The level of Tim-3+CD8+ T cells can serve as a potential marker for evaluating the severity of acute graft-versus-host disease after haplo-PBSCT

Early and accurate diagnosis of acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation is crucial for the prognosis of patients. This study identified a potential biomarker for the severity of aGVHD after human leukocyte antigen (HLA)-haploidentical peripheral blood hematopoietic stem cell transplantation (haplo-PBSCT). We included 20 healthy subjects and 57 patients who underwent haplo-PBSCT. Of these patients, 22 developed aGVHD after haplo-PBSCT. The results showed that patients with aGVHD had significantly increased levels of Tim-3+/Perforin+/Granzyme B+CD8+ T cells, but significantly decreased Galectin-9. The differences in Galectin-9 and Tim-3+/Granzyme B+CD8+ T cells between grade I-II aGVHD and III-IV aGVHD were also significant. In vitro, the apoptosis of CD8+ T cells from aGVHD patients was significantly increased after Tim-3/Galectin-9 pathway activation, which decreased Granzyme B secretion. As revealed by univariate analysis, the level of Tim-3+CD8+ T cells was a risk factor for severe aGVHD. ROC analysis demonstrated that high levels of Tim-3+CD8+ T cells had a significant diagnostic value for severe aGVHD, with an area under the curve of 0.854 and cut-off value of 14.155%. In conclusion, the binding of Tim-3 with exogenous Galectin-9 can promote apoptosis of CD8+ T cells and affect the secretion of Granzyme B. Tim-3+CD8+ T cells have the potential to serve as immunological markers for assessing the severity of aGVHD after haplo-PBSCT and identifying patients at a higher risk for severe aGVHD.

Clinical study of 19 cases of steroid-refractory gastrointestinal acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation with fecal microbiota transplantation / 中华血液学杂志

Objective: To investigate the clinical efficacy of fecal microbiota transplantation (FMT) for treating steroid-refractory gastrointestinal acute graft-versus-host disease (GI-aGVHD) . Methods: This analysis included 29 patients with hematology who developed steroid-refractory GI-aGVHD after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in Huaian Hospital Affiliated to Xuzhou Medical University from March 2017 to March 2022. Among them, 19 patients underwent FMT treatment (the FMT group) and 10 patients did not (the control group). The efficacy and safety of FMT were assessed, as well as the changes in intestinal microbiota abundance, lymphocyte subpopulation ratio, peripheral blood inflammatory cytokines, and GVHD biomarkers before and after FMT treatment. Results: ① Complete remission of clinical symptoms after FMT was achieved by 13 (68.4%) patients and 2 (20.0%) controls, with a statistically significant difference (P<0.05). Intestinal microbiota diversity increased and gradually recovered to normal levels after FMT and FMT-related infections did not occur. ②The proportion of CD3(+) and CD8(+) cells in the FMT group after treatment decreased compared with the control group, and the ratio of CD4(+), regulatory T cells (Treg), and CD4(+)/CD8(+) cells increased (all P< 0.05). The interleukin (IL) -6 concentration in the FMT group was lower than that in the control group [4.15 (1.91-5.71) ng/L vs 6.82 (2.40-8.91) ng/L, P=0.040], and the IL-10 concentration in the FMT group was higher than that in the control group [12.11 (5.69-20.36) ng/L vs 7.51 (4.10-9.58) ng/L, P=0.024]. Islet-derived protein 3α (REG3α) was significantly increased in patients with GI-aGVHD, and the REG3α level in the FMT group was lower than that in the control group after treatment [30.70 (10.50-105.00) μg/L vs 74.35 (33.50-139.50) μg/L, P=0.021]. Conclusion: FMT is a safe and effective method for the treatment of steroid-refractory GI-aGVHD by restoring intestinal microbiota diversity, regulating inflammatory cytokines, and upregulating Treg cells.

Preliminary study of shear wave dispersion imaging in the evaluation of hepatic acute graft-versus-host disease / 中华超声影像学杂志

Objective:To investigate the feasibility of using shear wave dispersion (SWD) imaging in evaluating acute graft-versus-host disease (aGVHD) of the liver.Methods:A total of 42 Wistar rats were used as receptors and 10 Fischer 344 rats were used as donors for bone marrow transplantation to establish aGVHD models. Six rats were randomly selected every week for clinical observation and scoring. Then, ultrasonic SWD was performed to obtain shear wave speed (SWS) and shear wave dispersion slope (SWDS). Then, the histological characteristics were used as a reference standard, and the rats were divided into two groups: the group without aGVHD (nGVHD group) and the group with aGVHD. The differences in the clinical score and SWD values between the two groups were compared, the meaningful indexes were screened by binary Logistic regression analysis, and the joint prediction parameters were obtained. The ROC curve was plotted and the diagnostic efficiency was compared. The correlations between SWS, SWDS, clinical score and pathological grade were analyzed.Results:Clinical score, SWS, and SWDS in aGVHD group were higher than those in the nGVHD group (all P<0.05). The correlation between SWDS and pathological grade ( r=0.774, P<0.001) was higher than those between SWS, clinical score and pathological grade ( r=0.579, P=0.005; r=0.452, P=0.034). Logistic regression showed that SWDS was a significant indicator. In addition, the AUC values determined by SWDS and predictive parameters were (0.859, 0.886), which were significantly higher than the AUC of the clinical score (0.760, P<0.05). Conclusions:SWD imaging technology may become a promising method to evaluate hepatic aGVHD.

Experience in diagnosis and treatment of acute graft-versus-host disease after liver transplantation of five cases / 西安交通大学学报(医学版)

【Objective】 To investigate our experience in the diagnosis and treatment of acute graft-versus-host disease (aGVHD) after liver transplantation in surgical ICU. 【Methods】 We retrospectively analyzed the general data, clinical manifestation, diagnosis, and treatment strategies of five patients with aGVHD after liver transplantation in The First Affiliated Hospital of Xi’an Jiaotong University from January 2000 to December 2019. 【Results】 The incidence rate of aGVHD was 5/850 (5.88 ‰), and all the five patients were male and aged 40-64 years (mean age 56 years). Diabetes, hepatocellular carcinoma, liver transplantation, transcatheter arterial chemoembolization (TACE), and high concentration of immune agents were the main risk factors associated with the development of aGVHD. The average time from surgery until clinical symptom of aGVHD was 15 to 32 days. In our patients with aGVHD, the most common symptom was fever (5/5), followed by skin rash (5/5), pancytopenia (5/5), diarrhea (3/5), and secondary pulmonary infection (3/5). However, liver functions were not remarkable affected. Diagnostic criteria for aGVHD in our center include acute onset, risk factors, typical clinical manifestation, and histopathology after exclusion of differential diseases. Our treatment strategies include high-dose methylprednisolone, stopping/reducing current immunosuppressive protocol, and antilymphocytic agents as second-line treatment. Empirical antibiotics and antifungal agents play a vital part in infections after transplantation. Hematopoietic cytokine was administered to treat pancytopenia. Patients also received supportive therapy, such as isolation and nutritional support, with the goal of benefiting the entire condition. Despite intensive treatment, two of five patients (40%) with aGVHD died due to sepsis and multiorgan failure. One case (20%) died of intracranial hemorrhage and one case (20%) died of tuberculosis. Only one case (20%) stayed alive after 1-year follow-up without complications. 【Conclusion】 The diagnosis of aGVHD relies on clinical suspicion and is confirmed by skin pathology. The patients with aGVHD had early onset (38.5 ℃), large rash range (>50%), complication of sepsis, and poor response to hematopoietic cytokine therapy indicate poor prognosis. Intensive treatment should be started immediately after aGVHD diagnosis. In conclusion, we strongly suggest an early identification, diagnosis, and vigorous treatment strategy, which is the key to improving the prognosis of aGVHD.

Study on the relationship between serum cytokines levels after chimeric antigen receptor (CAR)-T cell immunotherapy and the prognosis of bridged allogeneic hematopoietic stem cell transplantation in acute B lymphoblastic leukemia patients / 中华检验医学杂志

Objective:To investigate the relationship between the levels of serum cytokines and chemokines and the prognosis of patients with acute B-ALL after receiving chimeric antigen receptor (CAR)-T cell immunotherapy and acute graft-versus-host disease (aGVHD) in patients after bridging allogeneic hematopoietic stem cell transplantation (allo-HSCT).Methods:According to the case-control principle, Forty-two patients with B-ALL who received CD19-CAR-T cell immunotherapy bridged to allo-HSCT at Heibei Yanda Ludaopei Hospital from September 18, 2019 to May 9, 2022 were enrolled. Mann-Whitney U test was used to compare the changes of aGVHD-related cytokines and chemokine levels between CAR-T cell immunotherapy and bridging transplantation in different patients at the same time. Their plasma levels of cytokines and chemokines related to aGVHD were monitored at the day before CAR-T therapy and after CAR-T treatment at day 4, 7,14,21,28. The receiver operating characteristic curve was drawn to evaluate the predictive value of cytokines and chemokines in predicting the occurrence and the death of aGVHD patients. Kaplan-Meier method and Log-rank tests were used for Overall survival (OS) analysis. Results:Twenty-four of total 42 patients had aGVHD, of which 11 patients died and 31 patients survived. There was no significant difference in cytokines and chemokines between the aGVHD group and the non-aGVHD group on the day before CAR-T cell treatment. According to statistical analysis, the serum Elafin levels of aGVHD group was higher than that of non-aGVHD group at the 21st day [4 482 (2 811, 6 061) ng/L vs 2 466 (1 948, 3 375) ng/L, Z=3.145, P=0.001] and the 28st day [4 391 (2 808, 5594) ng/L vs 2 463 (1 658, 2 830) ng/L, Z=2.038, P=0.048] separately. At the 14th day, serum cytokines and chemokines levels between the two group were as follows,MIP-1 α [21.02 (12.36, 30.35) ng/L vs 5.56 (3.64, 10.79) ng/L], sCD25 [422.47 (257.99, 1 233.78) IU/ml vs 216.11 (133.75,457.39) IU/ml], Elafin [4 101 (2 393, 5 006) ng/L vs 2 155 (1 781, 3 033) ng/L], IL-6 [119.08 (23.97, 183.43) ng/L vs 8.39 (2.91, 17.42) ng/L] and IL-8 [13.56 (12.50, 24.52) ng/L vs 2.83 (1.73,6.87) ng/L] were at higher levels ( Z=2.653, P=0.007; Z=2.176, P=0. 030; Z=2.058, P=0.041; Z=3.329, P<0.001; Z=3.162, P=0.001). The KM survival curve showed that the cumulative survival rates of patients with higher serum levels of MIP-1α, sCD25, Elafin, IL-6 and IL-8 were lower than those with low levels at day 14, and the difference was statistically significant (χ 2=12.353, 4.890, 6.551, 10.563, 20.755, P<0.05). Conclusion:The outcomes of patients treated with CAR-T cell therapy bridged to allo-HSCT was correlated with serum MIP-1α, sCD25, Elafin, IL-6 and IL-8 levels after receiving CAR-T therapy. High concentrations of MIP-1α, sCD25, Elafin, IL-6 and IL-8 suggest poor prognosis and can be used as biomarkers to suggest appropriate clinical selection of therapy.

The Characteristics of T Lymphocyte Reconstitution after Haploid Hematopoietic Stem Cell Transplantation in SAA and Its Relationship with aGVHD / 中国实验血液学杂志

OBJECTIVE@#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).@*METHODS@#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.@*RESULTS@#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.@*CONCLUSION@#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.

Clinical Study of Intestinal Acute Graft-Versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation in Children / 中国实验血液学杂志

OBJECTIVE@#To observe the clinical characteristics, treatment and prognosis of intestinal acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in children and futher evaluate the occurring risk factors.@*METHODS@#The clinical data of 136 patients undergoing allo-HSCT in Wuhan Children's Hospital Affiliated to Tongji Medical College from August 2016 to August 2020 were retrospectively analyzed, clinical characteristics of children with intestinal aGVHD were observed. The risk factors of intestinal aGVHD were assessed by logistic regression while cumulative survival were analyzed by Kaplan-Meier method.@*RESULTS@#Among 136 patients intestinal aGVHD occurred in 24 (17.6%) cases, with 4 cases of grade II, 20 cases of grade III-IV, and the median occurrence time was 28(10-63) days. The clinical manifestations were diarrhea with intermittent abdominal pain, 17 cases with nausea and vomiting, 11 cases with fresh bloody stool, and 8 cases with skin rash before intestinal aGVHD. The average time for treatment was 33(11-100) days. 18 cases received electronic colonoscopy and histopathology examination. 20 out of 24 cases achieved remission after treatment, and the total effective rate was 83.3%. Finally, 9 out of 24 cases died during the follow-up time. Survival analysis showed that the cumulative survival rate of patients with intestinal aGVHD (15/24, 62.5%) were significantly lower than those without intestinal aGVHD (101/112, 90.2%) (Log-rank test, P=0.001). Univariate analysis showed that recipient age, sex, primary disease, donor age, donor sex, donor-recipient blood type, conditioning regimen, prophylaxis of GVHD, dosage of ATG, engraft time of blood platelet and neutrophils, and number of MNC/CD34+ were not risk factors for intestinal aGVHD (P>0.05). Only the type of HSCT (χ2=16.020, P=0.001) and matched degree of HLA (χ2=15.502, P=0.001) had statistical significance with intestinal aGVHD (P<0.05). Multivariate analysis showed that only HLA-mismatched unrelated donor was the risk factor for intestinal aGVHD for children (P=0.014，OR=16，95%CI 1.735-147.543).@*CONCLUSION@#Intestinal aGVHD is a risk factor for cumulative survial of patients who received allo-HSCT in children and HLA-mismatched unrelated donor is its independent risk factor.

Kinetics of MDSC in Patients Treated Steroids-Ruxolitinib as the First Line Therapy for aGVHD / 中国实验血液学杂志

OBJECTIVE@#To analyze the kinetic characteristics of lymphocyte subsets and myeloid-derived suppressor cell (MDSC) in patients who newly diagnosed intermediate- to high-risk aGVHD and treated with steroids-ruxolitinib as the first line therapy from a single-arm, open clinical trial (NCT04061876).@*METHODS@#We prospectively observed the efficacy of 23 patients having intermediate- to high-risk aGVHD and treated with steroids-ruxolitinib as the first line therapy. The kinetic characteristics of lymphocyte subsets and MDSC were monitored, and then we compared them in steroids-ruxolitinib group (n=23), free-aGVHD group (n=20) and steroids group (n=23).@*RESULTS@#Of the 23 patients, the CR rate was 78.26% (18/23) on day 28 after first-line treatment with steroids-ruxolitinib. On day 28 after treatment, patients had lower level of CD4+CD29+ T cells (P=0.08) than that of pre-treatment, whereas levels of other lymphocyte subsets in this study were higher than that of pre-treatment; CD4+CD29+ T cells in CR patients decreased, compared with refractory aGVHD patients. On day 28 of treatment, CD8+CD28- T cells (P=0.03) significantly increased in patients with aGVHD than that in patients without aGVHD, so did CD8+CD28- T / CD8+CD28+ T cell ratio (P=0.03). Compared with patients without aGVHD, patients with aGVHD had lower level of G-MDSC, especially on day 14 after allo-HSCT (P=0.04). Compared with pre-treatment, M-MDSC was higher in CR patients on day 3 and 7 post-treatment (P3=0.01, P7=0.03), e-MDSC was higher on day 28 post-treatment (P=0.01). Moreover, compared with CR patients, M-MDSC was lower in refractory aGVHD patients on day 3 post-treatment (P=0.01) and e-MDSC was lower on day 28 post-treatment (P=0.01). Compared with steroids group, MDSC in steroids-ruxolitinib group was higher, with the most significant difference in M-MDSC (P3=0.0351; P7=0.0142; P14=0.0369).@*CONCLUSION@#We found that patients newly diagnosed intermediate- to high-risk aGVHD receiving first-line therapy with steroids-ruxolitinib achieved high response rate. Moreover, the novel first-line therapy has a small impact on the immune reconstitution of patients after allo-HSCT. Elevated MDSC might predict a better response in aGVHD patients receiving this novel first-line therapy. M-MDSC responded earlier to steroids-ruxolitinib than e-MDSC, G-MDSC.

The value of regenerating islet-derived protein 3-alpha, soluble tumor suppressor factor 2 and tumor necrosis factor receptor 1 in the diagnosis and treatment of intestinal acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation in children / 中华实用儿科临床杂志

Objective:To investigate the clinical value of the expression levels of biological protein markers regenerating islet-derived protein 3-alpha(REG3α), soluble tumor suppressor factor 2(sST2) and tumor necrosis factor receptor 1(TNFR1) in peripheral blood in the diagnosis and efficacy evaluation of intestinal acute graft-versus-host disease (aGVHD) in children after allogeneic hematopoietic stem cell transplantation (allo-HSCT).Methods:Retrospective analysis of 50 children who underwent allo-HSCT, in the Department of Pediatrics, the First Affiliated Hospital of Zhengzhou University from January 2020 to February 2021 were enrolled, including 39 males and 11 females [median age: 8.5 (1-13) years]. The expression levels of above 3 biological proteins were detected before transplantation, 1 week, 2 weeks, 3 weeks, 5 weeks, 7 weeks, 9 weeks, 11 weeks and 13 weeks after transplantation, when intestinal aGVHD occured, and after treatment.Children with intestinal aGVHD were taken as the observation group, and children without intestinal aGVHD were taken as the control group.Whether differences in the expression levels of the 3 biological proteins in the peripheral blood of the 2 groups of children were statistically significant was analyzed.The receiver operating characteristic(ROC) curve was used to evaluate the diagnostic value of the above three biological proteins for intestinal aGVHD, and independent sample t test was performed to compare the expression levels of the 3 biological proteins before and after treatment in children with intestinal aGVHD. Results:(1) The concentrations of REG3α, sST2, and TNFR1 in the peripheral blood of the observation group were (33 985.42±24 631.33) ng/L, (139 899.66±115 825.65) ng/L, (3 041.65±2 418.72) ng/L, respectively, which were higher than the control group of (7 457.39±4 547.49) ng/L, (32 059.57±23 452.85) ng/L, (1 944.51±1 170.35) ng/L, the difference was statistically significant ( t=6.04, 5.19, 2.17, all P<0.05). (2) The area under ROC curve (AUC) of REG3α combined with sST2 in the diagnosis of intestinal aGVHD was 0.952 (95% CI: 0.851-0.992, P<0.001), the maximum Youden index was 0.894, the corresponding sensitivity was 83%, and the specificity was 99%.Its diagnostic value was better than REG3α, sST2 and TNFR1 ( Z=1.763, 1.332, 3.001, all P<0.05). (3) The concentrations of REG3α, sST2, and TNFR1 before treatment in the peripheral blood of children having received effective treatment were (31 343.01±25 364.71) ng/L, (146 629.52±110 501.04) ng/L and (2 489.00±859.70) ng/L, respectively, which were (12 104.37±11 704.60) ng/L, (93 539.55±81 920.93) ng/L and (2 048.15±813.47) ng/L after treatment, lower than those before treatment.The expression levels of REG3α and sST2 were significantly reduced ( t=-3.23, -2.10, all P<0.05), while the difference of the expression level of TNFR1 before and after treatment was not statistically significant ( P>0.05). Conclusions:REG3α and sST2 can be used as important reference indicators for clinical auxiliary diagnosis of intestinal aGVHD, and have good auxiliary diagnostic value.REG3α and sST2 can be used as objective indicators to evaluate the efficacy of clinical treatment of intestinal aGVHD.

Baicalin protects intestinal mucosal barrier and intervenes acute graft-versus-host disease by regulating autophagy / 中国组织工程研究

BACKGROUND: Acute graft-versus-host disease is one of the important complications of early death after hematopoietic stem cell transplantation. How to intervene in the course of graft-versus-host disease is a hot topic. OBJECTIVE: To explore the mechanism of baicalin regulating autophagy in the treatment of acute intestinal graft-versus-host disease. METHODS: Within 4 hours after 60Co irradiation in CB6F1 mice, mononuclear cell suspension (bone marrow + spleen) of Balb/c mice was immediately infused into the tail vein to establish haploid hematopoietic stem cell transplantation model. On the day after the establishment of the model, the rats in the model group were intragastrically given normal saline and the rats in the treatment group were intragastrically given baicalin at a dose of 30 mg/(kg·d) for 14 days. After treatment, clinical evaluation of acute graft-versus-host disease was conducted in mice. Pathological grade of acute graft-versus-host disease of small intestinal mucosa was analyzed. Autophagic vesicles in small intestinal mucosa were observed by transmission electron microscopy. Levels of reactive oxygen species in intestinal epithelial cells were detected by flow cytometry. The expression levels of autophagy related proteins LC3-II, LC3-I and Beclin1 were detected by western blot assay. RESULTS AND CONCLUSION: The scores of acute graft-versus-host disease and intestinal mucosa pathology grade in the treatment group were significantly better than those in the model group. Under transmission electron microscope, there were autophagic vesicles in the model group, but the mitochondrial structure was seriously damaged. In the treatment group, there were more autophagic vesicles and the mitochondrial structure was relatively intact. The level of reactive oxygen species in small intestinal epithelial cells in the treatment group was lower than that in the model group (P < 0.01). The expression level of LC3II/I and Beclin1 in the treatment group was significantly higher than that in the model group (P < 0.01). The results showed that baicalin could reduce the level of reactive oxygen species, increase the autophagy level of intestinal mucosal cells, protect intestinal mucosal barrier, and reduce the incidence rate of acute graft-versus-host disease after transplantation.

Third-party regulatory T cells prevent murine acute graft-versus-host disease

BACKGROUND/AIMS: Adoptive therapy with regulatory T (Treg) cells to prevent graft-versus-host disease (GVHD) would benefit from a strategy to improve homing to the sites of inflammation following hematopoietic stem cell transplantation (HSCT). Although donor-derived Treg cells have mainly been used in these models, third-party-derived Treg cells are a promising alternative for cell-based immunotherapy, as they can be screened for pathogens and cell activity, and banked for GVHD prevention. In this study, we explored major histocompatibility complex (MHC) disparities between Treg cells and conventional T cells in HSCT to evaluate the impact of these different cell populations on the prevention of acute GVHD, as well as survival after allogeneic transplantation. METHODS: To induce acute GVHD, lethally irradiated BALB/c (H-2d) mice were transplanted with 5 × 10⁵ T cell-depleted bone marrow cells and 5 × 10⁵ CD4+CD25– splenic T cells from C57BL/6 (H-2b) mice. Recipients were injected with 5 × 10⁵ cultured donor-, host-, or third-party-derived CD4+CD25+CD62L+ Treg cells (bone marrow transplantation + day 1). RESULTS: Systemic infusion of three groups of Treg cell improved clinicopathological manifestations and survival in an acute GVHD model. Although donor-derived Treg cells were immunologically the most effective, the third-party-derived Treg cell therapy group displayed equal regulation of expansion of CD4+CD25+- Foxp3+ Treg cells and suppressive CD4+IL-17+ T-helper (Th17) cells in ex vivo assays compared with the donor- and host-derived groups. CONCLUSIONS: Our findings demonstrate that the use of third-party Treg cells is a viable alternative to donor-derived Treg cellular therapy in clinical settings, in which human leukocyte antigen-matched donors are not always readily available.

Relationship of aGVHD with Treg, NK cells as well as their related cytokines in mice with allogeneic bone marrow transplantation / 西安交通大学学报(医学版)

Objective To investigate the relationship of acute graft versus host disease (aGVHD) and the regulatory T (Treg) cells,NK cells as well as their function-related cytokines such as IL-10,TGF-β,and perforin in mice with allogeneic bone marrow transplantation.Methods H-2 completely mismatched C57BL/6→ BALB/c aGVHD mice model was constructed.Flow cytometry analysis was used to detect the proportion of CD4+CD25 + Treg cells and NK-1.1+ NK cells in the spleen of aGVHD mice after transplantation.ELISA method was used to detect the serum levels of IL-10,TGF-β and perforin in the aGVHD mice intervened with CSA prophylactic or not.The normal C57BL/6 mice were used as controls.Results Compared with those of normal mice,the proportion of Treg cells in aGVHD mice after transplantation was decreased (mean 3.6％ vs.1.55％) and the proportion of NK cells increased (mean 3.3％ vs.11.5％).In the aGVHD mice treated with cyclosporine A,serum IL-10 expression level was significantly increased (treated group (125.79 ± 0.27)pg/mL,untreated group [(103.09 ± 3.27)pg/mL,P＜0.01)],TGF-β expression level was increased [(252.05 ±7.84)pg/mL vs.(241.61±15.41)pg/mL,P＞0.05],perforin expression level was significantly increased [(186.97 ± 4.68)pg/mL vs.(144.35 ± 14.42)pg/mL,P＜0.01].Conclusion ① The occurrence of aGVHD is correlated with the decreased number of Tree cells after transplantation in mice.Treg cell function-related cytokines IL-10 and TGF-β are involved in the treatment of aGVHD by cyclosporine A-mediated immunosuppression.②NK cells are involved in the occurrence of aGVHD after allogeneic bone marrow transplantation,and the increased level of perforin is related to the inhibition of aGVHD.

Relationship between the methylation status of STAT3 promoter DNA in peripheral blood CD4+ T cells from patients after allo-HSCT and aGVHD / 中南大学学报(医学版)

Objective:To study the relationship between acute graft versus host disease (aGVHD) and the methylation status of the STAT3 promoter in peripheral blood CD4+ T cells from patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT).Methods:We collected 40 patients who underwent allo-HSCT from HLA-identical sibling donors.Serum IL-10,TGF-β1,IL-17A and IL-17F levels were detected by ELISA.Foxp3 cytotoxic T-lymphocyte-associated protein 4 (CTLA4),IL-10,TG F-β 1,RO Rγt,IL-17A and IL-17F mRNA levels in CD4+ T cells were measured by real-time PCR.STAT3 expression levels were detected by real-time PCR and Western blot,and promoter DNA methylation was analyzed by bisulfite sequencing PCR (BSP).Results:IL-10 and TGF-β1 levels were significantly down-regulated,while IL-17A and IL-17F levels were significantly up-regulated in patients with aGVHD compared with patients without aGVHD.Foxp3,CTLA4,IL-10,TGF-β1 mRNA levels were significantly down-regulated,while RORγt,IL-17A,IL-17F mRNA levels were significantly up-regulated in patients with aGVHD compared with patients without aGVHD.STAT3 expression was increased,while STAT3 promoter DNA was hypomethylated in patients with aGVHD compared with those without aGVHD.The STAT3 mRNA level was negatively correlated with STAT3 promoter DNA methylation.Conclusion:The imbalance of Treg/fh17 in CD4+ T cells from patients after allo-HSCT is a key factor for triggering aGVHD,and the DNA hypomethylation of STAT3 promoter could promote its expression in CD4+ T cells and contribute to the imbalance.

Establishment of murine animal model for acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation / 局解手术学杂志

Objective To establish the murine animal model for acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation (HSCT). Methods T cell-depleted (TCD) BM cells from allogeneic C57BL/6 mice were harvested and prepared from the marrow of the femurs and tibias. Age of 4 to 6 weeks’ BALB/c mice received 900 cGy total body irradiation ( TBI) from a 137Cs source. Two to four hours later,the mice were injected intravenously ( i. v. ) 5 × 106 TCD-BM cells with 1. 25 × 106 or 2. 5 × 106 or 5 × 106 splenic cells from allogeneic C57BL/6 mice,and respectively divided them into experimental group 1,experimental group 2 and experimental group 3. TCD-BM only was used as the negative control group of GVHD. Ten mice were used for each group. The establishment of aGVHD model was evaluated with a clinical GVHD scoring system,survival rate and target-organ damage. Results On 7 to 14 days after transplanta-tion,the typical clinical GVHD manifestation of severe diarrhea,hogback,activity reduced and ruffling were observed in experimental group 2. Furthermore,the aGVHD target organs of colon,lung and liver were harvested and made histological paraffin sections,then the obviously path-ological tissue damages of GVHD were detected under microscope. And the survival rate was lowed down to 0 on 45 days after transplantation in experimental group 2. On the contrary,no obviously clinical manifestation of aGVHD were observed in the control group,experimental group 1 and group 3. On 60 days later, the survival rate was 80% in experimental group 1 and 100% in control group. However,no mice was sur-vived on 10 days later in group 3. Conclusion BALB/c mice aGVHD model after allogeneic HSCT is successfully established by injecting i. v. 5 × 106 TCD-BM cells with 2. 5 × 106 splenic cells from allogeneic C57BL/6 mice.

Relative tolerance of lung in murine model of acute graft-versus-host disease / 中华器官移植杂志

Objective To analyze the immune privilege of lung in acute graft-versus-host disease (aGVHD).Methods The models of aGVHD were established,and C57BL/6J→C57BL/6J model was used as control.The clinical scores and survival were observe& The pathological injuries were compared between the lung and traditional target organs (liver,small intestines and skin).The expression of IFN-γ in different organs after transplantation was detected by ELISA.Results Allogeneic hematopoietic stem cell transplantation (HSCT) mice had high 42-day survival rate (20 ％) post-transplantation,and recipients of allogeneic grafts showed classical symptoms and histological injury,and pathological changes of lung were not as serious as the liver,small intestine,skin at day 28 after transplantation.Syngeneic mice all survived at day 42 after transplantation,without GVHD symptoms and pathological changes.The mice in MHC-disparate mice (2 ∶ 1 and 4 ∶ 1 groups) died significantly faster at a median of 12 days after transplantation,with severe changes of clinical symptoms and pathology of classical organs,and MHC-disparate mice (4∶1 groups) had developed severe interstitial pneumonitis.The mean IFN-γ concentration in the lung of allogeneic HSCT mice was obviously increased in the first and second week after transplantation,the IFN-γ concentrations of target organs (liver,small intestines,skin) were slightly increased in the first and second week after transplantation,and there were statistically significant difference from lung (P＜0.05).Condmion There wasrelativeimmune privilege of lung in a murine model of aGVHD induced by HSCT,which was associated with the expression of MHCin the mice and IFN-gamma of lungs after transplantation.

Expression changes of CD47 on peripheral blood T-lymphocytes in aGVHD after allo-HSCT / 中华器官移植杂志

Objective To detect the change of CD47 on T-lymphocyte subsets marked by CD3 + CD4+ and CD3+ CD8+ in the acute graft versus host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and to explore its clinical significance.Methods The peripheral blood of 30 patients was collected before and every two weeks from hematopoietic reconstitution after allo-HSCT.The expression of CD47 on T-lymphocyte subsets marked by CD3+ CD4+ and CD3+ CD8+ was detected by flow cytometry,and the relationship between their expression and the occurrence of aGVHD was analyzed.Results The percentage of CD3+ CD4+-and CD3+ CD8+-marked T-lymphocyte subsets showed no statistically significant difference between aGVHD group and non-aGVHDgroup [(38.95 ± 6.18)％ vs.(37.38 ± 4.6)％,and (22.35 ± 3.32)％ vs.(20.34 ±4.56) ％,respectively] (P＞0.05).The expression levels of CD47 on T-lymphocyte subsets marked by CD3+ CD4+ and CD3+ CD8+ was obviously increased in aGVHD group when compared to before transplantation and non-aGVHD group after transplantation,and that had no changes in non-aGVHD group when compared to before conditioning regimen after transplantation.Mean fluorescence intensity of CD47 on T-lymphocyte subsets marked by CD3+ CD4+ and CD3+ CD8+ [(93.70 ± 14.88) and (70.09 ± 12.51)] in aGVHD group significantly increased as compared with non-aGVHD group [(71.27-± 11.32) and (53.93 ± 8.35)] (P＜0.05).There was no significant difference in the expression of CD47 on T-lymphocyte subsets marked by CD3+ CD4+ and CD3+ CD8+ before and after transplantation in non-aGVHD group (P＞0.05).Moreover the expression of CD47 on T-lymphocyte subsets marked by CD3 + CD4 + and CD3 + CD8 + changed correspondingly with the outcome of aGVHD in aGVHD group.Conclusion The change of CD47 on T-lymphocyte subsets marked by CD3+ CD4+ andCD3+ CD8+ is valuable to monitor the aGVHD after allo-HSCT,which may provide a new means for the early detection of aGVHD.

Angiogenic factors are associated with development of acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation / 华中科技大学学报（医学）（英德文版）

Acute graft-versus-host disease (aGVHD) is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the mechanisms of aGVHD are not well understood. We aim to investigate the roles of the three angiogenic factors: angiopoietin-1 (Ang-1), Ang-2 and vascular endothelial growth factor (VEGF) in the development of aGVHD. Twenty-one patients who underwent allo-HSCT were included in our study. The dynamic changes of Ang-1, Ang-2 and VEGF were monitored in patients before and after allo-HSCT. In vitro, endothelial cells (ECs) were treated with TNF-β in the presence or absence of Ang-1, and then the Ang-2 level in the cell culture medium and the tubule formation by ECs were evaluated. After allo-HSCT, Ang-1, Ang-2 and VEGF all exhibited significant variation, suggesting these factors might be involved in the endothelial damage in transplantation. Patients with aGVHD had lower Ang-1 level at day 7 but higher Ang-2 level at day 21 than those without aGVHD, implying that Ang-1 may play a protective role in early phase yet Ang-2 is a promotion factor to aGVHD. In vitro, TNF-β promoted the release of Ang-2 by ECs and impaired tubule formation of ECs, which were both weakened by Ang-1, suggesting that Ang-1 may play a protective role in aGVHD by influencing the secretion of Ang-2, consistent with our in vivo tests. It is concluded that monitoring changes of these factors following allo-HSCT might help to identify patients at a high risk for aGVHD.

Effect of granulocyte colony-stimulating-factor on acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation in a mu-rine model / 中国病理生理杂志

AIM:To explore the impact of granulocyte colony-stimulating factor (G-CSF) on acute graft-ver-sus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in a murine model and its possible mechanisms.METHODS:Male C57BL/6 (H-2b) and BALB/c (H-2d) mice were used as the allogeneic and syngeneic donor mice , respectively .Moreover , female BALB/c mice were used as recipient mice .The recipient mice were conditioned by a single dose ( 8 Gy ) of total body irradiation ( TBI ) .The recipient mice were randomly divided into 7 groups:TBI group, Syn-BMST control group, post-Syn-BMST G-CSF administration (Syn-BMST+G-CSF) group, allo-BMT control group, post-allo-BMT G-CSF administration (allo-BMT+G-CSF)group, allo-BMST control group and post-al-lo-BMST G-CSF administration (allo-BMST+G-CSF) group.The mice in control groups and G-CSF administration groups were subcutaneous injected with 0.1 mL normal saline (NS) and 0.1 mL NS containing 2μg G-CSF per day from 1st day, respectively.The effect of G-CSF on aGVHD was evaluated by clinical manifestations and pathological changes , as well as survival time of the mice in different groups .The serum levels of IL-2, IL-4, IFN-γand TNF-αin allo-BMST and allo-BMST+G-CSF groups were detected by ELISA at 10th day.Flow cytometry was used to analyze the immunophenotypes of splenocytes at 10th day.RESULTS:The mice in TBI group were all died for hematologic failure on 9~15 d after TBI.No effect of G-CSF on the survival of the mice underwent Syn-BMST and transplantation of single allogeneic marrow cells was observed.The mean survival days in allo-BMST group and allo-BMST+G-CSF group were (34.8 ±4.5) d and (19.8 ± 6.1) d’respectively (P<0.01).Moreover, post-transplant administration of G-CSF increased the spleen total nucleated cells count (SpTNC), NK cells subset, and DC1/DC2 ratio in the spleen with over 99%of donor chimerism rate at 10th day.No difference in the levels of serum IL-2, IL-4, IFN-γand TNF-αbetween the 2 group at 10th day was found.CON-CLUSION:The administration of G-CSF after allo-BMST significantly aggravates mouse aGVHD .The expansion of NK cells stimulated by G-CSF may be involved in the mechanism of generating alloreactivity against host cells .These results imply there may be potential risk of evoking or aggravating acute GVHD if G-CSF is administered in the early stage of clini-cal allo-HSCT.

Relationship between helper T cell 17/regulatory T cells balance and acute graft versus host disease after allogeneic hematopoieticstem cell transplantation / 中华器官移植杂志

Objective To investigate the relationship between the helper T cell 17 (TH 17)/ Regulatory T cells (Treg cells) balance in peripheral blood with acute graft-versus-host reaction (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT),as well as the impact of anti-thymocyte immunoglobulin (ATG) on helper T cells in peripheral blood.Method Seventyeight hematologic patients underwent allo-HSCT,conditioning with or without ATG.Ten healthy volunteers severed as a control group.The helper T and regulatory T cells in peripheral blood were detected by flow cytometry.Enzyme-linked irnmunosorbent assay (ELISA) was used to detect serum concentrations of interleukin(IL)-17,IL-21,IL22,IL23,γ interferon (IFN-γ),and transforming growth factor β1 (TGF-β1).Result The percentage of Treg cells,TH17 cells and ratio of TH17/Treg cells in patients without aGVHD showed no significant difference from the healthy controls (P＞ 0.05).As compared with control group and non aGVHD group,the ratio of Treg cells was increased,the percentage of TH 17 cells,and TH 17/Treg cells were significantly increased in 1-2-degree aGVHD group (P＜0.01).With increased degree of aGVHD,the difference as above was more significant in 3-4-degree aGVHD recipients (P＜0.01).In aGVHD group,the IL-17,IL-23,IL-21 and IFN-γ concentrations were higher than the healthy group (P＜0.01) and non-aGVHD group (P＜0.05).Serum TGF-β1 level in aGVHD group was significantly decreased as compared with healthy group and non-GVHD group (P＜0.05),while IL-22 concentrations showed no statistically significant difference among three groups (P＞0.05).In anti-thymocyte immunoglobulin (ATG) pretreatment group,the absolute count of peripheral blood lymphocytes was less than in healthy control group (P＜0.01).In ATG group,the absolute counts of TH1 cells,TH17 cells,CD3+ CD4+ cells and non-TH1/17 cells were less than in non-ATG group (P =0.0000),while the absolute counts of lymphocytes,CD3+ CD4-cells,and TH 1/17 cells were less than in non-ATG group,but there was significant difference (P＞0.05).Conclusion The balance of TH 17/Treg cells and related cytokines were closely associated with aGVHD after allo-HSCT,and ATG influences the reconstruction of TH 17 and Th1 cells at early stage.

Mesenchymal stem cell-induced CD8α+Jagged2high CD11bhigh regulatory dendritic cells prevent and treat mouse aGVHD / 中国病理生理杂志

AIM:To investigate the role of mesenchymal stem cell-induced regulatory dendritic cells ( MSC-DCregs) in mouse acute graft-versus-host disease( aGVHD) model.METHODS: Bone marrow cells from BALB/c ( H-2 d ) mice were isolated and were induced to differentiate into DCs.The DCs were selected by flow cytometry, and after 10 d co-culture with MSCs, they were induced to be MSC-DCregs.Male 8-week-old C57BL/6 (H-2b) mice were used as do-nor mice.The female 8-week-old BALB/c (H-2d) mice, who had received 100 cm source-skin distance, 30 cm ×30 cm radiation field, 700 cGy total body irradiation (TBI) pretreatment were used as recipient mice.The recipients were divided into 5 groups:control group, TBI group ( injected with medium only) , bone marrow transplantation group ( injected with 1 ×107 bone marrow cells), aGVHD group (injected with 1 ×107 bone marrow cells and 1 ×107 spleen cells), and MSC-DCregs group (injected with 1 ×107 bone marrow cells, 1 ×107 spleen cells and 1 ×106 MSC-DCregs).The white blood cell count, recipients’ chimerism, clinical evaluation of aGVHD, survival analysis and pathological changes were deter-mined.RESULTS:Hematopoieic recovery was seen at 10 d after transplantation.The recipients’ chimerism was parallel to the donors’ at 30 d.The median survival time of the mice in aGVHD group and MSC-DCregs group was 27 d and 33 d, and the survival rates at 30 d were 20% and 100% (P<0.01), respectively.The clinical scores of the mice in MSC-DCregs group were lower than those in aGVHD group ( P<0.01) .Moreover, the pathological changes in the skin and liver of the mice in MSC-DCregs group were less serious than those in aGVHD group.CONCLUSION: The MSC-DCregs in-duce an aGVHD tolerance in vivo, and further research of its mechanism is still in great necessary.