Graft-versus-host Disease Patients Research Articles

Unlocking protein-based biomarker potential for graft-versus-host disease following allogenic hematopoietic stem cell transplants.

Despite the numerous advantages of allogeneic hematopoietic stem cell transplants (allo-HSCT), there exists a notable association with risks, particularly during the preconditioning period and predominantly post-intervention, exemplified by the occurrence of graft-versus-host disease (GVHD). Risk stratification prior to symptom manifestation, along with precise diagnosis and prognosis, relies heavily on clinical features. A critical imperative is the development of tools capable of early identification and effective management of patients undergoing allo-HSCT. A promising avenue in this pursuit is the utilization of proteomics-based biomarkers obtained from non-invasive biospecimens. This review comprehensively outlines the application of proteomics and proteomics-based biomarkers in GVHD patients. It delves into both single protein markers and protein panels, offering insights into their relevance in acute and chronic GVHD. Furthermore, the review provides a detailed examination of the site-specific involvement of GVHD. In summary, this article explores the potential of proteomics as a tool for timely and accurate intervention in the context of GVHD following allo-HSCT.

Gut microbiota, microbiota-derived metabolites, and graft-versus-host disease.

Allogeneic hematopoietic stem cell transplantation is one of the most effective treatment strategies for leukemia, lymphoma, and other hematologic malignancies. However, graft-versus-host disease (GVHD) can significantly reduce the survival rate and quality of life of patients after transplantation, and is therefore the greatest obstacle to transplantation. The recent development of new technologies, including high-throughput sequencing, metabolomics, and others, has facilitated great progress in understanding the complex interactions between gut microbiota, microbiota-derived metabolites, and the host. Of these interactions, the relationship between gut microbiota, microbial-associated metabolites, and GVHD has been most intensively researched. Studies have shown that GVHD patients often suffer from gut microbiota dysbiosis, which mainly manifests as decreased microbial diversity and changes in microbial composition and microbiota-derived metabolites, both of which are significant predictors of poor prognosis in GVHD patients. Therefore, the purpose of this review is to summarize what is known regarding changes in gut microbiota and microbiota-derived metabolites in GVHD, their relationship to GVHD prognosis, and corresponding clinical strategies designed to prevent microbial dysregulation and facilitate treatment of GVHD.

Monocyte Subpopulations and Their Expression of HLA-DR and CD300e after Allogeneic Stem Cell Transplantation in Patients with or without GvHD

Introduction: Graft versus host disease (GvHD) is a complex immune process that can affect up to 50% of patients undergoing allogeneic stem cell transplantation (allo-SCT). Even though prophylactic immunosuppressive regimens have been established over the last decades, GvHD is still one of the main causes of morbidity and mortality among these patients. Monocytes are cells of the innate immune system, that represent, as professional antigen-presenting cells, a connection to the adaptive immune system, and play a role in several autoimmune disorders. They are classified according to the expression of CD14 and CD16 into classical (CD14++/CD16-), intermediate (CD14++/CD16+), and non-classical (CD14+/CD16++) monocytes. Previous research has shown higher levels of intermediate monocytes in pediatric patients with GvHD. Furthermore, non-classical monocytes in allogenic bone marrow grafts have also been correlated with the development of GvHD. The expression of other surface markers such as HLA-DR and CD300e (IREM-2) are related to monocyte function and engagement in inflammatory processes. Nevertheless, the role of CD300e in GvHD remains unclear. Here, we present a description of monocyte subgroups and their expression of HLA-DR and CD300e in allo-SCT patients with or without GvHD as well as in healthy subjects. Methods: Transversal observational study with patients undergoing allo-SCT. Peripheral blood of patients was collected once after engraftment. Both acute and chronic GvHD patients were enrolled. Five healthy volunteers served as controls. Flow cytometry was performed to determine the immunophenotype of monocytes according to their expression of CD14 and CD16 stratifying into classical, intermediate, and non-classical subgroups as described above. In addition, the expression of HLA-DR and CD300e was analyzed for all monocytes and individual subgroups. Clinical characteristics are described in the table below. Statistical analysis was performed using GraphPad Prism 9. Comparisons between groups were made using non-parametrical tests and tests for multiple comparisons (Mann-Whitney and Kruskal-Wallis respectively). Results: 29 patients were enrolled. Two patients with GvHD were excluded due to neutropenia. 19 patients presented GvHD grade II or higher. Of those, ten had acute GvHD, five had prolonged acute GvHD, two had late onset acute GvHD, and two had chronic GvHD. Eight patients did not show GvHD or had only mild skin disease (grade one). The mean age was 60 years for all patients, 62.5 and 58.5 years among GvHD and non-GvDH patients, respectively. The image summarizes patient characteristics and comparisons. We compared transplant recipients with and without GvHD which each other and with healthy controls. In single comparisons, patients with GvHD showed significantly lower percentages of total monocytes as well as CD14+/HLA-DR+, total HLA-DR+, and intermediate monocytes compared to patients without GvHD. In addition, we observed a trend toward lower levels of CD16+/HLA-DR+ and CD300e-expressing monocytes in GvHD patients. When compared to healthy controls, allogeneic recipients without GvHD showed significantly higher levels of total monocytes, intermediate monocytes, and CD16+/HLA-DR+ monocytes and lower levels of classical and non-classical monocytes. Compared to controls, patients with GvHD showed significantly lower levels of total monocytes expressing CD300e (also true in all subgroups) as well as lower levels of classical monocytes, but higher levels of intermediate and non-classical monocytes. P values are shown in Table 1. Means and ranges for all groups are represented in Image 1. Conclusions: In our cohort, patients with GvHD showed higher levels of intermediate and non-classical monocytes compared to controls, however, these populations were lower when compared to allogeneic recipients without GvHD. This could indicate a consumption process similar to that observed in COVID-19 patients and a direct involvement of these monocytes in the GvHD process. In addition, we report for the first time the involvement of CD300e in GvHD, which was significantly lower expressed in the GvHD patients, compared to controls. However, there was only a strong trend compared to transplanted non-GvHD patients. This indicates a possible contribution of CD300e in the pathogenesis of GvHD. Further studies are needed in order to confirm these findings.

Analysis of Hospitalization Outcomes in Bone Marrow Transplant Patients with Graft-Versus-Host Disease: A Nationwide Study

Background: Graft-versus-host disease (GVHD) is a well-known complication in patients with bone marrow transplantation. There is a need for additional data on the in-patient outcomes of GVHD in patients who have undergone bone marrow transplantation. Our analysis aims to evaluate the outcomes of GVHD in hospitalized patients with bone marrow transplantation. Method: In this retrospective study, we utilized data from the National Inpatient Sample (NIS) database from 2016 to 2019. Using ICD-10 codes, we identified hospitalizations involving bone marrow transplantation and categorized them into two groups: those with GVHD and those without GVHD. To account for potential confounding variables, we considered a comprehensive set of factors, including age, sex, race, zip code-based income quartile, hospital region, hospital teaching status, hospital division, hospital bed size, insurance status, and the Charlson comorbidity index score. We performed univariable logistic regression analysis to calculate unadjusted odds ratios. Subsequently, we conducted a multivariable logistic regression analysis to account for potential confounding effects and calculated adjusted odds ratios.(see Figure), where the model included only variables that were found to be associated with the outcome of interest in the univariable regression analysis at a significance level of P < 0.2. Proportions were compared using the Fisher exact test for categorical variables, and continuous variables were compared using the Student t-test. All P-values were two-sided, and the significance level was set at P < 0.05, indicating statistical significance. Results: From 2016 to 2019, data were collected from 13,999 hospitalizations involving bone marrow transplants. Among these cases, 836 hospitalizations were linked to GVHD patients. A comparison of patient characteristics showed that individuals with GVHD had a mean age of 51.61 years, while those without GVHD had a slightly higher mean age of 55.55 years. There were slight variations in gender and race distributions between the two groups, with a higher percentage of males and whites observed in the GVHD group compared to the group without GVHD. The analysis of outcomes between the two groups revealed significant differences. Patients with GVHD experienced notably longer mean lengths of stay (41.40 days vs. 21.31 days, P = 0.000) and incurred higher mean total hospital charges ($824,058 vs. $335,765, P = 0.000). Furthermore, after adjusting for confounding factors, GVHD posed a substantial risk to patients undergoing bone marrow transplantation. The adjusted odds ratio (aOR) for mortality in those with GVHD was 7.20 (95% CI: 5.54 to 9.36, P = 0.000) compared to those without GVHD, indicating a considerably higher risk of death. The coefficient for the length of stay was 19.36 days (95% CI: 17.29 to 21.42), and the coefficient for total hospital charges was $453,733 (95% CI: $396,577 to $510,889) in patients with GVHD compared to those without GVHD hospitalizations. Moreover, the presence of GVHD in patients was associated with elevated risks of various medical conditions. The adjusted odds ratios (aORs) for sepsis, pneumonia, acute respiratory failure, intubation and mechanical ventilation, Clostridium difficile infection, and acute kidney injury (AKI) in patients with GVHD were 2.79 (95% CI: 2.28 to 3.41, P = 0.000), 3.30 (95% CI: 2.57 to 4.24, P = 0.000), 5.10 (95% CI: 4.01 to 6.49, P = 0.000), 4.88 (95% CI: 3.75 to 6.34, P = 0.000), 1.45 (95% CI: 1.13 to 1.86, P = 0.003), and 3.57 (95% CI: 2.97 to 4.29, P = 0.000), respectively (see Figure). Conclusion: Patients with GVHD in patients with bone marrow transplantation experience higher mortality rates, extended hospital stays, and increased hospital charges. Additionally, they are at a significantly higher risk of developing severe medical conditions, including sepsis, pneumonia, acute respiratory failure, Clostridium difficile infection, and acute kidney injury (AKI). These findings highlight the importance of closely monitoring and effectively managing GVHD to enhance patient outcomes and alleviate the burden of complications associated with bone marrow transplantation. Nevertheless, further prospective studies are necessary to gain a deeper understanding and more comprehensive evaluation of the outcomes in these hospitalized patients.

Soluble Urokinase-Type Plasminogen Activator Receptor (suPAR) and Growth Differentiation Factor-15 (GDF-15) Leves Are Significantly Associated with Edothelial Injury Indices in Adult Allogenic Hematopoietic Cell Transplantation Recipients

Introduction: Endothelial dysfunction is a common denominator of graft-versus-host disease (GVHD) and transplant-associated thrombotic microangiopathy (TA-TMA). Among endothelial injury indices, the Endothelial Activation and Stress Index (EASIX) has been studied in allogeneic hematopoietic cell transplantation recipients (allo-HCT) recipients, while others including suPAR and GDF-15 have been determined only in other patient populations with hematologic diseases, such as in patients with Multiple Myeloma, AL- amyloidosis and Sickle Cell Disease. We hypothesized that suPAR and GDF-15 would reflect endothelial injury in allo-HCT recipients. Methods: We enrolled consecutive adult TA-TMA (classified according to current standardized criteria), acute and/or chronic graft-versus-host-disease (GVHD), control allogeneic hematopoietic cell transplantation (alloHCT) recipients and apparently healthy individuals of similar age and gender in a 1:1:1:1 ratio. Plasma was collected and stored immediately at -80 oC at the first day of confirmed TA-TMA or GVHD diagnosis and at a similar post-transplant period in control recipients. EASIX [lactate dehydrogenase (U/L) × creatinine (mg/dL)/thrombocytes (10⁹ cells per L)] was calculated at day 0, 30, 100 and at last follow-up. Soluble C5b-9/membrane attack complex, suPAR and GDF-15 levels were measured using immunoenzymatic techniques. Results: We studied 20 TA-TMA, 20 GVHD, 20 control alloHCT patients, and 20 healthy controls. We found significantly higher suPAR and GDF-15 levels in TA-TMA and GVHD patients compared to alloHCT and healthy controls (p<0.001, Bonferroni's correction). Then, we further analyzed characteristics of the alloHCT population. TA-TMA developed at a median of 125 post-transplant day (range 9-2931); whereas the first day of confirmed GVHD diagnosis was at a median of 78 post-transplant day (range 16-145). Both GDF-15 and suPAR concentrations were associated with EASIX at day 100 (r=0.351, p=0.012 and r=0.338, p=0.015, respectively) and last follow-up (r=0.473, p<0.001 and r=0.410, p=0.020, respectively). Among the laboratory values used to calculate EASIX (LDH, creatinine, platelets), suPAR was associated with creatinine and platelets at day 100 and last follow-up; while GDF-15 only with platelets at both time points, suggesting that the association with EASIX is not driven by laboratory values per se. Interestingly, only suPAR and not GDF-15 levels was associated with soluble C5b-9 levels (p=0.013), a marker reflecting high risk in TA-TMA. Conclusion: Our study shows for the first time that suPAR and GDF-15 reflect endothelial injury in allo-HCT recipients. In accordance with other patient populations, suPAR emerges as a marker of renal dysfunction, characterizing high-risk in endothelial injury syndromes and in particular, TA-TMA. However, prior to their clinical usefulness, these biomarkers must undergo through rigorous validation in multiple cohorts.

Inflammatory Change of FoxP3 + Regulatory T Cells in Human Acute Graft-Versus-Host Disease: Implication in Immunopathogenesis

FoxP3 + regulatory T (Treg) cells are a subset of CD4 + T cells that are requisite for control of immune responses in various settings, thereby preventing excessive inflammation and maintaining homeostasis of the immune system. Paradoxically, in several autoimmune disorders, it has been suggested that Treg cells undergo inflammatory conversion to produce effector cytokines promoting tissue inflammation. Although Treg cells are known to play an important role in the pathogenesis of acute graft-versus-host disease (GvHD) following allogeneic stem cell transplantation (SCT), such inflammatory changes have not been focused on so far especially in the setting of human acute GvHD. Herein, in a prospective cohort of patients with hematologic malignancies who were treated with allogeneic SCT, we investigated the quantitative and qualitative changes of Treg cells in subjects with acute GvHD, including production of inflammatory cytokines from Treg cells. We also studied a cellular mechanism for production of effector cytokines by Treg cells and clinical significance of inflammatory changes of Treg cells. We first determined the frequency of Treg cells in the peripheral blood of patients with acute GvHD and compared it with that of patients who did not develop acute GvHD. Percentage of CD25 hiCD127 loFoxP3 + Treg cells among CD4 + T cells was significantly lower in patients with acute GvHD. When we analyzed for the composition of Treg cell subsets in more detail, we observed that the proportion of CD45RA -FoxP3 hi (actively suppressive) Treg cells was decreased and the proportion of CD45RA -FoxP3 lo (cytokine-producing non-suppressive) Treg cells was increased in patients with acute GvHD. Next, we assessed the production of inflammatory cytokines from Treg cells following T cell receptor (TCR) stimulation of peripheral blood lymphocytes with anti-CD3/CD28 antibody, using intracellular cytokine staining. As a result, we found that a remarkable proportion of CD4 +CD25 hiFoxP3 + Treg cells of acute GvHD patients produced TNF-α upon TCR stimulation. Interestingly, a significant proportion of TNF-α-producing Treg cells also secreted IL-17A simultaneously. These findings are consistent with the increased CD45RA -FoxP3 lo fraction among the Treg cell subsets. To explore a potential mechanism underlying inflammatory change of Treg cells, we quantified a variety of soluble factors in systemic circulation before and after onset of acute GvHD. The concentration of IL-6 and IL-21 was significantly higher in the serum drawn after onset of acute GvHD compared to baseline, suggesting that these Th17-polarizing cytokines might be responsible for the inflammatory conversion of Treg cells during acute GvHD. We further checked the suppressive activity of TNF-α-producing CD4 +CD25 hiFoxP3 + Treg cells of acute GvHD patients. TNF-α + Treg cells of acute GvHD patients expressed lower level of FoxP3 than the TNF-α - counterpart. Similarly, the percentage of CD39 + cells was lower in TNF-α + Treg cells than in the TNF-α - counterpart. In fact, Treg cell suppressive activity during acute GvHD was significantly reduced compared with that of pre-SCT control when non-Treg CD4 + T cells or CD8 + T cells were used as responder cells. Finally, we examined the clinical significance of TNF-α-producing CD4 +CD25 hiFoxP3 + Treg cells in acute GvHD patients. The frequency of TNF-α + cells among circulating Treg cells significantly correlated with the severity of acute GvHD, with higher proportion of TNF-α + Treg cells in patients with the higher grade of acute GvHD. This result indicates that the pathologic conversion of Treg cells to produce TNF-α contributes to immune-mediated tissue injury during acute GvHD. Taken together, during human acute GvHD, Treg cells undergo functional alteration to produce proinflammatory cytokines including TNF-α and IL-17A and to exhibit the reduced suppressive activity. TNF-α production by Treg cells is closely associated with the clinical severity of acute GvHD.

Graft-Versus-Host Disease (GvHD) and Steroid-Refractory GvHD after Allogeneic Hematopoietic Stem Cell Transplantation: A Large Real-World EBMT-Based Epidemiology and Treatment Pattern Study

Introduction: Graft-versus-host disease (GvHD) is a multi-systemic disorder affecting patients who undergo allogenic hematopoietic stem cell transplantation (allo-HSCT) and is associated with considerable morbidity and mortality both in its acute (a) and chronic (c) forms, particularly in patients with steroid-refractory (SR)-GvHD (Flinn AM and Gennery AR. Fac Rev 2023).Given the lack of real-life data on GvHD, this study was conducted using a large European database with the objective to provide evidence on the epidemiology and treatment patterns in patients with aGvHD, cGvHD, and SR-GvHD. Methods: This retrospective, observational cohort study was conducted between July 1, 2014 and December 31, 2020 in allo-HSCT patients registered to the European Society for Blood and Marrow Transplantation (EBMT) database. Male or female patients aged ≥12 years at index (≥18 years for SR-GvHD and ruxolitinib [RUX] cohorts) who had received ≥1 allo-HSCT during this study period (January 1, 2017 to July 1, 2019 for SR-GvHD and RUX cohorts) were included in the study population. The EBMT cohort included all allo-HSCT patients, irrespective of GvHD development post-transplant (Tx). The primary objective was to assess the cumulative incidence of aGvHD and cGvHD in patients from the EBMT cohort who met the inclusion criteria. Overall survival of patients up to 60 months was also assessed in the EBMT cohort. The SR-GvHD cohort comprised a sub-selection of GvHD patients (from selected EBMT centers) who were diagnosed with SR-aGvHD, SR-cGvHD, or both, some receiving RUX as treatment; from the SR-GvHD cohort, a sub-selection of patients who had been treated with RUX for aGvHD and/or cGvHD, irrespective of treatment line, were included in the RUX cohort. Results:In the EBMT database, 60,140 patients had received an allo-HSCT. Most patients (94.5%) underwent 1 allo-HSCT, the remaining had >1 procedure. The EBMT cohort included a higher proportion of male patients between 50 and 69 years (Table 1). The main indication for allo-HSCT was acute leukemia. At the time of Tx, most patients were in their early disease stage and had unrelated donors(Table 1). The cumulative incidence of aGvHD was 27.9% and 27.6%, 4 months after first and second Tx, respectively. The cumulative incidence of cGvHD was 37.4% and 29.7%, 60 months after first and second Tx, respectively. aGvHD was of grade 2-4 in 27.1% of patients, and the most affected organ was skin (33.6%, grade 2-4: 19.5%) followed by liver (4.0%, grade 2-4: 2.3%) and gut (4.6%, grade 2-4: 2.8%); however, organ involvement was not collected in many patients. The cGvHD was limited (13.6%), extensive (14.7%) or of unknown grade (3.1%). Graft failure occurred in 5.9% of patients. The overall survival of EBMT cohort patients at 60 months was 53.1% with a median follow-up time of 32.9 months. The most frequent cause of death was relapse/progression (30.6%) followed by infection (27.1%) and GvHD (19.1%). The EBMT registry contains more SR-GvHD patients but only for subset of 292 SR-GvHD patients, additional information was obtained, of which 37 (34.6%) patients were treated with RUX and 70 (65.4%) with other second-line (2L) treatment for SR-aGvHD. For SR-cGvHD, 59 (31.9%) received RUX and 126 (68.1%) other 2L treatment. Baseline characteristics were comparable between SR-GvHD and the EBMT cohort (Table 1). Within SR-GvHD treatment groups, overall, the baseline characteristics were comparable across RUX and other 2L, and consistent with the EBMT and SR-GvHD cohorts. Compared with other 2L treatment, a slightly higher proportion of RUX-treated patients were females in SR-aGvHD cohort and males in SR-cGvHD cohort. While the most common first-line treatment for aGvHD in both RUX and the other group was methylprednisolone, for SR-cGvHD, it was prednisone+cyclosporine in RUX and prednisone in the other group. Conclusion:This large retrospective real-world evidence study from the EBMT reports on patients with GvHD after allo-HSCT. The cumulative incidence of aGvHD at 4 months post-Tx and cGvHD at 60 months was similar after the first or the second Tx. Though this study provides valuable insights on the epidemiology and treatment patterns of SR-GvHD patients, interpretation of these data is limited due to the small sample size of RUX and other treatment groups compared with the EBMT cohort.

Patterns of Graft-Versus-Host Disease (GvHD) Prevention and Management in the Eastern Mediterranean (EM) Region: A Worldwide Network for Blood & Marrow Transplantation (WBMT) Survey

Introduction: GvHD is a significant cause of morbidity and mortality in recipients of allogeneic hematopoietic cell transplant (HCT). The prevention, management, and treatment of GvHD vary in different regions worldwide. This WBMT study aims to better understand the current clinical practices of GvHD prevention and management around the globe. This study presents our survey results in the Eastern Mediterranean Region. Methodology: This is a cross-sectional survey-based study involving hematopoietic cell transplant (HCT) centers in the EM region. Participants of the survey were programs' directors or their designees. The survey comprised two parts; the first part aimed to study the practices of GvHD prevention, whereas the second part aimed to study GvHD management and treatment. The survey was distributed and filled out between December 2022 and May 2023. Results: Thirty participants from 28 institutions responded. Participants were from eleven different countries in the EM region. Fourteen participants (50%) filled it for combined pediatrics and adult HCT programs. The remaining participants filled it for programs performing HCT, either only in adult (27%) or pediatrics (23%) patients. The majority of participants indicated that cyclosporine and methotrexate combination is the most used GvHD prophylaxis therapy in both myeloablative and reduced intensity HCT in their centers. Cyclosporine was the predominantly used calcineurin inhibitor (CNI), followed by tacrolimus. All centers using cyclosporine indicated that they monitor its level, with 200-300 mcg/L being the target in 66% of the survey. Cyclosporine was typically given for 2-12 months after HCT, with around 80% of centers stopping it at 3-4 months post-HCT in recipients with malignant indications and around 90% of centers stopping it around 6-12 months post-HCT in recipients with non-malignant indications. When tacrolimus is used, results showed that it is typically used for similar duration to cyclosporine. Twenty-nine participants reported using post-transplant cyclophosphamide (PTCy) in their centers, with seven centers (24%) reporting its use in indications other than haploidentical transplants. Additionally, the results show that PTCy is typically used in combination with other medications, with 53% of participants reporting that it is used along with mycophenolate mofetil and cyclosporine and only one participant reported its use as a single agent. Seven participants (23%) reported that PTCy is typically used in doses less than 50 mg/kg. In patients with established GvHD, steroids were most commonly used alone as a first-line therapy in acute and chronic GvHD. The majority of centers report re-introducing or continuing the immunosuppressive agent used in GvHD prevention, with few centers indicating that they switched to another CNI. 64% and 55% of centers indicated that they have a standard-operating procedure (SOP) for steroid-refractory acute GvHD (aGvHD) and chronic GvHD (cGvHD), respectively. Only two centers reported involving steroid-refractory GvHD patients in clinical trials. Second-line therapy for steroid-refractory aGvHD and cGvHD varies significantly between centers depending on the type of GvHD and organ involved. In steroid-refractory aGvHD, ruxolitinib was the most frequently listed agent to be considered in the second line, regardless of the involved organ. Ruxolitinib was followed by Extracorporeal photopheresis in Skin aGvHD, Budesonide in lower GI tract aGVHD, and Mycophenolate Mofetil in liver aGvHD. In cGvHD, ruxolitinib was also the most frequently listed medication. However, more significant variability between centers was noted in treating organ-specific cGvHD. Conclusion: GvHD prevention and management practices vary between the different EM region centers. Some variations could be related to access to some medications. This might indicate the need for more evidence-based practices in preventing and managing GvHD and possibly adjusting the guidelines according to access and availability of some medications. WBMT is currently working on understanding the global patterns of GvHD practices in other regions and, ultimately, a global comparative study of all data combined.

High Diversity but Monodominance of Multidrug-Resistant Bacteria in Immunocompromised Pediatric Patients with Acute Lymphoblastic Leukemia Developing GVHD Are Not Associated with Changes in Gut Mycobiome.

Graft-versus-host disease (GvHD) is a severe complication after hematopoietic stem cell transplantation (HSCT). Our study focused on identifying multidrug-resistant (MDR) gut bacteria associated with GvHD-prone guts and association with gut microbiota (GM) diversity, bacteriome, and mycobiome composition in post-HSCT patients. We examined 11 pediatric patients with acute lymphoblastic leukemia (ALL), including six with GvHD, within three time points: seven days pre-HSCT, seven days post-, and 28 days post-HSCT. The gut microbiome and its resistome were investigated using metagenomic sequencing, taxonomically classified with Kraken2, and statistically evaluated for significance using appropriate tests. We observed an increase in the abundance of MDR bacteria, mainly Enterococcus faecium strains carrying msr(C), erm(T), aac(6')-li, dfrG, and ant(6)-la genes, in GvHD patients one week post-HSCT. Conversely, non-GvHD patients had more MDR beneficial bacteria pre-HSCT, promoting immunosurveillance, with resistance genes increasing one-month post-HSCT. MDR beneficial bacteria included the anti-inflammatory Bacteroides fragilis, Ruminococcus gnavus, and Turicibacter, while most MDR bacteria represented the dominant species of GM. Changes in the gut mycobiome were not associated with MDR bacterial monodominance or GvHD. Significant α-diversity decline (Shannon index) one week and one month post-HSCT in GvHD patients (p < 0.05) was accompanied by increased Pseudomonadota and decreased Bacteroidota post-HSCT. Our findings suggest that MDR commensal gut bacteria may preserve diversity and enhance immunosurveillance, potentially preventing GvHD in pediatric ALL patients undergoing HSCT. This observation has therapeutic implications.

Quality Control Analysis of Mesenchymal Stem/Stromal Cells During Investigational New Drug Application for GvHD Administration in China.

Graft-versus-host disease (GvHD), including the acute and chronic types (aGvHD, cGvHD), arise as the dominating secondary disease in patients with unsatisfying consequences of allogeneic hematopoietic stem cell transplantation (HSCT). Approximately half of GvHD patients were steroid-resistant, with a two-year overall survival rate lower than 20%. Worse still, there are no standardized criteria for an optimal second-line therapy for steroid-resistant aGVHD patients. Notably, pioneering investigators have highlighted the ameliorative or therapeutic effects of human umbilical cord-derived mesenchymal stem/stromal cells (hUC-MSCs) upon GvHD largely attributed to their unique hematopoietic-supporting and immunomodulatory properties. Of note, quality control (QC) is the prerequisite to assure the safety and quality of hUC-MSCs before investigational new drug (IND) applications and large-scale clinical applications. Herein, we summarize the state-of-the-art updates upon IND-associated QC and clinical trials of hUC-MSCs during allogeneic HSCT in China. Meanwhile, the supervisory policy and medical ethics of current licensed MSC products for GvHD administration and the concomitant opportunities and challenges have also been discussed.

FRI100 Lipoprotein X In Graft Vs. Host Disease Affecting Liver

Abstract Disclosure: D. Vedamurthy: None. C. Vitali: None. M. Cuchel: None. D. Soffer: None. D. Rader: None. Introduction: Lipoprotein-X (LpX) is an abnormal lipoprotein rich in phospholipid and unesterified cholesterol (UC) found in patients with cholestatic liver disease, lecithin-cholesterol acyl transferase (LCAT) deficiency, and after lipid emulsion infusion. LpX has been detected in patients with graft-versus-host disease (GVHD) of the liver. Cholestasis leads to the accumulation of bile salts and UC in the circulation, promoting the formation of LpX. This condition is often not well-recognized, partly because the detection of LpX is technically challenging and requires specialized laboratory assays. Case Description: A 66-year lady with acute myeloblastic leukemia (AML) underwent an allogeneic matched unrelated donor stem cell transplant (SCT) which was complicated by graft-versus-host disease (GVHD) affecting the liver, eyes, lungs, and skin. Past medical history includes hypertension. Family history was significant for coronary artery disease in the mother. She is a reformed smoker. Her post-transplant course was highly complicated with infections, GVHD affecting the liver, eyes, lungs, and skin, and vascular events including myocardial infarction and stroke. Laboratory testing revealed abnormal liver enzymes (ALT=268, AST=173, ALKP =402 U/L), and a markedly abnormal lipid profile (TC=500, Non-HDL-C=358, TG= 345 mg/dL). Her thyroid panel and viscosity were normal, but apolipoprotein B levels were discordantly low (77mg/dL). The ratio of ApoB: Non-HDL cholesterol was abnormally low (0.215). She was started on ezetimibe, evolocumab, and omega-3 fatty acids by her neurovascular team for cardiovascular risk reduction. A physical exam revealed a thin, frail woman with mild oral and nail GVHD changes. The rest of the exam was notable for the abdomen showing chronic patchy hyperpigmentation with sclerodermatous changes anteriorly and posteriorly. She had no significant focal neurologic deficits. Her lipid levels were improved with therapy, but TC, non-HDL-C, UC, and triglycerides levels were still elevated (TC=353, non-HDL=258, UC=163, TG=414 mg/dL), UC: TC ratio was high (0.46). FPLC separation of TRL-depleted plasma revealed the presence of LpX. LpX is not very atherogenic, and we recommended continuing current medical therapy as the serum viscosity was within normal limits. Conclusion: The presence of LpX should be suspected in the setting of GVHD accompanied by markedly elevated TC. LpX may cause hyperviscosity, which can induce end-organ ischemia. While the direct assessment of LpX is challenging and requires specialized techniques, ApoB and non-HDL-C levels are available in most clinical laboratories, and a very low ApoB: Non-HDL-C ratio in GVHD patients may indicate the presence of LpX. The treatment goal is to resolve the underlying causes of cholestasis. If hyperviscosity is present, Lp-X can be acutely treated by plasma exchange or lipoprotein apheresis. Presentation: Friday, June 16, 2023

Novel therapies for graft versus host disease with a focus on cell therapies.

Graft versus host disease (GVHD) can occur at any period post allogeneic hematopoietic stem cell transplantation as a common clinical complication contributing to significant morbidity and mortality. Acute GVHD develops in approximately 30-50% of patients receiving transplants from matched related donors. High doses of steroids are used as first-line treatment, but are unsuccessful in around 40% of patients, resulting in the diagnosis of steroid-refractory acute GVHD. Consensus has yet to develop for the management of steroid-refractory acute GVHD, and prognosis at six months has been estimated at around 50%. Thus, it is critical to find effective treatments that increase survival of steroid-refractory acute GVHD. This article describes the currently known characteristics, pathophysiology, and treatments for GVHD, with a special focus on recent advances in cell therapies. In particular, a novel cell therapy using decidua stromal cells (DSCs) was recently shown to have promising results for acute GVHD, with improved effectiveness over previous treatments including mesenchymal stromal cells. At the Karolinska Institute, severe acute GVHD patients treated with placenta-derived DSCs supplemented with either 5% albumin or 10% AB plasma displayed a one-year survival rate of 76% and 47% respectively. Furthermore, patients with steroid-refractory acute GVHD, displayed survival rates of 73% with albumin and 31% with AB plasma-supplemented DSCs, compared to the 20% survival rate in the mesenchymal stromal cell control group. Adverse events and deaths were found to be attributed only to complications of hematopoietic stem cell transplant and GVHD, not to the study intervention. ASC Therapeutics, Inc, in collaboration with the Karolinska Institute, will soon initiate a phase 2 multicenter, open-label study to further assess the efficacy and safety of intravenous DSC treatment in sixty patients with Grade II-IV steroid-refractory acute GVHD. This novel cell therapy represents a promising treatment to combat the poor prognosis that steroid-refractory acute GVHD patients currently face.

Targeting PRMT1 prevents acute and chronic graft-versus-host disease

Graft-versus-host disease (GVHD) is a common complication after allogeneic hematopoietic stem cell transplantation. Recent studies have reported that protein arginine methyltransferase 1 (PRMT1) is essential for the differentiation and proliferation of T and B cells. Therefore, it is possible that PRMT1 may play a critical role in GVHD. In this study, we observed that PRMT1 expression was upregulated in CD4+ T and B cells from chronic GVHD (cGVHD) patients and mice. However, the prophylactic use of a PRMT1 inhibitor significantly prevented cGVHD in mice by reducing the percentage of T helper (Th)17 cells, germinal center B cells, and plasma cells. The PRMT1 inhibitor also controlled acute GVHD (aGVHD) in mice by decreasing the percentage of Th17 cells. Moreover, inhibiting PRMT1 also weakened Th17 cell differentiation, B cell proliferation, and antibody production in cells from cGVHD patients. Additionally, further studies revealed that PRMT1 regulated B cell proliferation and antibody secretion by methylating isocitrate dehydrogenase 2 (IDH2). We observed asymmetric di-methylation of IDH2 by PRMT1 at arginine 353 promoted IDH2 homodimerization, which enhanced IDH2 activity, further increasing B cell proliferation and antibody production. Collectively, this study provides a rationale for the application of PRMT1 inhibitors in the prevention of aGVHD and cGVHD.

Gut microbiota trajectory in β-thalassemia major children who underwent allogeneic hematopoietic stem cell transplantation.

The gut microbiota of patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) changes, leading to complications such as acute graft-versus-host disease (GVHD). This study aimed to evaluate the human microbiota composition before and after HSCT in β-thalassemia major (β-TM) children. Twenty-two β-TM children who received allo-HSCT between December 2018 and March 2020 were enrolled. They were followed up for more than 100 days after HSCT, and their gut microbiota information and disease data were recorded at five-time points. The dominant bacteria were Bacteroidetes and Firmicutes at the phylum level and Lachnospiraceae at the family level before and after HSCT. In the differential analysis, Ruminococcaceae constantly decreased after HSCT. Besides, Rothia mucilaginosa was the most abundant 2 months after HSCT compared to before it. Additionally, GVHD patients presented decreased levels of Bacteroidetes compared to those without GVHD. Moreover, Blautia levels significantly decreased in critically ill GVHD patients. The gut microbiota of the 22 β-TM children showed a clear trend of destruction and reconstruction within 100 days after HSCT. The extra-oral infections and inflammations of Rothia mucilaginosa, a Gram-positive bacterium of the normal oropharyngeal tract microbiota, might play an important role in the recovery process of HSCT. Finally, decreased Bacteroidetes levels were associated with GVHD onset.

Estimating the Lifetime Medical Cost Burden of an Allogeneic Hematopoietic Cell Transplantation Patient

Allogeneic hematopoietic cell transplantation (allo-HCT) has the potential for curative outcomes for a variety of hematologic malignancies. Current allo-HCT studies often describe the outcomes and costs in the near term; however, research on the lifetime economic burden post-allo-HCT remains limited. This study was conducted to estimate the average total lifetime direct medical costs of an allo-HCT patient and the potential net monetary savings from an alternative treatment associated with improved graft-versus-host disease (GVHD)-free, relapse-free survival (GRFS). A disease-state model was constructed using a short-term decision tree and a long-term semi-Markov partitioned survival model to estimate the average per-patient lifetime cost and expected quality-adjusted life years (QALYs) for an allo-HCT patient from a US healthcare system perspective. Key clinical inputs included overall survival, GRFS, incidence of both acute and chronic GVHD, relapse of the primary disease, and infections. Cost results were reported as ranges based on varying the percentage of chronic GVHD patients that remained on treatment after 2 years (15% or 39%). Over a lifetime, the average per-patient medical cost of allo-HCT was estimated to range from $942,373 to $1,247,917. The majority of the costs were for chronic GVHD treatment (37% to 53%), followed by the allo-HCT procedure (15% to 19%). The expected lifetime QALYs of an allo-HCT patient were estimated as 4.7. Lifetime per-patient treatment costs often exceed $1,000,000 for allo-HCT patients. Innovative research efforts focused on the reduction or elimination of late complications, particularly chronic GVHD, may provide the greatest value to improved patient outcomes.

Effects of Extracorporeal Photopheresis on Quality of Life and the Course of Diseases in Patients With Mycosis Fungoides and Graft-Versus-Host Disease: A Single-Center Analysis.

The aim of the study was to systematically analyze the influence of extracorporeal photopheresis (ECP) on the quality of life (LQ) and the course of the disease in patients with Mycosis Fungoides (MF), as well as with Graft-versus-Host Disease (GvHD). LQ was monitored retrospectively by using the dermatology life quality index (DLQI) and Skindex-29 test before ECP onset and after the last ECP. Disease parameters were assessed by objective criteria i.e. number of associated medical drugs taken, intervals between therapeutic cycles, gradual change of the disease, and eventual side-effects and complications of ECP therapy. Fifty-one patients were treated with ECP during 2008-19; 19 out of 51 died, and follow-up was not completed in 13 patients. Finally, treatment protocols of 671 ECP procedures were evaluated in 19 patients (10 MF; 9 GvHD).MF and GvHD subpopulations did not differ in the individual scores of LQ questions, either before the outsetor after the last ECP. DLQI and Skindex-29 scores were ameliorated by the ECP therapy (p= 0.001 and p< 0.001, respectively) due to improvement of individual scores of feelings, daily/social activities (p< 0.05), and functionality (p≤ 0.05). The median interval between ECP cycles was extended from two to eight weeks (p= 0.001). Needs of GvHD patients for drugs being received for the underlying disease were reduced (p= 0.035). Two of the 10 MF patients worsened from stage IIA to IIIA. Severe or minor side effects leading to a therapy interruption were not recorded. Patients with GvHD experienced a notable decrease in the administration of drugs for their underlying condition, and there were no instances of severe side effects that resulted in the discontinuation of treatment. ECP is safe and effective for the treatment of MF and GvHD.

Differential protein expression in endothelial cells exposed to serum from patients with acute graft-vs-host disease, depending on steroid response.

Graft-versus-host disease (GVHD) is a complication of allogeneic haematopoietic cell transplantation. Endothelial injury is crucial as pathophysiological substrate for GVHD. GVHD first-line treatment is high-dose corticosteroids, although some patients are steroid-refractory. Through the present study, we compared the endothelial proteomic profiles in response to serum from steroid-refractory acute GVHD (SR-aGVHD) and steroid-sensitive acute GVHD (SS-aGVHD) patients. Blood samples from SR-aGVHD (n= 4) and SS-aGVHD (n= 8) patients were collected at aGVHD diagnosis. Endothelial cell cultures were exposed (48 h) to patients' serum. Protein extraction and proteomic analysis were performed. Differences were statistically evaluated by multivariate analysis. Forty-four proteins contributed to separate all samples into the two study groups, among which 15 participated significantly (p< 0.05), 10 exhibiting a fold change >1.2. Differentially expressed proteins were mainly associated with oxidative phosphorylation (Cytochrome C oxidase subunit 6B1, CX6B1), inflammation and angiogenesis (Apolipoprotein D, APOD), cell survival (Rapamycin-insensitive companion of mTOR, RICTR), and oxidative stress (Riboflavin kinase, RIFK). This pilot study used a novel approach to distinguish the aGVHD response to steroid treatment. The proteins differentially expressed could constitute potential biomarkers for steroid-treatment response. These findings signify a step forward to identify the mechanisms of response to steroids, of high clinical relevance considering the SR-aGVHD elevated mortality.

The composition of the intestinal microbiota after allogeneic haematopoietic stem cell transplantation and its association with graft versus host disease as assessed by 16Sribosomal ribonucleic acid.

To observe the evolution of the intestinal microbiota in patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and discuss the relationship between the intestinal microbiota and graft-versus-host disease (GVHD). In this study, 11 patients who underwent allo-HSCT in the Aerospace Central Hospital from January 2021 to October 2021 were selected, along with 11 donors. Fecal specimens were collected 7 times: at admission, after pre-treatment, and every 3 weeks after transplantation from patients and once from donors. The composition of the intestinal microbiota and its association with GVHD after allogeneic hematopoietic stem cell transplantation were analyzed by 16S rRNA sequencing. Of the 11 patients, 5 developed GVHD, and 6 did not. The diversity of the intestinal microbiota among GVHD patients first increased and then decreased after transplantation, while that among non-GVHD patients first increased and then tended to be stable. The diversity of the intestinal microbiota among GVHD patients was lower than that among non-GVHD patients before pre-treatment and after transplantation. The taxa diversity of the intestinal microbiota in the non-GVHD group was better than that in the GVHD group before allo-HSCT, and the difference was statistically significant (P<0.05 for OTUs and CHAO1 index). The taxa abundance of Enterococcaceae 2.16% (2.13%, 2.22%) before allo-HSCT was significantly higher than that in the non-GVHD group 1.33% (0.27%, 1.52%), and the difference was statistically significant (P=0.004). There was no significant difference between the GVHD group and the non-GVHD group in the diversity of the intestinal microbiota of donors (P<0.05). The characteristics of the intestinal microbiota in the final sample of patients in the GVHD group were similar to the preoperative structure of the intestinal microbiota. In conclusion: The decrease in the diversity of the intestinal microbiota after HSCT may be a risk factor for the occurrence of GVHD. The presence of Enterococcaceae in the intestinal microbiota may be associated with an increased risk of developing GVHD. The intestinal microbiota reconstitute to be close to the intestinal microbiota composition of the donors in the non-GVHD group.

Chimeric Antigen Receptor Engineering of Mesenchymal Stromal Cells (CAR-MSC) Enhance Immunosuppression and Outcomes in Graft Versus Host Disease (GvHD) Preclinical Models

Mesenchymal stromal cells (MSCs) have attracted attention as a treatment for graft vs host disease (GvHD) given their immunosuppressive and off-the-shelf properties. Although MSCs are well-tolerated in GvHD patients, therapeutic efficacy is inconsistent due to insufficient immunosuppression at inflamed tissue sites. We therefore proposed engineering MSCs with chimeric antigen receptors (CAR) to enhance immunosuppressive efficacy at sites of inflammation. In GvHD, donor T cells attack host epithelial tissues in the skin, liver, and intestinal tract in part through the interaction of T cell integrins with E-cadherin (Ecad) expressed on epithelia. We thus hypothesized that redirecting MSCs with an Ecad-targeted CAR (EcCAR-MSCs) would lead to enhanced antigen-specific immunosuppression at these sites of inflammation. Therefore, we transduced adipose-derived MSCs with CAR containing anti-Ecad (human/canine cross-reactive) scFv with CD28ζ signaling domain generating EcCAR-MSCs at high efficiency (>90%). CD28 signal was selected for its ability to activate downstream immunosuppressive factors To test their immunosuppressive function, we cocultured EcCAR-MSCs for 24 hours with activated donor T cells with or without CAR antigen-specific soluble Ecad stimulation. Cocultures with antigen-stimulated EcCAR-MSCs led to significant T cell suppression (p=.0023) as compared to unstimulated EcCAR-MSCs and untransduced MSCs (UTD-MSCs). To verify enhanced immunosuppression found in vitro and test therapeutic efficacy in vivo, we tested EcCAR-MSCs in GvHD xenograft models. GvHD was induced in NOD-SCID-γ-/- mice via intravenous injection of human peripheral blood mononuclear cells (25 x 106 cells/mouse). We then treated mice with EcCAR-MSCs (1x106 cells), UTD-MSCs, or vehicle control intraperitoneally. Mice were monitored for weight loss, clinical GvHD symptom score (diarrhea, activity, posture, fur, and skin integrity), human T cell counts in peripheral blood, and survival outcomes. EcCAR-MSCs ameliorated weight loss (Fig.1), clinical GvHD score (3 vs 5.2 score, p=.0014), T cell suppression (60 vs 450cells/uL, p=.0039), and Treg induction (6.7 vs 2.1% in CD4+ cells, p=.0009), leading to improved overall survival (Fig. 2). Mechanisms underlying superior immunosuppression in EcCAR-MSCs upon CAR stimulation were interrogated via RNA sequencing, serum cytokine assays, and immunophenotype analyses. RNA sequencing evaluated gene signatures across Ecad-stimulated and unstimulated EcCAR-MSC and UTD-MSC groups. CAR CD28-specific stimulation was supported by the upregulation of downstream immunosuppressive gene targets such as NFkB (p=.0002) and JUN (p=.0082) found only in Ecad-stimulated EcCAR-MSCs. Furthermore, gene set enrichment analysis supported TNFαactivation via NFκB (p<.0001) and IL-10 anti-inflammatory pathway (p=.036) enrichment in EcCAR-MSCs upon Ecad stimulation. To corroborate transcriptional enrichments, serum cytokines were analyzed. Here, mice treated with stimulated EcCAR-MSCs vs UTD-MSCs revealed elevated anti-inflammatory serum cytokines, such as IL-10 (76 vs 13pg/mL, p<.0001), TNFα (35 vs 10pg/mL, p<.0001), and G-CSF (95 vs 50pg/mL, p=.0064). Immunophenotypic analyses revealed upregulated T cell inhibitory receptor expression on stimulated EcCAR-MSCs vs UTD-MSCs, including programmed death-1 (12 vs 0.7%, p=.0087) and galectin-9 (67 vs 17%, p<.0001). Collectively, our mechanistic studies support enhanced EcCAR-MSC immunosuppression through immunosuppressive gene pathway enrichment, increased anti-inflammatory cytokine secretion, and upregulated inhibitory receptors upon antigen-specific stimulation. Finally, to demonstrate EcCAR-MSC safety, we ruled out unwanted MSC differentiation by flow cytometry (Maintenance of CD105+, CD90+, CD73+, CD34-, and CD14- stemness phenotype) and RNAseq (stem cell pathway enrichment, p<.0001) . Safety of EcCAR-MSCs were also verified in large animal models using healthy canines through intraperitoneal EcCAR-MSC injection (2x106 cells/kg), with no reported weight loss, hematological toxicity, or organ damage observed 28 days following administration. In summary, our findings position CAR-MSCs as a safe and novel therapeutic platform to enhance MSC antigen-specific immunosuppressive functionality for improved treatment outcomes in GvHD and other autoimmune diseases. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Microbial Bile Acid Metabolism Shapes Effector T Cell Responses during Inflammation in Mouse and Human

Microbial transformation of bile acids (BAs) regulates intestinal immune homeostasis but their effect on T cell-driven pathologies is unknown. We previously reported that gut dysbiosis in patients undergoing allogeneic hematopoietic cell transplantation (allo-HCT) is associated with increased graft-versus-host disease (GVHD), in which graft-derived T cells recognize the host as foreign. We hypothesized that the dramatic changes in intestinal microbiome composition during GVHD would lead to changes in the BA pool, which would alter immunoregulatory mechanisms governing T cell responses. Using a preclinical MHC-disparate GVHD model (B6 into BALB/c) and quantifying BAs with liquid chromatography-mass spectrometry, we found that GVHD was associated with a significant decrease in total BA concentration with a preferential loss of microbiota-dependent unconjugated primary BA (PBAs) and secondary BA (SBAs) (p=0.0002 and p<0.001, n=10/group). Metagenomic sequencing revealed changes in microbiota composition, including reduced abundance of Lactobacillus johnsonii and expansion of Enterococcus faecalis in GVHD mice (FDR=0.03, n=5/group) as previously reported. These changes were accompanied by a reduction in the abundance of bile salt hydrolase (BSH) genes (p=0.008) encoded by the gut microbiome (mainly Lactobacillus johnsonii). BSH catalyzes the deconjugation of PBAs, which is the gateway reaction leading to SBAs formation, suggesting that GVHD affects the BA pool by compromising early steps in microbial BA metabolism. To investigate the effects of GVHD on host metabolism, we profiled the liver transcriptome by bulk RNA-sequencing analysis. Pathways involved in BA metabolism were reduced in the livers of GVHD mice, which was associated with reduced levels of transcripts for genes involved in BA synthesis (Abcd3, Hsd17b11, Cyp7b1, Hsd17b6) and secretion (Abcg8, Slc22a18). As BAs regulate their synthesis via farnesoid X receptor (FXR) in a negative feedback loop, these results suggested that FXR activation may be increased because of loss of SBAs. Indeed, SBAs counteracted the activation of FXR by the natural agonist chenodeoxycholic acid, in FXR reporter assay and FXR TR-FRET coactivator assay, suggesting that bacterial transformation of BAs generally decreases FXR activity in vivo. Pharmacological activation of FXR (B6 into BALB/c, treatment with selective FXR agonist GW4064 day -1 to day 7, p<0.0001, n=20/group) showed increased mortality, whereas genetic ablation of FXR in donor T cells decreased GVHD and improved OS in two preclinical models (B6 into BALB/c, p<0.0001, n=30/group, Fig. 1a and B6 into 129, p<0.0001, n=20/group). Mice transplanted with TΔFXRcells had reduced GVHD-related pathology compared to recipients of TWT on day 28 post allo-HCT (p=0.006, n=10/group). Furthermore, we found reduced IFNγ production by CD4+ and CD8+ TΔFXR cells in the lamina propria of the large intestine on day 14 post-transplant (p=0.01 and p=0.02, n=10/group). Finally, we set out to corroborate our preclinical findings in allo-HCT patients. We compared 63 fecal samples from allo-HCT patients with lower gastrointestinal GVHD obtained at peri-GVHD onset (+/- 10 days) with 61 samples from control allo-HCT patients without GVHD, who were matched for age, graft type and HLA compatibility. Samples from controls were selected +/-10 days from pseudo-GVHD day that was assigned based on their paired GVHD patient. Most of the samples were exposed to the SBA ursodeoxycholic acid (UDCA, 93%). Similar to our findings in mice, we found that SBAs (non-UDCA, Fig. 1b) and unconjugated PBAs, as well as BSH abundances were decreased in GVHD patients compared to controls (p=0.007, p=0.008, and p=0.02 respectively). Notably, we found that important immunomodulatory SBAs were reduced in GVHD patients compared to controls, including 3-oxolithocholic acid, isolithocholic acid and ω-muricholic acid (p=0.01, p=0.004, and p<0.001). Lastly, in 747 allo-HCT recipients, we found that UDCA exposure was associated with a lower incidence of Enterococcus sp. domination (p=0.001), lower aGVHD cumulative incidence (p=0.005) and improved OS (p=0.017) as previously reported. Our results in mice and humans suggest that loss of SBA and increased FXR activation may amplify T-cell driven disease such as GVHD and provide a rationale to target the host-microbiota BA network to improve clinical outcomes in these patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal