non-aGVHD Group Research Articles

Suppression of P53 Pathway is an Important Factor Inducing Acute Graft-versus-Host Disease Through T Cell Activation Based on Bioinformatics Analysis.

Acute graft-versus-host disease (aGVHD) poses a significant impediment to achieving a more favourable therapeutic outcome in allogeneic hematopoietic stem cell transplantation (allo-HSCT). The tumour suppressor p53 plays a pivotal role in preventing aGVHD development. However, whether P53 pathway which contains p53 family members and other related genes participates in aGVHD development remains an unsolved question. Transcriptomic data was obtained from Gene Expression Omnibus (GEO) database. Gene set enrichment analysis was applied to determine the enrichment degree of signaling pathways. CIBERSORT and ssGSVA were used to evaluate immune cell compositions. Univariate and multivariate logistic regression analysis were performed to examine the independent diagnostic variables. qRT-PCR was utilized to validate the genes expression levels in our cohort. A total number of 102 patients (42 aGVHD patients vs 60 non-aGVHD patients) were obtained after integrating two datasets in GEO database (GSE73809 and GSE4624). P53 pathway was remarkably suppressed in T cells from aGVHD patients and negatively associated with activated T cells as well as T cells activation related signaling pathways, including T-cell receptor (TCR), mTORC1, MYC and E2F target pathways. A risk model for aGVHD built by four genes (DDIT3, FBXW7, TPRKB and TOB1) in P53 pathway, exhibiting high differentiate and predictive value. DDIT3 and FBXW7 mRNA expression levels significantly decreased in peripheral blood mononuclear cells (PBMCs) from aGVHD patients compared with non-aGVHD group in our patient cohort, consisting with bioinformatics analysis. P53 pathway plays a potential role in impeding T cell activation through suppressing its related signaling pathways, thereby preventing aGVHD development. P53 pathway may emerge as a promising therapeutic target in aGVHD treatment.

Effect of CD8+ CD28- T Cells on Acute Graft-Versus-Host Disease after Haploidentical Hematopoietic Stem Cell Transplantation

To investigate the effect of CD8+ CD28- T cells on acute graft-versus-host disease(aGVHD) after haploidentical hematopoietic stem cell transplantation(haplo-HSCT). The relationship between absolute count of CD8+ CD28- T cells and aGVHD in 60 patients with malignant hematological diseases was retrospectively analyzed after haplo-HSCT, and the differences in the incidence rate of chronic graft-versus host disease(cGVHD), infection and prognosis between different CD8+ CD28- T absolute cells count groups were compared. aGVHD occurred in 40 of 60 patients after haplo-HSCT, with an incidence rate of 66.67%. The median occurrence time of aGVHD was 32.5(20-100) days. At 30 days after the transplantation, the absolute count of CD8+ CD28- T cells of aGVHD group was significantly lower than that of non-aGVHD group (P =0.03). Thus the absolute count of CD8+ CD28- T cells at 30 days after transplantation can be used to predict the occurrence of aGVHD to some extent. At 30 days after transplantation, the incidence rate of aGVHD in the low cell count group (CD8+ CD28- T cells absolute count < 0.06/μl) was significantly higher than that in the high cell count group (CD8+ CD28- T cells absolute count ≥0.06/μl,P =0.011). Multivariate Cox regression analysis further confirmed that the absolute count of CD8+ CD28-T cells at 30 days after transplantation was an independent risk factor for aGVHD, and the risk of aGVHD in the low cell count group was 2.222 times higher than that in the high cell count group (P =0.015). The incidence of cGVHD, fungal infection, EBV infection and CMV infection were not significantly different between the two groups with different CD8+ CD28- T cells absolute count. The overall survival, non-recurrent mortality and relapse rates were not significantly different between different CD8+ CD28- T cells absolute count groups. Patients with delayed CD8+ CD28- T cells reconstitution after haplo-HSCT are more likely to develop aGVHD, and the absolute count of CD8+ CD28- T cells can be used to predict the incidence of aGVHD to some extent. The absolute count of CD8+ CD28- T cells after haplo-HSCT was not associated with cGVHD, fungal infection, EBV infection, and CMV infection, and was also not significantly associated with the prognosis after transplantation.

Quantitative analysis of contrast-enhanced ultrasonography in rat models of hepatic acute graft-versus-host disease.

Hepatic acute graft-versus-host disease (aGVHD) is a major life-threatening complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We hypothesized that contrast-enhanced ultrasound (CEUS) could serve as a new imaging biomarker in early diagnosis of hepatic aGVHD by detecting liver microcirculation. Thirty Wistar rats received allo-HSCT were finally included after excluding 9 rats, and they were randomly divided into 5 groups (1- to 5-week groups, 6 per group). Six rats were used for the control group without any intervention. We observed the clinical scores, serum liver enzyme levels and liver CEUS parameters of rats in each group. Hepatic aGVHD was finally confirmed by histopathologic analysis. The diagnostic performance of CEUS parameters in detecting GVHD was evaluated by comparing the area under the receiver operating curve (AUC) values. After HSCT, the rats developed ruffling of fur, maculopapular rash, weight loss, accompanied by increased clinical scores. Serum liver enzymes were significantly higher than those in the control group from the third week, especially alkaline phosphatase, while CEUS parameters, peak intensity (PI) and mean transit time (MTT), changed in the second week (P<0.001). Compared with non-aGVHD group, the PI was significantly decreased while time to peak and MTT were prolonged in aGVHD group. CEUS parameters were more strongly correlated with pathological grade than serology. PI was an independent predictor for hepatic aGVHD. The AUC of CEUS parameters for diagnosing hepatic aGVHD was 0.933 (95% CI: 0.779-0.992), which was higher than that of clinical scores (AUC =0.748, 95% CI: 0.557-0.888, P=0.032) and serological markers (AUC =0.902, 95% CI: 0.737-0.980, P=0.694). CEUS exhibits promising applications as a quantitative method to detect hepatic aGVHD and early liver damage.

The Characteristics of T Lymphocyte Reconstitution after Haploid Hematopoietic Stem Cell Transplantation in SAA and Its Relationship with aGVHD

To investigate the recovery characteristics of T cell subsets in patients with severe aplastic anemia (SAA) who received haploid hematopoietic stem cell transplantation(HSCT) and its relationship with acute graft-versus-host disease(aGVHD). The clinical data of 29 SAA patients who received haploid hematopoietic stem cell transplantation in the department of hematology, Shanxi Bethune Hospital from June 2018 to January 2022 were retrospectively analyzed. The absolute counts of CD3+T, CD4+T, CD8+T lymphocytes and the ratio of CD4+T/CD8+T lymphocytes in all patients before transplantation, 14, 21, 30, 60, 90 and 120 days after transplantation were analyzed. The proportion of T lymphocytes was compared in the non-aGVHD group, the grade Ⅰ-Ⅱ aGVHD group and the grade III-IV aGVHD group. The counts of all T cells in 27 patients were far below the normal level at 14 and 21 days after transplantation, but there was obvious heterogeneity. There was a certain relationship between T cell immune reconstitution and conditioning regimen, age, and immunosuppressive treatment before transplantation. CD3+T cells showed a steady upward trend at 30, 60, 90, and 120 days after transplantation, and returned to the normal levels at 120 days after transplantation; faster recovery of CD4+T cells was closely related to aGVHD, which was at 30, 60, 90, 120 days after transplantation showed a slow upward trend, and which was still far below the normal level of 120 days after transplantation. CD8+T cell counts began to recover at 14 and 21 days after transplantation, and the recovery was earlier than the CD4+T cells, and its recovery speed was rapid 30 and 60 days after transptantation, which showed an upward trend and exceeded the normal levels 90 days after transplantation. Since CD8+ T cells reconstituted quickly, while the CD4+ T cells reconstitution was slowly, which made the long-term CD4+T/CD8+T cell ratio after transplantation was inverted . Compared with the non-aGVHD group, the absolute counts of CD3+T, CD4+T, and CD8+T cells in the aGVHD group were significantly higher than those in the non-aGVHD group at each time period after transplantation. In the aGVHD group, grade Ⅲ-Ⅳ aGVHD occurred more frequently in the early post-transplantation period (within 14-21 days), the grade Ⅰ-Ⅱ aGVHD group mostly occurred within 30-90 days after transplantation, and CD3+T, CD4+T, CD8+T cell counts in the grade Ⅲ-Ⅳ aGVHD group were significantly higher than those in the grade Ⅰ-Ⅱ aGVHD group; and the greater the proportion of CD4+T, the more severe the degree of aGVHD. The speed of T cell immune reconstitution after SAA haploid transplantation is different, which is related to the conditioning regimen, age, and immunosuppressive therapy before transplantation. The rapid recovery of CD4+ T cells is closely related to the occurrence of aGVHD.

Activated CD4 + T lymphocyte is a potential biomarker for acute graft-vs.-host disease after hematopoietic stem cell transplantation in children with transfusion-dependent β-thalassemia.

BackgroundAcute graft-vs.-host disease (aGVHD) is still one of the most common and life-threatening complications of allogeneic hematopoietic stem cell transplantation (HSCT). Whether or not the level of activated T lymphocytes rises before the onset of aGVHD is unknown. We explored the possibility of T lymphocytes as biomarkers for early prediction of aGVHD in children with transfusion-dependent β-thalassemia (TDTβ).MethodsWe retrospectively analyzed the characteristics of T lymphocyte subsets before and 14 days after HSCT in children with TDTβ who developed aGVHD. Data from 95 children (Age ≤ 14 years) who underwent allogeneic HSCT from January 2020 to December 2021 were collected. Patients were divided into non-aGVHD group (n = 55) and aGVHD group (n = 40), and aGVHD group was divided into two subgroups: grade I aGVHD (n = 16) and grade II-IV aGVHD (n = 24). Receiver operating characteristic curve (ROC) analysis was performed to predict aGVHD.ResultsBefore preconditioning in non-aGVHD and aGVHD groups, there was no significant difference in all lymphocyte subsets and ratio of CD4 + /CD8 + T cells. On day 14 post-transplantation in non-aGVHD and aGVHD groups, the absolute concentrations per μl blood of T cells, CD4 + T cells, CD8 + T cells, activated CD4 + T cell and NK cells, were 69.73 (14.70, 137.77) and 140.36 (65.06, 293.42), 10.00 (2.35, 23.59) and 35.91 (12.41, 68.71), 37.25 (5.82, 84.36) and 89.99 (35.83, 180.81), 0.52 (0.17, 2.20) and 4.08 (0.91, 11.12), 43.86 (15.00, 91.31) and 26.35 (15.19, 49.39), respectively. On day + 14 (14 days post-transplantation), the differences in all cell subsets and the ratio of CD4 + /CD8 + T cells were not statistically significant between grade I aGVHD and grade II-IV aGVHD subgroups. The absolute concentrations of CD8 + T cells in grade I aGVHD were significantly higher than in grade II-IV aGVHD [128.21 (61.11, 258.91) vs. 60.81 (21.59, 176.38), P = 0.057]. AUC of NK cells, CD8 + T cells, T cells, CD4 + T cells, and CD4 + CD25 + T cells were 0.6275, 0.6839, 0.7068, 0.7241, and 0.7589, and cut-off values were 73.75 (97.50, 34.55), 146.90 (37.50, 94.55), 187.30 (45.00, 90.91), 18.95 (70.00, 72.73), and 3.24 (52.50, 87.27), respectively. The AUC of the combined CD4 + CD25 + T cells and CD8 + T cells, CD4 + CD25 + T cells and T cells, CD4 + CD25 + T cells and CD4 + T cells, CD4 + CD25 + T cells and NK cells, respectively, were 0.7500, 0.7598, 0.7750, and 0.8050.ConclusionOur findings demonstrate that level of activated CD4 + T cells on day + 14 (post-HSCT) is a valuable biomarker for predicting aGVHD in children with TDTβ and CD8 + T cells could likely be a biomarker for severe aGVHD.

The absence of AhR in CD4+ T cells in patients with acute graft-versus-host disease may be related to insufficient CTCF expression

BackgroundAcute graft-versus-host disease (aGVHD) is a life-threatening complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Accumulating evidence suggests that imbalanced Treg/Th17 ratio accelerates the progression of aGVHD. The aryl hydrocarbon receptor (AhR) is a basic helix-loop-helix transcription factor activated through cognate ligand binding. Current evidence supports that AhR plays a critical regulatory role in the differentiation of Treg and Th17 cells. However, the relationship between AhR and aGVHD remains unclear.ResultsOur results showed that AhR expression was downregulated significantly in CD4+ T cells from patients with aGVHD compared with the non-aGVHD group. We also discovered that after activating AhR deficient CD4+ T cells, the expression levels of the activation markers-CD40L, CD134 and CD137 and cell proliferation activity were significantly higher than those of AhR-expressing CD4+ T cells. Restoring the expression of AhR in aGVHD CD4+ T cells resulted in significantly increased percentage of Tregs and associated gene transcripts, including Foxp3, IL-10 and CD39. In contrast, Th17 cell amounts and the transcription of related genes, including RORγt, IL-17A and IL-17F, were significantly reduced. We confirmed that CTCF recruited EP300 and TET2 to bind to the AhR promoter region and promoted AhR expression by mediating histone H3K9/K14 hyperacetylation and DNA demethylation in this region. The low expression of CTCF caused histone hypoacetylation and DNA hypermethylation of the AhR promoter, resulting in insufficient expression in aGVHD CD4+ T cells.ConclusionsCTCF is an important inducer of AhR transcription. Insufficient expression of CTCF leads to excessive AhR downregulation, resulting in substantial CD4+ T cell activation and Th17/Treg ratio increase, thereby mediating the occurrence of aGVHD.

Circulating endothelial cells and endothelial progenitor cells as potential predictors of acute GVHD after allogeneic hematopoietic stem cell transplantation.

Acute graft-versus-host disease (aGVHD) is a major cause of treatment-related mortality after allogeneic hematopoietic stem cell transplantation. Endothelial cell damage may trigger the initiation of aGVHD. Endothelial damage and repair were evaluated by counting circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs) in 17 allogeneic hematopoietic stem cell transplantation patients at pre-conditioning, day 0, day 7, day 14, day 30, and day 60 by multicolor flow cytometry. Von Willebrand factor activity was simultaneously measured. Eight patients developed aGVHD and were compared to non-aGVHD patients. Patients' characteristics were not different, except for previous treatment courses. There was no difference in von Willebrand factor activity between the two groups. Both CEC and EPC counts were decreased on day 7 and day 14 and then increased thereafter. The CEC count on day 7 was significantly lower in the aGVHD group than in the non-aGVHD group (p=.0401). Restoration of the EPC count on day 60 was significantly suppressed in the aGVHD group (p=.0464). The CEC count on day 7 could predict aGVHD development (AUC 0.8214, p=.0372). The present results showed that CEC count on day 7 could be a predictor of aGVHD.

Interleukin-7 Receptor Gene Heterozygous Mutations in Recipients Significantly Increase Acute Graft-Versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation

Acute graft-versus-host disease (aGVHD) is one of major complications of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Genetic polymorphisms of interleukin-7 receptor (IL-7R) have an impact on transplant-related mortality (Z Shamim et al. BMT 2006). Our previous studies indicated that hematological and immunological hereditary predisposition genes have potential influence on major complications of allo-HSCT in patients with hematological malignancies (unpublished data). In present study, the influence of hematological and immunological hereditary predisposition genes on aGVHD after allo-HSCT in the patients with hematological malignancies was investigated. Between February 2018 and August 2019, 92 patients with hematological malignancies who underwent allo-HSCT in Beijing Boren Hospital were enrolled. The median age was 17 (1 to 45) years old. The diagnoses included acute myeloid leukemia (37, 40.2%), acute lymphoblastic leukemia (36, 39.1%), non-Hodgkin's lymphoma (13, 14.1%), and myelodysplastic syndrome (6, 6.5%). The disease status before transplant was complete remission in 54 (58.7%), non-remission in 23 (25%), and relapse in 15 (16.3%). Before transplants, blood samples were collected from the patients, their parents and potential related donors to detect hematological and immunological hereditary predisposition genes with whole exon sequencing and validation by sanger sequencing. The donors were from identical siblings (4, 4.3%) or matched unrelated volunteers (8, 8.7%) or haploidentical family members (80, 86.9%). Myeloablative conditioning regimens with either total body irradiation/fludarabine-based or busulfan/fludarabine-based were applied. Anti-thymocyte globulin was used in haploidentical and unrelated transplants. GVHD prophylaxis was with cyclosporine, short-term methotrexate and mycophenolate mofetil. Methylprednisolong 1-2mg/kg daily was used for aGVHD treatment and basiliximab was administrated in severe aGVHD. Forty-eight patients (52.2%) developed aGVHD as aGVHD group and 44 patients (47.8%) have no aGVHD as non-aGVHD group. IL7R gene heterozygous mutations in recipients significantly increased overall aGVHD and also severe aGVHD after allo-HSCT. IL7R gene heterozygous mutations were found in 7/48 patients in aGVHD group, but 0/44 in non-GVHD group (P = 0.041). The incidence of grade III-IV aGVHD in patients with IL7R gene heterozygous mutations was significantly higher than that without IL7R gene heterozygous mutations. (5/7 vs. 7/41, P=0.007). Among the patients with IL7R gene heterozygous mutations in aGVHD group, 5/7 patients (71.4%) had grade III-IV aGVHD. Three of them with grade III aGVHD had the same heterozygous mutation site: c.c1241t; p.t414m and rest 2 patients with grade IV aGVHD had the same heterozygous mutation site: c.g314a; p. S105n. Our preliminary results have shown that IL7R gene heterozygous mutations in recipients have significantly increased the risk of overall aGVHD and severe aGVHD after allo-HSCT in the patients with hematological malignancies. Larger sample size is needed to address this issue. Disclosures No relevant conflicts of interest to declare.

Downregulation of p53 by Insufficient CTCF in CD4+ T Cells Is an Important Factor Inducing Acute Graft-Versus-Host Disease.

Recent evidence indicates that p53 plays a protective role against various systemic autoimmune diseases by suppressing pro-inflammatory cytokine production and reducing the number of pathogenic T cells. However, whether abnormal p53 expression participates in the development of acute graft-versus-host disease (aGVHD) remains unclear. In this study, we demonstrated that p53 was downregulated in CD4+ T cells from patients with aGVHD compared with the non-aGVHD group. Furthermore, we confirmed that low expression of CCCTC-binding factor (CTCF) in CD4+ T cells from aGVHD cases is an important factor affecting histone H3K9/K14 hypoacetylation in the p53 promoter and p53 downregulation. Restoring CTCF expression in CD4+ T cells from aGVHD patients increased p53 amounts and corrected the imbalance of Th17 cells/Tregs. Taken together, these results provide novel insights into p53 downregulation in CD4+ T cells from aGVHD patients.

Cytotoxicity of Donor Natural Killer Cells to Allo-Reactive T Cells Are Related With Acute Graft-vs.-Host-Disease Following Allogeneic Stem Cell Transplantation.

Objectives: The mechanism and immunoregulatory role of human natural killer (NK) cells in acute graft-vs.-host-disease (aGVHD) remains unclear. This study quantitatively analyzed the cytotoxicity of donor NK cells toward allo-reactive T cells, and investigated their relationship with acute GVHD (aGVHD).Methods: We evaluated NK dose, subgroup, and receptor expression in allografts from 98 patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT). A CD107a degranulating assay was used as a quantitative detection method for the cytotoxic function of donor NK cells to allo-reactive T cells. In antibody-blocking assay, NK cells were pre-treated with anti-DNAM-1(CD226), anti-NKG2D, anti-NKP46, or anti-NKG-2A monoclonal antibodies (mAbs) before the degranulating assay.Results: NK cells in allografts effectively inhibited auto-T cell proliferation following alloantigen stimulation, selectively killing alloantigen activated T cells. NKG2A− NK cell subgroups showed higher levels of CD107a degranulation toward activated T cells, when compared with NKG2A− subgroups. Blocking NKG2D or CD226 (DNAM-1) led to significant reductions in degranulation, whereas NKG2A block resulted in increased NK degranulation. Donor NK cells in the aGVHD group expressed lower levels of NKG2D and CD226, higher levels of NKG2A, and showed higher CD107a degranulation levels when compared with NK cells in the non-aGVHD group. Using univariate analysis, higher NK degranulation activities in allografts (CD107ahigh) were correlated with a decreased risk in grade I–IV aGVHD (hazard risk [HR] = 0.294; P < 0.0001), grade III–IV aGVHD (HR = 0.102; P < 0.0001), and relapse (HR = 0.157; P = 0.015), and improved overall survival (HR = 0.355; P = 0.028) after allo-HSCT. Multivariate analyses showed that higher NK degranulation activities (CD107ahigh) in allografts were independent risk factors for grades, I–IV aGVHD (HR = 0.357; P = 0.002), and grades III–IV aGVHD (HR = 0.13; P = 0.009).Conclusions: These findings reveal that the degranulation activity of NK in allografts toward allo-activated T cells was associated with the occurrence and the severity of aGVHD, after allogeneic stem cell transplantation. This suggested that cytotoxicity of donor NK cells to allo-reactive T cells have important roles in aGVHD regulation.

Intestinal Microbiota Can Predict Acute Graft-versus-Host Disease Following Allogeneic Hematopoietic Stem Cell Transplantation.

The intestinal microbiome plays an important role in the development of acute graft-versus-host disease (aGVHD). However, whether intestinal microbiota can predict the development of aGVHD has been reported only rarely. Here we conducted a prospective study of microbiota in 141 patients after allogeneic hematopoietic stem cell transplantation. We found lower microbiota diversity in the aGVHD group compared with the non-aGVHD group at day 0 and day 15 ± 1 (P = .018 and .009, respectively). Diversity was negatively associated with conditioning intensity (P = .017, day 0; P = .045, day 15) and β-lactam antibiotic administration (P = .004, day 15). Intensified conditioning and β-lactam antibiotics were associated with a lower regulatory T (Treg)/T helper 17 (Th17) cell ratio at day 15 (P = .030 and .047, respectively). At day 15, the levels of the inflammatory factors (tumor necrosis factor α, interleukin [IL]-6, IL-17A, IL-1β, and lipopolysaccharide) were higher in the intensified conditioning group compared with the standard group (P < .05). The accumulated intestinal microbiota (AIM) score was defined as microbiota diversity and gradient of the 4 bacterials (Lachnospiraceae, Peptostreptococcaceae, Erysipelotrichaceae, and Enterobacteriaceae) at day 15 post-transplantation. The AIM score was positively correlated with aGVHD grade (r = .481, P < .001), and the AIM score could be predictive of the development of aGVHD (grade II-IV aGVHD: area under the curve [AUC], .75, P < .001; grade III-IV aGVHD: AUC, .84, P < .001). These findings suggest that intestinal microbiota and conditioning might induce aGVHD by inflammatory factors and the Treg/Th17 balance. The constitution of the intestinal microbiota at neutrophil engraftment may predict the development of aGVHD.

Intestinal Microbiota at Neutrophil Engraftment Can Predict Acute Graft-Versus-Host Disease in the Recipients of Allogeneic Hematopoietic Stem Cell Transplantation

Backgrounds: An increasing of researches have demonstrated that intestinal microbiome plays an important role in the development of acute graft-versus-host disease (aGVHD), and loss of microbiota diversity is associated with aGVHD. But whether microbiota might be as a biomarker to predict aGVHD is undetermined. This study investigates the prediction of intestinal microbiota at neutrophil engraftment for occurrence of aGVHD.Methods: We prospectively collected stool and blood samples from 166 patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) at pre-conditioning, day 0 and day 15 post-transplantation. The microbiota in stool were detected by 16S rRNA gene sequencing; the inflammatory factors (TNF-α, IL-6, IL-17A, IL-1β and Lipopolysaccharide (LPS)) in blood by multiplex immunoassays and dynamic turbidimetric methods; and T lymphocyte subsets were tested by flow cytometry. AGVHD was determined by retrospective review of the clinical charts.Results: 141 patients were enrolled into analysis, including 83 in grade 0-I aGVHD (non-aGVHD group) and 58 in grades II-IV aGVHD (aGVHD group). The microbiota diversity was lower in aGVHD group than that in non-aGVHD both at day 0 and day 15 post-transplantation (P = 0.018, 0.009, respectively). The diversity was associated with conditioning (Intensified vs. Standard: medain, 3.39 vs. 3.04 (P = 0.017, day 0); medain, 3.17 vs.1.89 (P = 0.045, day 15)) and β-lactam antibiotics (β-lactam vs. No β-lactam: medain, 3.33 vs. 2.48, P = 0.004 (day 15)). At day 15 post-transplantation, Treg/Th17 cells ratio in blood positively correlated with the microbial diversity(r = 0.217, P = 0.010), but negatively correlated with the levels of TNF-α, IL-17A and LPS (r = -0.226, -0.216, -0.291; P = 0.007, 0.010, < 0.001, respectively). Based on the accumulated microbiota Score of the diversity and the special discriminated constitution (Enterobacteriaceae, Lachnospiraceae, Peptostreptococcaceae and Erysipelotrichaceae) between aGVHD and non-aGVHD groups at day 15, the area under the receiver operator characteristic curve (AUC) of the Score could be used to predict aGVHD (II-IV aGVHD: AUC = 0.75, P < 0.001; III-IV aGVHD: AUC = 0.84, p < 0.001). The Score also correlated with the grades of aGVHD (r = 0.481, P < 0.001).Conclusions: The intestinal microbiota diversity is affected by both conditioning and antibiotics. The constitution of microbiota at neutrophil engraftment was a predictive marker for the subsequent development and grades of aGVHD.Keywords: intestinal microbiota, marker, acute graft-versus-host disease, hematopoietic stem cell transplantation DisclosuresNo relevant conflicts of interest to declare.

Intestinal Microbiota at Engraftment Influence Acute Graft-Versus-Host Disease via the Treg/Th17 Balance in Allo-HSCT Recipients.

Animal models have indicated that intestinal microbiota influence acute graft-versus-host disease (aGVHD) by modulating immune homeostasis. But, in humans, the mechanism by which the microbiota induces aGVHD remains unclear. In this study, we investigated the relationship between the intestinal microbiota and T cell subsets in patients who undergo allogeneic hematopoietic stem cell transplantation (allo-HSCT) to explore the mechanism by which microbiota induced aGVHD. Based on aGVHD, this study was categorized into two groups: grades II–IV aGVHD (aGVHD group, n = 32) and grade 0–I aGVHD (non-aGVHD group, n = 49). The intestinal microbiota was detected by 16S rRNA gene sequencing, and the T cell subsets and histone 3 (H3) acetylation in CD4+ T cells in the peripheral blood was assayed by flow cytometry at the time of engraftment. The aGVHD group had greater low microbial diversity than the non-aGVHD group (56.3 versus 24.5%, p = 0.004). The bacterial community was depleted of Clostridia (e.g., the Lachnospiraceae and Ruminococcaceae families) and enriched for Gammaproteobacteria (e.g., the Enterobacteriaceae family) in the aGVHD group compared with the non-aGVHD group. The relative abundance of Lachnospiraceae and Ruminococcaceae was positively correlated with the Treg/Th17 ratio counts (r = 0.469 and 0.419; p < 0.001 and <0.001, respectively), whereas Enterobacteriaceae was negatively correlated with the Treg/Th17 ratio (r = −0.277; p = 0.012). The level of acetylated H3 in CD4+ T cells was not only correlated with Lachnospiraceae/Ruminococcaceae, but also with the Treg/Th17 ratio (r = 0.354; p = 0.001). In conclusions, our results suggest that decreased Lachnospiraceae and Ruminococcaceae and increased Enterobacteriaceae, correlate with a Treg/Th17 imbalance, which might be through acetylated H3 in CD4+ T cells. These findings suggest that intestinal microbiota might induce aGVHD by influencing the Treg/Th17 balance.

Changes in body composition in children with acute graft-versus-host disease within the first 100 days after hematopoietic stem cell transplantation.

Acute graft-versus-host-disease (aGVHD) is one of the main complications of hematopoietic stem cell transplantation (HSCT). This study investigated the changes in body composition of pediatric patients with aGVHD during the first 100 days after HSCT. Fifty-five children receiving HSCT were divided into two groups (aGVHD and non-aGVHD). Body mass index Z-scores (BMI-z), arm muscle area index (AMAI), fat mass index (FMI), and fat-free mass index (FFMI) were measured on the day of transplantation (H0), and on the 30th (H30), 60th (H60), and 100th day (H100) after the transplantation. The correlative factors on body composition were evaluated. In the aGVHD group, the rates of absolute change of BMI-z at H30, H60, and H100 showed a significant increase as compared to that at H0, especially at H30 which was remarkably higher than that of the non-aGVHD group (P = 0.008). AMAI showed a continuous decrease from H0 to H100 in the aGVHD group; also FFMI was found to be lower than that of the non-aGVHD group during the first 100 days after transplantation, however, no significant differences were found between the two groups. At H60 and H100, FFMI in the aGVHD group was lower than that in the non-aGVHD group (P = 0.014, P = 0.032, respectively). Glucocorticoid treatment and the occurrence of mucositis were the key factors for changes in body composition in the aGVHD group. Changes in body composition are characterized by a lean reduction in body mass and increase in adipose tissues in the early stage of post-transplantation in the aGVHD children. Glucocorticoid treatment and occurrence of mucositis are the two important factors that were found to affect body composition after HSCT.

Significance of Thl7/Treg Ratio in aGVHD Occurence after Allo-HSCT

To explore the relationship between Th17 and Treg levels and aGVHD occurence in patients after allo-HSCT. Thirty-nine patients received allo-HSCT were divided into 2 groups: aGVHD group(17 cases) and non-aGVHD group (22 cases). For aGVHD group, the peripheral blood was collected before aGVHD occurence, in occurence and after aGVHD improvement; for non-aGVHD group, the peripheral blood was collected after 1, 2 and 3 months of transplantation. In addition, 16 healthy donors were used as controls, their peripheral blood was collected before mobilization. The Th17 and Treg counts as well as Th17/Treg ratio were detected by flow cytometry. Among patients with aGVHD after transplantation, the Th17 count increased, and the Treg count decreased, the Th17/Treg ratio increased before aGVHD occurence, as compared with the patients without aGVHD(P<0.05); but after aGVHD occurence, the Th17 count decreased, Treg count increated, and the Th17/Treg ratio decreased as compared with that before aGVHD occurence (P<0.05). After aGVHD was improved, the Th17/Treg ratio decreased as compared with level before aGVHD occurence (P<0.05). After aGVHD was improved, the Th17/Treg ratio was no statistical different from healthy donors (P>0.05). Among patients without aGVHD after transplantation, the Th17/Treg ratio at 2 and 3 months after transplantation was no statistical different from that of healthy donos(P>0.05). The Th17 and Treg levels recoverel showly after transplantation, but the Th17/Treg ratio recoveres after 2 months in the patients after transplantation. The Th17 cells may initiate aGVHD; when aGVHD happened, the Treg level increases, which may regulate the aGVHD ontcome through inhibiting the Th17 cells. The detection of Th17/Treg ratio after transplantation can predict the occurence and outcome of aGVHD.

CD200/CD200R Expression Levels and Its Significance after Allogeneic Hematopoietic Stem Cell Transplantation

CD200 and its receptor CD200R are both type-1 membrane glycoproteins, which are members of the immunoglobulin superfamily (IgSF). Besides the inhibitory effect on macrophages, CD200/CD200R also play an important role in regulating the regulatory T cells, allergicreaction, autoimmune diseases, allograft, neurological diseases and other autoimmune-related diseases, etc. To investigate the role of CD200 and its receptor in the graft versus host disease (GVHD). Experimental samples were divided into aGVHD group, non-aGVHD group, cGVHD group and non-cGVHD group, the healthy persons were used as normal controls. Firstly, the expression levels of CD200 and CD200R on CD19+ cell, CD3+ cell and dendritic cell (CD19- CD14- CD1c+) surfaces in each group were detected by using flow cytometry, so as to determine whether there were expression differences among each groups. Then, the mRNA levels of each groups were tested by using real-time quantitative polymerase chain reaction for finding the differences of mRNA expression level among each group. Finally, the peripheral blood mononuclear cells of the patients and healthy controls were co-cultured with anti-CD200R1 antibody for 48 hours, and the interleukin-10 level in the co-culture system was tested by using enzyme linked immunosorbent assay for verifying the function of CD200/CD200R. The CD200 expression level on CD19+ cell surface in the aGVHD group and non-aGVHD group was both lower than that in healthy control group; that in the non-aGVHD group was higher than that in the aGVHD group. The CD200 expression level on CD19+ CD200+ cells in the non-cGVHD group were higher than that in the cGVHD group and healthy control group. There were no significant differences of CD200 and CD200R expression levels on CD3 cells and dendritic cells among all groups. The CD200 mRNA expression levels in the aGVHD group and cGVHD group were both lower than the healthy control group. The CD200 mRNA expression level was lower in the aGVHD group than in the non-aGVHD group, and was lower in the cGVHD group than in the non-cGVHD group. There was no significant difference of the CD200R mRNA expression level among all groups. After the peripheral blood mononuclear cells of the patients and healthy controls were co-cultured with anti-CD200R1 antibody for 48 hours, the interleukin-10 concentration decreased with the increasing of anti-CD200R1 antibody concentrations in the co-culture system. The CD200/CD200R may play a role in the pathogenesis of GVHD after allo-HSCT.

Circulating Levels of Adipokines Predict the Occurrence of Acute Graft-versus-host Disease.

Currently, detecting biochemical differences before and after allogeneic stem cell transplantation (SCT) for improved prediction of acute graft-versus-host disease (aGVHD) is a major clinical challenge. In this pilot study, we analyzed the kinetics of circulating adipokine levels in patients with or without aGVHD before and after allogeneic SCT. Serum samples were obtained and stored at -80℃ within 3 hours after collection, prior to conditioning and at engraftment after transplantation. A protein array system was used to measure the levels of 7 adipokines of patients with aGVHD (n=20) and without aGVHD (n=20). The resistin level at engraftment was significantly increased (p<0.001) after transplantation, regardless of aGVHD occurrence. In the non-aGVHD group, the concentrations of the hepatocyte growth factor (HGF) (mean values±SD; 206.6±34.3 vs. 432.3±108.9 pg/ml, p=0.040) and angiopoietin-2 (ANG-2) (mean values±SD; 3,197.2±328.3 vs. 4,471.8±568.4 pg/ml, p=0.037) at engraftment were significantly higher than those of the pre-transplant period, whereas in the aGVHD group, the levels of adipokines did not change after transplantation. Our study suggests that changes in serum HGF and ANG-2 levels could be considered helpful markers for the subsequent occurrence of aGVHD.

Relation of Treg and iNKT cell reconstruction with aGVHD after allogeneic hematopoietic stem cell transplantation in children

This study was aimed to explore the relation of Treg and invariant natural killer T (iNKT) cell reconstruction with acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in children. According to the occurrence or absence of aGVHD, 29 pediatric patients who underwent allo-HSCT were firstly divided into two groups non-aGVHD and aGVHD group,then those patients with aGVHD were divided into steroid effective group and steroid resistant group according to their reaction to the steroid treatment. Flow cytometry was used to detect the frequency of Treg cells and iNKT cells in the peripheral blood of the recipients at different time after allo-HSCT(d 15, d 30, d 60, d 90, the time of aGVHD onset and two weeks after steroid treatment). The result showed that the frequencies of Treg cells and the iNKT/T ratio on day 15 in non-aGVHD group were significantly higher than those in the aGVHD group (P < 0.05). It is concluded that a combined monitoring strategy of Treg and iNKT cell reconstruction early after allo-HSCT may facilitate the diagnosis and treatment of aGVHD in children.

Elevated Fas/FasL system and endothelial cell microparticles are involved in endothelial damage in acute graft-versus-host disease: A clinical analysis

Acute graft-versus-host disease (aGVHD) is the major cause of early morbidity and mortality in allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients, but its mechanism remains not well understood. This study firstly compared dynamic change of Fas/Fas ligand (FasL) system in plasma and also in the surface of endothelial cell microparticles (EMPs) in aGVHD patients, pre-aGVHD patients (7, 14, 21 and 28 days after transplantation), non-aGVHD patients and normal controls. Plasma soluble FasL (sFasL) was detected by ELISA. Fas, FasL and EMPs were evaluated by Confocal microscopy and flow cytometric analysis respectively. Significantly higher levels of sFasL and EMPs were detected in the patients at the time of aGVHD attack. In addition, sFasL exhibited significant elevation but EMPs exhibited simultaneously decrease within the early phase (+21d) after allo-HSCT in pre-aGVHD group compared with non-aGVHD group. At +21d, there was a significant difference of sFasL and EMPs between pre-aGVHD group and non-aGVHD group. Moreover our present study firstly reported that EMPs expressed higher Fas antigen (Fas+-EMPs) in the patients with aGVHD. Therefore it can be inferred that EMPs may play protective roles through Fas in endothelial cell damage of aGVHD after allo-HSCT. This suggested monitoring EMPs and sFasL in the early phase after HSCT may be useful for the early diagnosis and forecast of aGVHD and it also provided new clues in understanding the pathogenesis of endothelial cell injury during aGVHD after allo-HSCT and meanwhile help identifying new drugs to circumvent it in clinic.