytometry and computational modeling, Ziegler et al. find that B cell receptor clustering and positive feedback through SYK and LYN drive signaling hypersensitivity, bistability, and hysteresis in chronic lymphocytic leukemic B cells. Super-resolution microscopy confirms membrane auto-aggregation in leukemic B cells, and variability in signaling dysfunction predicts disease severity.

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.

The intestinal microbiota is essential for the fermentation of dietary fibers into metabolites, notably the short-chain fatty acids (SCFA) butyrate, acetate and propionate. SCFA can bind to G-protein-coupled receptors such as GPR109a to activate downstream anti-inflammatory effects. GPR109a is the main butyrate receptor and we have previously demonstrated that butyrate can ameliorate gastrointestinal injury during graft versus host disease (GVHD). Therefore, we hypothesized that GPR109a is important in the pathophysiology of intestinal GVHD. Using a MHC-mismatched model of GVHD, we demonstrated a lower incidence of disease when mice received wild type (WT) bone marrow (BM) and T cells from a GPR109a−/− mouse. It is known that GPR109a is highly expressed on myeloid cell populations such as dendritic cells and macrophages; however, expression on T cells is not entirely resolved. At baseline, we observed low expression of GPR109a on T cells, which increases upon T-cell receptor stimulation. T cell development in GPR109a−/− mice appears normal, however, 7 days after transplant, we found significantly fewer CD4+ and CD8+ T cells in the intestines of mice that received WT BM and GPR109a−/− T cells. While frequency of Th1 and Th17 cells were similar between the groups, we saw significantly more regulatory T cells in the intestines and spleen of GPR109a−/−T cell recipients. This reduction of GVHD was accompanied by maintenance of the gut commensal Clostridium and increased butyrate, which is known to protect the intestinal epithelium and increases the regulatory T cell pool. Thus, we propose a novel role for GPR109a in the expansion and activation of T cells during GVHD.

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, patients may develop toxicity due to cytokine release syndrome (CRS) or neurotoxicity. We hypothesized that the composition of the intestinal microbiota prior to CAR T cell infusion is correlated with efficacy and toxicity. We collected fecal samples from recipients of CAR T cells at Memorial Sloan Kettering Cancer Center (MSKCC) pre-CAR T cell infusion. Microbiota composition was profiled by 16S sequencing. Shotgun metagenomic sequencing was performed on a subset of the samples. Clinical response to assess efficacy was classified as complete response (CR) or no CR. Toxicity was defined as CRS or neurotoxicity of Grade 1 to 4. Linear discriminant analysis effect size (LEfSe) identified differentially abundant bacteria between groups with a linear discriminant analysis (LDA) score threshold >2.5. We analyzed baseline samples from 25 patients treated at MSKCC. The patients were adult recipients of CAR T cells who varied in regard to conditioning regimen, CAR construct and underlying diagnosis, which included hematologic and solid malignancies. LEfSe revealed increased representation of bacterial taxa in the microbiome of CR versus no CR, with Oscilliospiraceae, Ruminococcacaeae and Lachnospiraceae enriched in CR and Peptostreptococcaceae more abundant in no CR (A). A higher abundance of Lachnospiraceae was found in those who experienced toxicity, while Peptostreptococcaceae was more abundant in patients who did not have toxicity (B). We explored shotgun metagenomic sequences from 19 of 25 samples that were functionally annotated using the shortBRED pipeline. We inspected abundances of genes assigned to three pathways that we hypothesized may be immunologically relevant: B vitamin synthesis, bile acid biosynthesis, and short-chain fatty acid production. We observed increased abundance of genes associated with B vitamin biosynthesis in patients who had no CR (C) or toxicity (D). We observe differential abundance of microbiota in patients who achieved a CR or experienced toxicity as compared to those who did not achieve a CR or experience toxicity. We observe that increased B vitamin gene abundance is associated with no CR or toxicity. Overall, this data indicates that features of the microbiota may correlate with outcomes to CAR T cell therapy.

Intestinal microbiota composition is associated with important allo-HCT outcomes including relapse, GVHD, immune reconstitution, and infections. Intestinal diversity assessed peri-neutrophil-engraftment is predictive of TRM and overall survival. These observations were made in single-center studies of post-HCT stool samples. We hypothesized that the pre-HCT microbiota is also a determinant of post-HCT outcomes. 1,922 stool samples were collected ∼weekly from 991 allo-HCT pts at 4 centers (Cohorts 1 and 2 in the US; cohort 3 in Europe; cohort 4 in Japan). The patients varied in diagnosis, graft sources, and conditioning intensity. All samples were 16S sequenced and analyzed at a central lab. Pre-HCT a-diversity (inverse Simpson) values in 4 cohorts were 1.7-to-2.5-fold lower than those of healthy volunteers (A, p We next asked how similar these pre-HCT communities are across geography. Bray-Curtis distances between cohorts were much smaller than the changes observed over time during transplantation (C, p To better characterize these low-diversity phenotypes, we defined domination as a microbiota injury in which any taxon comprised >30% of bacterial abundance. The dominating taxa belonged to multiple genera, most commonly Enterococcus (E). In all 4 cohorts, the cumulative incidence of monodomination was >50% by d+0 and >87% by d+28 (Fig F). In the largest cohort, low-diversity states were associated with exposure to broad-spectrum antibiotics, conditioning intensity, and low calorie intake. In conclusion, we demonstrate that allo-HCT pts from 4 institutions on 3 continents presented with pre-HCT 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 that 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.

Mucositis is one of the most significant toxicities in cancer patients undergoing cytotoxic treatment. It can have a negative impact on both quality of life and health economics. Severe oral mucositis can contribute to hospitalization, need for narcotic analgesics, total parentral nutrition, suboptimal delivery of anti-neoplastic treatment, and morbidity and mortality. Palifermin, a recombinant derivative of human keratinocyte growth factor, is the first active agent approved by the FDA for the prevention of severe oral mucositis in patients undergoing haematopoietic stem cell transplantation (HSCT). Several studies have also shown significant reduction in the incidence, severity and/or duration of oral mucositis in other high-risk settings such as concurrent chemoradiotherapy (CT/RT) for patients with head and neck cancer, and use of mucotoxic chemotherapeutic agents such as doxorubicin in sarcoma and fluorouracil for the treatment of colorectal cancer. The reduction in mucositis has translated into amelioration of symptoms and improvement in daily functioning as measured by patient-reported outcome in multiple studies. The clinical response to palifermin appears to be related in part to epithelial proliferation and mucosal thickening. Palifermin also has other potential clinical applications including the acceleration of immune reconstitution and inhibition of graft-versus-host disease in patients undergoing HSCT, and mitigation of dysphagia in lung cancer patients treated with concurrent CT/RT. Palifermin is generally well tolerated with mild-to-moderate skin and oral adverse events. Future studies may expand the use of palifermin into other areas that would benefit from its cytoprotective and regenerative effects.

Despite its critical role in supplying naïve T cells to the circulation, the thymus is particularly sensitive to immune injury, such as that caused by cytoreductive chemo- or radiation therapy, shock, infection and graft versus host disease (GVHD). Crucially, insufficient thymic recovery has been directly correlated with increased risk of opportunistic infections and poor clinical outcomes in recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Prolonged immune deficiency is particularly pronounced in older patients whose thymi are already significantly impaired due to age-related thymic involution. Previous preclinical studies have revealed several strategies that can enhance thymic function and immune reconstitution after transplant, including sex steroid ablation (SSA), growth factors (growth hormone, GH, keratinocyte growth factor, KGF, insulin-like growth factor 1, IGF-1, interleukin-7, IL-7) and ex vivo generated precursor T cells (preT). In addition, recent studies have shown that other approaches, such as interleukein-22 (IL-22) and nutritional changes, may represent additional candidates to enhance thymic regeneration. In this review we provide updates on these strategies and comment on their potential to be translated into clinical therapies.

Age-associated changes of T and NK cell (T/NK) potential of human hematopoietic stem cells are unknown. In this study, we enumerate and characterize T/NK precursors among CD34+Lin− cell populations circulating in normal human adult peripheral blood (PB) by a limiting-dilution assay using coculture with OP9-DL1 stroma cells expressing Notch 1 ligand, Delta–like 1. The frequency of T cell precursors in CD34+Lin− cells was found to decrease with donor age, whereas the ratio of NK to T cell precursor frequency (NK/T ratio) increased with age, suggesting that lymphoid differentiation potential of PB progenitors shifts from T to NK cell lineage with aging. Clonal analyses of CD34+Lin− cells showed that differences in the NK/T ratio were attributable to different distributions of single- and dual-lineage T/NK precursor clones. Because nearly all of the clones retained monocyte and/or granulocyte differentiation potentials in coculture with OP9-DL1 cells, T/NK precursors in PB are considered to be contained in the pool of T/NK/myeloid multipotent progenitors. The age-associated increase in NK over T cell commitment might occur in precursor cells with T/NK/myeloid potential.

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

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

Coordinating the balance between haematopoietic stem cell (HSC) quiescence and self-renewal is crucial for maintaining haematopoiesis lifelong. Equally important for haematopoietic function is modulating HSC localization within the bone marrow niches, as maintenance of HSC function is tightly controlled by a complex network of intrinsic molecular mechanisms and extrinsic signalling interactions with their surrounding microenvironment. In this study we demonstrate that nuclear factor erythroid 2-related factor 2 (Nfe2l2, or Nrf2), well established as a global regulator of the oxidative stress response, plays a regulatory role in several aspects of HSC homeostasis. Nrf2 deficiency results in an expansion of the haematopoietic stem and progenitor cell compartment due to cell-intrinsic hyperproliferation, which was accomplished at the expense of HSC quiescence and self-renewal. We further show that Nrf2 modulates both migration and retention of HSCs in their niche. Moreover, we identify a previously unrecognized link between Nrf2 and CXCR4, contributing, at least partially, to the maintenance of HSC function.

Consolidation with allogeneic hematopoietic stem cell transplantation (allo-HSCT) provides a survival benefit to patients with acute lymphoblastic leukemia (ALL). We have previously reported comparable survival and relapse rates after T cell–depleted (TCD) allo-HSCT compared with unmodified transplantations for acute myelogenous leukemia, myelodysplastic syndrome, and non-Hodgkin lymphoma with significantly decreased graft-versus-host disease (GVHD). We performed a 56-patient retrospective study to evaluate TCD allo-HSCT for the treatment of ALL after myeloablative total body irradiation–based therapy. The 2-year and 5-year overall survival rates for patients with ALL after TCD allo-HSCT were 0.39 (95% confidence interval [CI], 0.26-0.52) and 0.32 (95% CI, 0.19-0.44), respectively, and the 2-year and 5-year disease-free survival rates were 0.38 (95% CI, 0.25-0.50) and 0.32 (95% CI, 0.20-0.44). There was a trend toward improved survival of patients who underwent TCD allo-HSCT in first complete remission compared with those who did so in other remission states. The cumulative incidence of grade II-IV acute GVHD at 1 year was 0.20 (95% CI, 0.10-0.31), and no patients developed grade IV acute GVHD. The cumulative incidence of chronic GVHD in 41 evaluable patients at 2 and 5 years was 0.15 (95% CI, 0.04-0.26), and that of extensive chronic GVHD at 2 and 5 years was 0.05 (95% CI, 0-11.6). We demonstrate OS and DFS rates that compare favorably to unmodified allo-HSCT with lower rates of GVHD.

INTRODUCTION Slow and incomplete reconstitution of cellular and humoral immunity has long been recognized as a weak link limiting the success of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Delayed posttransplantation reconstitution of the adaptive immune system is associated with increased infection, relapse, secondary malignancies, and overall mortality in recipients of an allo-HSCT [1,2]. In particular, reconstitution of the T cell compartment can take 1-2 years, and deficiencies in the T cell repertoire can persist even longer. Deficiencies in T cell reconstitution are dependent on patient age, conditioning regimen, use of in vitro or in vivo T cell depletion to decrease graft-versus-host disease (GVHD), HLA disparity between donor and recipient, and GVHD prophylaxis, as well as the occurrence of GVHD. T cell recovery after allo-HSCT occurs through 2 unrelated mechanisms: (1) peripheral expansion of infused donor T cells and, to a lesser extent, residual host T cells, and (2) de novo T cell development in the thymus. Peripheral expansion is associated with a narrow repertoire, whereas de novo T cell development can reconstitute a full T cell repertoire. However, damage from conditioning and GVHD, in combination with age-associated involution, results in poor posttransplantation thymic function. Consequently, this review takes a “bench-to-bedside” approach, evaluating strategies aimed at enhancing not only the peripheral expansion of the tumor and virus-specific immune response, but also the recovery of thymic function (Figure 1).

In allogeneic stem cell transplantation (ASCT), systemic broad-spectrum antibiotics are frequently used for treatment of infectious complications, but their effect on microbiota composition is still poorly understood. This retrospective analysis of 621 patients who underwent ASCT at the University Medical Center of Regensburg and Memorial Sloan Kettering Cancer Center in New York assessed the impact of timing of peritransplant antibiotic treatment on intestinal microbiota composition as well as transplant-related mortality (TRM) and overall survival. Early exposure to antibiotics was associated with lower urinary 3-indoxyl sulfate levels (P .001) and a decrease in fecal abundance of commensal Clostridiales (P = .03) compared with late antibiotic treatment, which was particularly significant (P = .005) for Clostridium cluster XIVa in the Regensburg group. Earlier antibiotic treatment before ASCT was further associated with a higher TRM (34%, 79/236) compared with post-ASCT (21%, 62/297, P = .001) or no antibiotics (7%, 6/88, P .001). Timing of antibiotic treatment was the dominant independent risk factor for TRM (HR, 2.0; P ≤ .001) in multivariate analysis besides increase age (HR, 2.15; P = .004), reduced Karnofsky performance status (HR, 1.47; P = .03), and female donor-male recipient sex combination (HR, 1.56; P = .02) A competing risk analysis revealed the independent effect of early initiation of antibiotics on graft-versus-host disease-related TRM (P = .004) in contrast to infection-related TRM and relapse (not significant). The poor outcome associated with early administration of antibiotic therapy that is active against commensal organisms, and specifically the possibly protective Clostridiales, calls for the use of Clostridiales-sparing antibiotics and rapid restoration of microbiota diversity after cessation of antibiotic treatment.