Interferon regulatory factor 4 plays a pivotal role in the development of aGVHD-associated colitis.

Interferon regulatory factor 4 (IRF4) is a master transcription factor that regulates T helper cell (Th) differentiation. It interacts with the Basic leucine zipper transcription factor, ATF-like (BATF), depletion of which in CD4+ T cells abrogates acute graft-versus-host disease (aGVHD)-induced colitis. Here, we investigated the immune-regulatory role of Irf4 in a mouse model of MHC-mismatched bone marrow transplantation. We found that recipients of allogenic Irf4-/- CD4+ T cells developed less GVHD-related symptoms. Transcriptome analysis of re-isolated donor Irf4-/- CD4+ T helper (Th) cells, revealed gene expression profiles consistent with loss of effector T helper cell signatures and enrichment of a regulatory T cell (Treg) gene expression signature. In line with these findings, we observed a high expression of the transcription factor BTB and CNC homolog 2; (BACH2) in Irf4-/- T cells, which is associated with the formation of Treg cells and suppression of Th subset differentiation. We also found an association between BACH2 expression and Treg differentiation in patients with intestinal GVHD. Finally, our results indicate that IRF4 and BACH2 act as counterparts in Th cell polarization and immune homeostasis during GVHD. In conclusion, targeting the BACH2/IRF4-axis could help to develop novel therapeutic approaches against GVHD.

Bacteria and bacteriophage consortia are associated with protective intestinal metabolites in patients receiving stem cell transplantation.

The microbiome is a predictor of clinical outcome in patients receiving allogeneic hematopoietic stem cell transplantation (allo-SCT). Microbiota-derived metabolites can modulate these outcomes. How bacteria, fungi and viruses contribute to the production of intestinal metabolites is still unclear. We combined amplicon sequencing, viral metagenomics and targeted metabolomics from stool samples of patients receiving allo-SCT (n = 78) and uncovered a microbiome signature of Lachnospiraceae and Oscillospiraceae and their associated bacteriophages, correlating with the production of immunomodulatory metabolites (IMMs). Moreover, we established the IMM risk index (IMM-RI), which was associated with improved survival and reduced relapse. A high abundance of short-chain fatty acid-biosynthesis pathways, specifically butyric acid via butyryl-coenzyme A (CoA):acetate CoA-transferase (BCoAT, which catalyzes EC 2.8.3.8) was detected in IMM-RI low-risk patients, and virome genome assembly identified two bacteriophages encoding BCoAT as an auxiliary metabolic gene. In conclusion, our study identifies a microbiome signature associated with protective IMMs and provides a rationale for considering metabolite-producing consortia and metabolite formulations as microbiome-based therapies.

Multimodal immune cell phenotyping in GI biopsies reveals microbiome-related T cell modulations in human GvHD.

Acute graft-versus-host disease (aGvHD) is a significant complication after allogeneic hematopoietic stem cell transplantation (aHSCT), but major factors determining disease severity are not well defined yet. By combining multiplexed tissue imaging and single-cell RNA sequencing on gastrointestinal biopsies from aHSCT-treated individuals with fecal microbiome analysis, we link high microbiome diversity and the abundance of short-chain fatty acid-producing bacteria to the sustenance of suppressive regulatory T cells (Tregs). Furthermore, aGvHD severity strongly associates with the clonal expansion of mainly CD8 T cells, which we find distributed over anatomically distant regions of the gut, persistent over time, and inversely correlated with the presence of suppressive Tregs. Overall, our study highlights the pathophysiological importance of expanded CD8 T cell clones in the progression of aGvHD toward more severe clinical manifestations and strongly supports the further development of microbiome interventions as GvHD treatment via repopulation of the gut Treg niche to suppress inflammation.

Intestinal IgA-positive plasma cells are highly sensitive indicators of alloreaction early after allogeneic transplantation and associate with both graft-versus-host disease and relapse-related mortality.

Intestinal immunoglobulin A (IgA) is strongly involved in microbiota homeostasis. Since microbiota disruption is a major risk factor of acute graft-versus-host disease (GvHD), we addressed the kinetics of intestinal IgA-positive (IgA+) plasma cells by immunohistology in a series of 430 intestinal biopsies obtained at a median of 1,5 months after allogeneic stem cell transplantation (allo-SCT) from 115 patients (pts) at our center. IgA+ plasma cells were located in the subepithelial lamina propria and suppressed in the presence of histological aGvHD (GvHD Lerner stage 0: 131+/-8 IgA+ plasma cells/mm2; stage 1-2: 108+/-8 IgA+ plasma cells/mm2; stage 3-4: 89+/-16 IgA+ plasma cells/mm2; P=0.004). Overall, pts with IgA+ plasma cells below median had an increased treatment related mortality (P=0.04). Time courses suggested a gradual recovery of IgA+ plasma cells after day 100 in the absence but not in the presence of GvHD. Vice versa IgA+ plasma cells above median early after allo-SCT were predictive of relapse and relapse-related mortality (RRM): pts with low IgA+ cells had a 15% RRM at 2 and at 5 years, while pts with high IgA+ cells had a 31% RRM at 2 years and more than 46% at 5 years; multivariate analysis indicated high IgA+ plasma cells in biopsies (hazard ratio =2.7; 95% confidence interval: 1.04-7.00) as independent predictors of RRM, whereas Lerner stage and disease stage themselves did not affect RRM. In contrast, IgA serum levels at the time of biopsy were not predictive for RRM. In summary, our data indicate that IgA+ cells are highly sensitive indicators of alloreaction early after allo-SCT showing association with TRM but also allowing prediction of relapse independently from the presence of overt GvHD.

Restrictive Versus Permissive Use of Broad-spectrum Antibiotics in Patients Receiving Allogeneic Stem Cell Transplantation and With Early Fever Due to Cytokine Release Syndrome: Evidence for Beneficial Microbiota Protection Without Increase in Infectious Complications.

BACKGROUND: Intestinal microbiome contributes to the pathophysiology of acute gastrointestinal (GI) graft-versus-host disease (GvHD) and loss of microbiome diversity influences the outcome of patients after allogeneic stem cell transplantation (SCT). Systemic broad-spectrum antibiotics have been identified as a major cause of early intestinal dysbiosis. METHODS: In 2017, our transplant unit at the university hospital in Regensburg changed the antibiotic strategy from a permissive way with initiation of antibiotics in all patients with neutropenic fever independent of the underlying cause and risk to a restrictive use in cases with high likelihood of cytokine release syndrome (eg, after anti-thymocyte globulin [ATG] therapy). We analyzed clinical data and microbiome parameters obtained 7 days after allogeneic SCT from 188 patients with ATG therapy transplanted in 2015/2016 (permissive cohort, n = 101) and 2918/2019 (restrictive cohort, n = 87). RESULTS: Restrictive antibiotic treatment postponed the beginning of antibiotic administration from 1.4 ± 7.6 days prior to 1.7 ± 5.5 days after SCT (P = .01) and significantly reduced the duration of antibiotic administration by 5.8 days (P < .001) without increase in infectious complications. Furthermore, we observed beneficial effects of the restrictive strategy compared with the permissive way on microbiome diversity (urinary 3-indoxylsulfate, P = .01; Shannon and Simpson indices, P < .001) and species abundance 7 days post-transplant as well as a positive trend toward a reduced incidence of severe GI GvHD (P = .1). CONCLUSIONS: Our data indicate that microbiota protection can be achieved by a more careful selection of neutropenic patients qualifying for antibiotic treatment during allogeneic SCT without increased risk of infectious complications.

Chronic Active Epstein-Barr Virus (EBV) Infection Controlled by Allogeneic Stem Cell Transplantation and EBV-Specific T Cells.

We report sustained remission of chronic active Epstein-Barr virus (EBV) infection in a 27-year-old female patient treated with third-party EBV-specific T cells followed by allogeneic hematopoietic stem cell transplantation (HSCT). The viremia cleared after administration of anti-T-lymphocyte globulin for graft-versus-host disease (GvHD) prophylaxis. Subsequent expansion of EBV-infected host T cells was controlled by transfusion of donor-derived EBV-specific T cells.

Impact of chronic graft-versus-host-disease on intensive care outcome in allogeneic hematopoietic stem cell recipients.

Chronic graft-vs-host-disease (cGvHD) is the most relevant long-term complication after allogeneic stem cell transplantation (HSCT) with major impact on non-relapse mortality, but data on intensive care unit (ICU) outcome are missing. In this retrospective, multicenter study we analyzed 174 adult HSCT recipients with cGvHD requiring intensive care treatment. Skin, pulmonary, liver, and intestinal involvement were present in 76.7%, 47.1%, 38.1% and 24.1%, respectively, and a total of 63.2% had severe cGvHD. Main reasons for ICU admission were respiratory failure (69.7%) and sepsis (34.3%). Hospital- and 3-year OS rates were 51.7% and 28.6%, respectively. Global severity of cGvHD did not impact short- and long-term survival. However, patients with severe liver cGvHD or the overlap subtype had a reduced hospital survival, while severe pulmonary cGvHD was associated with worse long-term survival. In multivariate analysis need for invasive ventilation (HR 1.08 (95% CI 1.02-1.14)) or hemodialysis (HR 1.73 (95% CI 1.14-2.62)) and <1 year since HSCT (HR 1.56 (95% CI 1.03-2.39)) were independently associated with a poorer survival. While the global severity of cGvHD does not per se affect patients' survival after intensive care treatment, pre-existing severe hepatic, intestinal or pulmonary cGvHD is associated with worse outcomes.

Reg3α concentrations at day of allogeneic stem cell transplantation predict outcome and correlate with early antibiotic use.

Intestinal microbiome diversity plays an important role in the pathophysiology of acute gastrointestinal (GI) graft-versus-host disease (GVHD) and influences the outcome of patients after allogeneic stem cell transplantation (ASCT). We analyzed clinical data and blood samples taken preconditioning and on the day of ASCT from 587 patients from 7 German centers of the Mount Sinai Acute GVHD International Consortium, dividing them into single-center test (n = 371) and multicenter validation (n = 216) cohorts. Regenerating islet-derived 3α (Reg3α) serum concentration of day 0 correlated with clinical data as well as urinary 3-indoxylsulfate (3-IS) and Clostridiales group XIVa, indicators of intestinal microbiome diversity. High Reg3α concentration at day 0 of ASCT was associated with higher 1-year transplant-related mortality (TRM) in both cohorts (P < .001). Cox regression analysis revealed high Reg3α at day 0 as an independent prognostic factor for 1-year TRM. Multivariable analysis showed an independent correlation of high Reg3α concentrations at day 0 with early systemic antibiotic (AB) treatment. Urinary 3-IS (P = .04) and Clostridiales group XIVa (P = .004) were lower in patients with high vs those with low day 0 Reg3α concentrations. In contrast, Reg3α concentrations before conditioning therapy correlated neither with TRM nor disease or treatment-related parameters. Reg3α, a known biomarker of acute GI GVHD correlates with intestinal dysbiosis, induced by early AB treatment in the period of pretransplant conditioning. Serum concentrations of Reg3α measured on the day of graft infusion are predictive of the risk for TRM of ASCT recipients.

Downregulation of the vitamin D receptor expression during acute gastrointestinal graft versus host disease is associated with poor outcome after allogeneic stem cell transplantation.

The vitamin D receptor (VDR) is critical in regulating intestinal homeostasis and emerging evidence demonstrates that VDR deficiency is a critical factor in inflammatory bowel disease pathology. However, no clinical data exist regarding the intestinal expression of VDR in patients after allogeneic haematopoietic stem cell transplantation (HSCT). Analyzing intestinal biopsies from 90 patients undergoing HSCT with mortality follow-up, we demonstrated that patients with severe acute gastrointestinal graft versus host disease (GI-GvHD) showed significant downregulation of VDR gene expression compared to mild or no acute GI-GvHD patients (p = 0.007). Reduced VDR expression was already detectable at acute GI-GvHD onset compared to GvHD-free patients (p = 0.01). These results were confirmed by immunohistochemistry (IHC) where patients with severe acute GI-GvHD showed fewer VDR+ cells (p = 0.03) and a reduced VDR staining score (p = 0.02) as compared to mild or no acute GI-GvHD patients. Accordingly, low VDR gene expression was associated with a higher cumulative incidence of treatment-related mortality (TRM) (p = 1.6x10-6) but not with relapse-related mortality (RRM). A multivariate Cox regression analysis identified low VDR as an independent risk factor for TRM (p = 0.001, hazard ratio 4.14, 95% CI 1.78-9.63). Furthermore, VDR gene expression significantly correlated with anti-microbial peptides (AMPs) gene expression (DEFA5: r = 0.637, p = 7x10-5, DEFA6: r 0 0.546, p = 0.001). In conclusion, our findings suggest an essential role of the VDR in the pathogenesis of gut GvHD and the prognosis of patients undergoing HSCT.

Low Intestinal IL22 Associates With Increased Transplant-Related Mortality After Allogeneic Stem Cell Transplantation.

The role of IL-22 in adult patients undergoing allogeneic stem cell transplantation (SCT) is of major interest since animal studies showed a protective and regenerative effect of IL-22 in graft versus host disease (GvHD). However, no clinical data exist on the tissue expression. Here we demonstrate that patients not suffering from transplant-related mortality (TRM) show significantly upregulated IL22 expression during histological and clinical GI-GvHD (p = 0.048 and p = 0.022, respectively). In contrast, in GvHD patients suffering from TRM, IL22 was significantly lower (p = 0.007). Accordingly, lower IL22 was associated with a higher probability of TRM in survival analysis (p = 0.005). In a multivariable competing risk Cox regression analysis, low IL22 was identified as an independent risk factor for TRM (p = 0.007, hazard ratio 2.72, 95% CI 1.32 to 5.61). The expression of IL22 seemed to be microbiota dependent as broad-spectrum antibiotics significantly diminished IL22 expression (p = 0.019). Furthermore, IL22 expression significantly correlated with G-protein coupled receptor (GPR)43 (r = 0.263, p = 0.015) and GPR41 expression (r = 0.284, p = 0.009). In conclusion, our findings reveal an essential role of IL-22 for the prognosis of patients undergoing allogeneic SCT.

Prolonged Suppression of Butyrate-Producing Bacteria Is Associated With Acute Gastrointestinal Graft-vs-Host Disease and Transplantation-Related Mortality After Allogeneic Stem Cell Transplantation.

BACKGROUND: Butyrogenic bacteria play an important role in gut microbiome homeostasis and intestinal epithelial integrity. Previous studies have demonstrated an association between administration of short-chain fatty acids like butyrate and protection from acute graft-vs-host disease (GvHD) after allogeneic stem cell transplantation (ASCT). METHODS: In the current study, we examined the abundance and butyrogenic capacity of butyrate-producing bacteria in 28 healthy donors and 201 patients after ASCT. We prospectively collected serial stool samples and performed polymerase chain reaction analysis of the butyrate-producing bacterial enzyme butyryl-coenzyme A (CoA):acetate CoA-transferase (BCoAT) in fecal nucleic acid extracts. RESULTS: Our data demonstrate a strong and prolonged suppression of butyrogenic bacteria early in the course of ASCT. In a multivariable analysis, early use of broad-spectrum antibiotics before day 0 (day of transplantation) was identified as an independent factor associated with low BCoAT copy numbers (odds ratio, 0.370 [95% confidence interval, .175-.783]; P = .009). Diminished butyrogens correlated with other biomarkers of microbial diversity, such as low 3-indoxylsulfate levels, reduced abundance of Clostridiales and low inverse Simpson and effective Shannon indices (all P < .001). Low BCoAT copy numbers at GvHD-onset were correlated with GI-GvHD severity (P = .002) and associated with a significantly higher GvHD-associated mortality rate (P = .04). Furthermore, low BCoAT copy numbers at day 30 were associated with a significantly higher transplantation-related mortality rate (P = .02). CONCLUSIONS: Our results are consistent with the hypothesis that alterations in the microbiome play an important role in GvHD pathogenesis and that microbial parameters such as BCoAT might serve as biomarkers to identify patients at high risk of lethal GI-GvHD.

GPR Expression in Intestinal Biopsies From SCT Patients Is Upregulated in GvHD and Is Suppressed by Broad-Spectrum Antibiotics.

Microbiota can exert immunomodulatory effects by short-chain fatty acids (SCFA) in experimental models of graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (allo-SCT). Therefore we aimed to analyze the expression of SCFAs sensing G-protein coupled receptor GPR109A and GPR43 by quantitative PCR in 338 gastrointestinal (GI) biopsies obtained from 199 adult patients undergoing allo-SCT and assessed the interaction of GPR with FOXP3 expression and regulatory T cell infiltrates. GPR expression was strongly upregulated in patients with stage II-IV GvHD (p=0.000 for GPR109A, p=0.01 for GPR43) and at the onset of GvHD (p 0.000 for GPR109A, p=0.006 for GPR43) and correlated strongly with FOXP3 and NLRP3 expression. The use of broad-spectrum antibiotics (Abx) drastically suppressed GPR expression as well as FOXP3 expression in patients' gut biopsies (p=0.000 for GPRs, FOXP3 mRNA and FOXP3+ cellular infiltrates). Logistic regression analysis revealed treatment with Abx as an independent factor associated with GPR and FOXP3 loss. The upregulation of GPRs was evident only in the absence of Abx (p=0.001 for GPR109A, p=0.014 for GPR43) at GvHD onset. Thus, GPR expression seems to be upregulated in the presence of commensal bacteria and associates with infiltration of FOXP3+ T regs, suggesting a protective, regenerative immunomodulatory response. However, Abx, which has been shown to induce dysbiosis, interferes with this protective response.

Evaluation of Elafin as a Prognostic Biomarker in Acute Graft-versus-Host Disease.

Acute graft-versus-host disease (GVHD) is a major cause of mortality in patients undergoing hematopoietic cell transplantation (HCT) for hematologic malignancies. The skin is the most commonly involved organ in GVHD. Elafin, a protease inhibitor overexpressed in inflamed epidermis, was previously identified as a diagnostic biomarker of skin GVHD; however, this finding was restricted to a subset of patients with isolated skin GVHD. The main driver of nonrelapse mortality (NRM) in HCT recipients is gastrointestinal (GI) GVHD. Two biomarkers, Regenerating islet-derived 3a (REG3α) and Suppressor of tumorigenesis 2 (ST2), have been validated as biomarkers of GI GVHD that predict long-term outcomes in patients treated for GVHD. We undertook this study to determine the utility of elafin as a prognostic biomarker in the general population of acute GVHD patients in whom GVHD may develop in multiple organs. We analyzed serum elafin concentrations as a predictive biomarker of acute GVHD outcomes and compared it with ST2 and REG3α in a large group of patients treated at multiple centers. A total of 526 patients from the Mount Sinai Acute GVHD International Consortium (MAGIC) who had received corticosteroid treatment for skin GVHD and who had not been previously studied were analyzed. Serum concentrations of elafin, ST2, and REG3α were measured by ELISA in all patients. The patients were divided at random into equal training and validation sets, and a competing-risk regression model was developed to model 6-month NRM using elafin concentration in the training set. Additional models were developed using concentrations of ST2 and REG3α or the combination of all 3 biomarkers as predictors. Receiver operating characteristic (ROC) curves were constructed using the validation set to evaluate the predictive accuracy of each model and to stratify patients into high- and low-risk biomarker groups. The cumulative incidence of 6-month NRM, overall survival (OS), and 4-week treatment response were compared between the risk groups. Unexpectedly, patients in the low-risk elafin group demonstrated a higher incidence of 6-month NRM, although the difference was not statistically significant (17% versus 11%; P = .19). OS at 6 months (68% versus 68%; P > .99) and 4-week response (78% versus 78%; P = .98) were similar in the low-risk and high-risk elafin groups. The area under the ROC curve (AUC) was 0.55 for elafin and 0.75 for the combination of ST2 and REG3α. The addition of elafin to the other 2 biomarkers did not improve the AUC. Our data indicate that serum elafin concentrations measured at the initiation of systemic treatment for acute GVHD did not predict 6-month NRM, OS, or treatment response in a multicenter population of patients treated systemically for acute GVHD. As seen in previous studies, serum concentrations of the GI GVHD biomarkers ST2 and REG3α were significant predictors of NRM, and the addition of elafin levels did not improve their accuracy. These results underscore the importance of GI disease in driving NRM in patients who develop acute GVHD.