| Objective This study aims at understanding the quantity and functional markers expression of regulatory T cells(Tregs) between severe aplastic anemia(SAA) patients and normal people, establishing an in vitro cell culture system, and evaluating the proliferation ability and ex-vivo suppressive function of Treg cells in SAA patients.Methods The SAA patients who were previously untreated, recovering SAA patients(R-SAA), and normal controls in Tianjin Medical University General Hospital from January 2014 to January 2016 were included in the study. Part I Circulating CD4~+ CD25~+ CD127 dim Tregs of untreated SAA patients, recovering SAA patients, and normal controls were identified by flow cytometry. The expression of biomarkers on Treg cell surface, including CTLA-4, Perforin, CD39, CD73, GITR and LAT were analyzed based on isotype control with mouse Ig G1. Correlation of Tregs quantity and functional biomarkers expression with clinical parameters were analyzed. Part II CD4~+CD25~+CD127dim Treg subsets were sorted from the blood and bone marrow of SAA patients and normal controls, and expanded in vitro in the presence of anti-CD3/CD28 beads with high concentration of recombinant human interleukin 2(r IL-2). Their phenotype was determined by flow cytometry. Mixed lymphocytes culture with Teff were performed to assess the function of ex-vivo expanded Treg cells. Culture supernatants obtained from Tregs proliferation and suppression assays were analyzed for IL-10 and IFN-γ by ELISA. Part III The Tregs were sorted by magnetic activated cell sorting system(MACS), and the mRNA expression of CTLA-4 were analyzed by Quantitative real-time PCR. Bone marrow mononuclear cells from SAA patients were cultured and induced became myeloid dentritic cells. After that, the DC was co-cultured with Tregs from SAA or normal controls. The expression of CD80 and CD86 on DCs were detected by flow cytometry. Part IV The expression of TRAIL on Treg cells was analyzed by flow cytometry. Tregs were sorted by MACS, and the mRNA expression of TRAIL were analyzed by Quantitative real-time PCR.Results Part I Patients with SAA had a significant decreased frequency of Tregs, and a reduction of CTLA-4 expression and an increment of perforin expression in Treg compared with healthy controls, while expression of CD39, CD73, GITR and LAT in Tregs from SAA patients is not changed compared with Tregs from healthy controls. The quantity of Tregs was positively correlated with therapeutic effect. Part II The Treg cells were expanded to approximately 10-fold in 1-2 weeks and retained the Treg cell phenotypic characteristics. Tregs from SAA patients even have more powerful proliferation ability than that from normal control. Furthermore, the ex-vivo expanded Tregs have a high expression of functional molecular marker CTLA-4. In ex-vivo immunosuppression experiment, the expansion rate of Teff from the four co-culture groups, including(control Teff: control Treg),(control Teff: SAA Treg),(SAA Teff: control Treg) and(SAA Teff: SAA Treg), did not show any significant difference. The IFN-γ secretion by Teff was suppressible by expanded Tregs from SAA patients. Part III The relative CTLA-4 mRNA level of Tregs from SAA, R-SAA and normal control group were(1.32 ± 1.05),(1.00 ± 1.46) and(1.73 ± 1.42), respectively. Wherein the R-SAA group was significantly lower than normal control(P <0.05). The SAA group was a little higher than R-SAA and lower than normal control, without significant difference. CD80/CD86 expression of SAA m DC cultured alone was 20.43%/ 47.80%. While SAA m DC: SAA Treg co-cultured group was 18.33%/ 43.93%, and SAA m DC: normal Treg group was 19.59%/ 45.98%. After co-cultured with Treg, CD80/CD86 expression of SAA m DC was slightly decreased. Part IV The expression of TRAIL in Treg cells from SAA patients was(23.83 ± 12.19)%, which was slightly higher than R-SAA group(19.71 ± 10.09)% and normal control(20.09 ± 9.57)%. The relative TRAIL mRNA level of Tregs from SAA, R-SAA and normal control group were(44.07 ± 81.56),(108.51 ± 253.19) and (15.50 ± 36.23), respectively.Conclusions(1) All of SAA patients had significantly decreased quantities of Tregs compared with normal controls, no matter the percentage and absolute Tregs numbers. Moreover, the quantities of Tregs in SAA patients improved significantly after intensive immune suppressive treatment(IST).(2) In SAA patients, CTLA-4 could be responsible for Treg abnormalities, but suppression mediated by perforin, CD39, CD73 and GITR, and survival capability of single Treg cell may not be injured. Just because of the inadequate number of whole Treg cells, suppression of whole Tregs in SAA patients cannot be made up to normal condition.(3) CD4~+ CD25~+ CD127 dim Treg cells from SAA patient can be sorted and expanded successfully in vitro, and used efficiently to suppress effective T cells proliferation, paving the way for future clinical trials.(4) The relative CTLA-4 mRNA level of Tregs from SAA, R-SAA and normal control group has the same tendercy with CTLA-4 protein expression. After co-cultured with Treg, CD80/CD86 expression of SAA m DC was slightly decreased, which need more data to support.(5) In the pathogenesis of SAA, apoptosis mechanism mediated by TRAIL of Treg cell may not be injured. |
PDF Full Text Request