Objective To study the changes of the expression of adhesion molecules on peripheral blood CD34+ cells during recombinant human granulocyte colony stimulating factor(rhG-CSF) mobilization,and to study the influence of rhG-CSF on donors.Methods Fifteen healthy blood donors were subcutaneously injected with rhG-CSF(10?g?kg-1?d-1)for 4 to 6 days.The expressions of very late antigen 5(VLA-5,CD49e) and L-selectin(CD62L) on CD34+ cells were examined by flow cytometry before mobilization,the fourth day during mobilization and the seventh day after mobilization.Results The percentage of CD34+ cells,especially CD34+CD49e+ cells,increased significantly during mobilization,reaching the peak on the forth day,but declined to normal level when the mobilization stopped.The percentage of CD34+CD62L+ cells didn't show any significant change during the mobilization.Conclusion rhG-CSF could increase the percentage of CD34+CD49e+ cells in peripheral blood,but the percentage was reduced one week after the mobilization.rhG-CSF doesn't affect the percentage of CD34+CD62L+ cells.

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.

AIM:To investigate the expression and potential role of complement 5a receptor (C5aR) in chro-nic graft-versus-host disease (cGVHD).METHODS:The expression of C5aR on lymphocytes and the frequency of CD4+CD25+ Foxp3+ regulatory T cells (Tregs) in 20 cGVHD patients and 9 healthy donors was detected by flow cytometry.The correlation between the expression of C5aR and the percentage of Tregs in the cGVHD patients was analyzed.In addition, the splenocytes from the mice were cultured in vitro, and stimulated these splenocytes with recombinant mouse C5a protein (rmC5a).The peripheral blood mononuclear cells (PBMCs) from cGVHD patients were cultured in vitro, which was inhibited by C5aR antagonist (C5aRA).The frequency of Tregs in these splenocytes and the PBMCs were evaluated by flow cytometry.RESULTS:The expression of C5aR on the lymphocytes was significantly increased in the cGVHD patients compared with the healthy donors, while the percentage of Tregs was markedly lower in the cGVHD patients.The expression of C5aR was negatively correlated with the percentage of Tregs.Furthermore, the development of Tregs was suppressed by rmC5a stimulation, but was promoted by C5aRA in vitro.CONCLUSION:C5aR elevation is associated with Treg reduction in cGVHD, indicating that C5aR may play a potential role in suppressing Tregs, resulting in the incidence of cGVHD.

Objective: To investigate the effects of artesunate treatment on chronic graft-versus-host disease (cGVHD). Methods: Recipient BALB/c mice received 8 × 10⁶ bone marrow cells with 8×10⁶ spleen cells from B10D2 mice. Artesunate solubilized in acetone was injected intraperitoneally every day at the dose of 1 mg/kg at Day 28 after BMT. The clinical scores, survival and histopathological damage were analyzed. The frequency of Th17 and Tregs in PB and spleens from the mice were evaluated by flow cytometry. In addition, CD4⁺ T cells from the spleens of mice were cultured in vitro, then stimulated with artesunate, the frequency of Th17 and Tregs in these splenocytes were evaluated by flow cytometry. Results: Artesunate administration diminished clinical and histopathological damage, and improved the survival of cGVHD mice[(46.57±7.83)% vs (55.71±6.99)%, χ²=5.457, P=0.020]; Artesunate contributed to Tregs development [(4.45±0.04)% vs (8.40±0.23)%, t=15.679, P<0.001; (6.62±0.24)% vs (10.48±0.48)%, t=6.587, P=0.003] while decreased Th17 cells [(1.51±0.18)% vs (0.58±0.19)%, t=7.233, P<0.001; (1.48±0.38)% vs (0.71±0.18)%, t=3.653, P=0.011] expressions in both PB and spleens, and decreased the Th17/Treg ratio (0.34±0.05 vs 0.09±0.03, t=7.621, P=0.002; 0.19±0.03 vs 0.06±0.02, t=6.993, P=0.002). Moreover, artesunate suppressed the Th17 cells expressions [(0.82±0.37) % vs (3.39±1.22) %, t=4.044, P=0.007] and contributed to Tregs development [(34.63±1.29) % vs (14.28±1.69) %, t=19.119, P<0.001], and also decreased the Th17/Treg ratio (0.24±0.09 vs 0.02±0.01, t=4.780, P=0.003) in vitro. Conclusions Artesunate suppressed the Th17 cells expressions and contributed to Tregs development, which provided new sights into the development of a novel drug for cGVHD, e.g., artemisinin.

Objective To investigate the levels of γδ T cells and their subpopulations in bone marrow(BM)of patients with myelodysplastic syndrome(MDS),it aims to explore the immune deficiency status of BM microenvi-ronment in MDS patients.Methods BM samples were collected from MDS patients before and after treatment,as well as from normal donors.Multicolor flow cytometry was utilized to detect bone marrow γδ T cells and subpopulation levels.The changes of the T cell subsets after treatment were also analyzed.Results The levels of BM γδ T cells and follicular helper γδ T cells from MDS patients were significantly lower than those of normal donors(P<0.05).Among γδ T cells at different stages of differentiation,only the frequencies of na?ve γδ T cells from MDS patients decreased significantly(P = 0.037),and there was no significant difference observed about central memory,effector memory,and terminally differentiated γδ T cells in MDS patients compared to normal donors(P>0.05).Although there was a slight decrease in PD1+γδ T cells and an increase in TIM3+γδ T cells,these differences were not statistically significant(P>0.05).In patients who achieved a curative effect,the proportions of γδ T cells and naive γδ T cells increased significantly after treatment,and the effector memory γδ T cells decreased significantly after treatment(P<0.05).After treatment,85.71%(6/7)of MDS patients showed a decrease in γδ+TIM3+ T cell levels to varying degrees.Conclusions The levels of γδ T cells and their subpopulations in the BM microenvironment of patients with MDS exhibit varying degrees of abnormalities.However,in patients who receive effective treatment,these abnormal γδ T cells can recover.By detecting the levels of γδ T cells and subpopulations,we can gain insights into the immune deficiency status of MDS.This information might serve as an indicator to assess treatment efficacy and provide valuable insights for anti-tumor immunotherapy.

Objective: To monitor the WT1 mRNA level and its dynamic changes in patients with myelodysplastic syndromes (MDS) after hypomethylating agents (HMA) , as well as to assess the significance of WT1 mRNA levels and its dynamic changes in evaluating the efficacy of HMA and distinguishing the disease status of heterogeneous patients with stable disease (SD) . Methods: Bone marrow or peripheral blood samples of 56 patients with MDS who underwent hypomethylating agents (≥4 cycles) from November 2009 to March 2018 were tested by real-time quantitative polymerase chain reaction (PCR) to detect the expression of WT1 mRNA, and to observe the correlation between the dynamic changes of WT1 mRNA expression and clinical efficacy and prognosis of patients. Results: WT1 mRNA expression levels of MDS patients decreased significantly after 3 cycles of hypomethylating agent treatment. Besides, the WT1 mRNA expression levels of patients increased significantly after diseases progression. According to the dynamic changes of WT1 mRNA expression levels during SD, 45 cases could be further divided into increased group and non-increased group. In those SD patients with increased WT1 mRNA expression level, the ratio of suffering disease progression or transformation to AML was 95.65% (22/23) , whereas the ratio turned to be 9.09% (2/22) for the non-increased group (χ²=33.852, P<0.001) . Compared with those SD patients reporting no increase in WT1 mRNA expression level, the overall survival[17 (95%CI 11-23) months vs not reached, P<0.001] and progression-free survival [13 (95%CI 8-18) months vs not reached, P<0.001] of those SD patients reporting increase in WT1 mRNA expression level were significantly shorter. Conclusion WT1 mRNA expression level is a useful indicator to assess the efficacy of hypomethylating agents in MDS patients. Especially in patients with SD, detection of the changes in WT1 mRNA expression level is able to predict disease progression and help to make clinical decision.