Objective
To establish a method to isolate the CD4+CD25+ regulatory T cells (Tregs) and to identify the purity and function of these cells.
Methods
The peripheral blood (8 mL) were collected from the great saphenous vein of 10 rhesus monkeys (4 females and 6 males, aged 4-5 years, and weighing 5-8 kg). The mononuclear cells were isolated with density gradient centrifugation. CD4+ T cells were separated by the Magnetic cell sorting (MACS) negative selection and MACS positive selection. The cell yield rate, the cell viability, and the cell purity were compared between MACS negative selection and MACS positive selection. In CD4+ MACS negative selection, the anti-biotin MicroBeads and biotin-antibody cocktai in CD4+CD25+ Tregs isolation kit non-human primate were used, and in MACS positive selection, the anti-APC MicroBeads in CD4+CD25+ Tregs isolation kit non-human primate and CD4-APC were used. The CD4+ T cells separated by positive selection were selected to obtain CD4+CD25 Tregs with CD25 MicroBeads. The purity, activity, the FoxP3 level, and the suppressive function to concanavalin A (ConA) activated autologous CD4+CD24- effective T cells (Teffs) of CD4+CD25+ Tregs were detected by flow cytometry.
Results
After CD4+ T cells were separated by MACS negative selection and MACS positive selection, the cell viabilities were all up to 95%, showing no significant difference (P gt; 0.05). The cell yield rate and purity of CD4+ T cells by positive selection were significantly higher than those of CD4+ T cells by negative selection (P lt; 0.05). CD4+CD25+ Tregs can be successfully isolated by MACS double positive selection. The classifying purity was 76.2% ± 8.6%; the cell activity was 93.3% ± 4.7%; and the level of FoxP3 was 74.2% ± 6.9%. The CD4+CD25+ Tregs had suppressive effect on ConA activated autologous CD4+CD25- Teffs.
Conclusion
MACS double positive selection can be used to isolate high-purity CD4+CD25+ Tregs from the peripheral blood of rhesus monkeys and the process does not influence the activity of CD4+CD25+ Tregs.
Objective To investigate the percentage of CD4 + CD25 + Treg cells and expression of Foxp3 mRNA in asthmatic patients and the impacts of inhaled steroids.Methods The percentages of CD4 +CD25 + Treg cells was assayed by flow cytometry and the expression of Foxp3 mRNA was detected by RT-PCR in peripheral blood mononuclear cells from the patients with chronic persistent asthma before and after steroids inhalation in comparison with healthy control. The forced expired volumin one second/predicted value( FEV1% pred) and peak expired flow( PEF) were measured by spirometry. Results The level of CD4 + CD25 + Treg cells and the expression of Foxp3 mRNA were lower in asthmatics before steroids treatment than those in control ( P lt; 0. 05) which were increased significantly after steroids treatment ( P lt; 0. 05) .FEV1% pred and PEF were declined significantly than those in control but improved markedly after treatment ( P lt; 0. 05) . Conclusions The insufficiency of amount and function of immue-suppressive CD4 + CD25 +Treg cells may play a role in the pathogenesis of asthma. Inhaled steroids can improve the lung function of asthmatics by upregulating the level of CD4 + CD25 + Treg and Foxp3.
【 Abstract】 Objective To construct a lentiviral expression vector carrying Nogo extra cellular peptide residues 1-40(NEP1-40) and to obtain NEP1-40 efficient and stable expression in mammalian cells. Methods The DNA fragment ofNEP1-40 coding sequence was ampl ified by PCR with designed primer from the cDNA l ibrary including NEP1-40 gene, and then subcloned into pGC-FU vector with in-fusion technique to generate the lentiviral expression vector, pGC-FU-NEP1-40. The positive clones were screened by PCR and the correct NEP1-40 was confirmed by sequencing. Recombinant lentiviruses were produced in 293T cells after the cotransfection of pGC-FU-NEP1-40， and packaging plasmids of pHelper 1.0 and pHelper 2.0. Green fluorescent protein (GFP) expression of infected 293T cells was observed to evaluate gene del ivery efficiency. NEP1-40 protein expression in 293T cells was detected by Western blot. Results The lentiviral expression vector carrying NEP1-40 was successfully constructed by GFP observation， and NEP1-40 protein expression was detected in 293T cells by Western blot. Conclusion The recombinant lentivirus pGC-FU-NEP1-40 is successfully constructed and it lays a foundation for further molecular function study of NEP1-40.
Objective
To summarize research progress of the mechanism of natural killer cells (NK cells) acted in regulating the T cell immunity in chronic infectious disease.
Method
Literatures about recent studies concerning how NK cells act as a regulator for T cells in chronic infectious disease were reviewed according to the results obtained from PubMed, Embase, CNKI, CBM, and Wanfang databases.
Results
NK cells that acted as regulators of T cell immunity could affect T cell immune responses through influencing antigen presentation, secreting cytokine, and presenting lytic activities, thus playing an important role in the immunological therapy of chronic infectious diseases.
Conclusion
NK cells are critical for T cell immune regulation, which could provide noval strategies for immunological therapy of chronic infectious disease, transplantation-related immune rejection, and autoimmune disease.
Objective
To investigate the influence of T helpers 17 (Th17) cells, regulatory T (Treg) cells and their related cytokines on postoperative atrial fibrillation (POAF) after coronary artery bypass graft (CABG).
Methods
A total of 132 consecutive patients undergoing CABG between May 2013 and July 2016 were recruited. There were 82 males and 50 females with the age ranging from 39-76 years. Venous blood samples were collected within 2 hours after surgery. The expression of Th17 cells, Treg cells and their related cytokines in the peripheral blood was determined.
Results
POAF occurred in 35 patients (a POAF group) and 97 patients did not develop POAF (a No POAF group). Compared to the No POAF group, the proportion of Th17 cells and Th17/Treg ratio in the peripheral blood significantly increased in the POAF group (P>0.05) while proportion of Treg cells remained no significant change (P>0.05). The expression of Th17-related cytokines (IL-6, IL-8 and IL-17) all obviously increased in the POAF group (P>0.05). However, no significant difference was found in the expression of Treg-related cytokines (IL-10 and TGF-β) between the two groups (P>0.05).
Conclusion
Th17/Treg is unbanlanced in POAF patients and regulation of this imbalance may decrease the occurrence of POAF.
Objective
To compare the difference in the expressions of forkhead box protein 3 (FoxP3) and adenosine 2a receptor (A2aR) in gastric cancer tissues and its adjacent tissues, and to investigate the relationship between the elevated expression of FoxP3/A2aR and clinicopathological features in gastric cancer.
Methods
Gastric cancer tissues and their adjacent tissues from 52 patients with gastric cancer were collected, who underwent surgery in the Affiliated Hospital of Xuzhou Medical University from July 2015 to November 2016, immunohistochemical staining was used to detect the expressions of FoxP3 and A2aR.
Results
① The high-expression rate of FoxP3 in gastric cancer tissues was 69.2% (36/52), which was higher than that of adjacent tissues (11.5%, 6/52), P<0.001. The high-expression rate of A2aR in gastric cancer tissues was 69.2% (36/52), which was higher than that of adjacent tissues (25.0%, 13/52),P<0.001. ② The expression of FoxP3 was positively correlated with the expression of A2aR in gastric cancer tissues (r=0.76, P<0.05). ③ In gastric cancer tissues, high-expressions of FoxP3 and A2aR were not related to gender, age, diameter of tumor, tumor location, degree of differentiation, gross type, and histological type (P>0.05), but both associated with TNM stage, T stage, number of lymph node metastasis, and distant metastasis (P<0.05), the high-expression rates of FoxP3 and A2aR in patients with stage Ⅲ+Ⅳ were higher than those of patients with stage Ⅰ+Ⅱ, the high-expression rates of FoxP3 and A2aR in patients with stage T3+T4 were higher than those of patients with stage T1+T2, the high-expression rates of FoxP3 and A2aR in patients with distant metastasis were higher than those of patients without distant metastasis, and the high-expression rates of FoxP3 and A2aR increased gradually with the increase in the number of lymph node metastasis.
Conclusion
There are high expressions of FoxP3 and A2aR in gastric cancer tissues, and both of them may play important role in promoting the occurrence and development of gastric cancer.
ObjectivesTo systematically review the clinical response rate of CD19 chimeric antigen receptor modified-T cells (CD19CART) in the treatment of B cell hematological malignancies.MethodsPubMed, EMbase, CNKI, WanFang Data and VIP databases were searched to collect cohort studies about CD19CART in the treatment of B cell hematological malignancies from 2000 to 2016. Two reviewers independently screened literature, extracted data and assessed the risk of bias of included studies. Then, a single rate meta-analysis was performed by R software and SPSS 16.0 software.ResultsA total of 13 prospective cohort studies were included. The results of single group rate meta-analysis showed that the overall pooled response rate of CD19 CART was 68% (95%CI 0.51 to 0.82). The 6 months and 1-year PFS after CD19 CART infused by Kaplan-Meier were 46% (95%CI 0.35 to 0.56) and 24% (95%CI 0.16 to 0.34), respectively. The median duration was 180 days (95%CI 138 to 222). The COX regression model showed lymphodepletion to be the only influence factor of PFS.ConclusionsCD19 CART has a good clinical response rate in the treatment of B cell hematological malignancies. Lymphodepletion is the only important impact on the response rate and PFS. Due to limited quality and quantity of included studies, more high quality studies are required to verify the above conclusions.
The pathogenesis of Vogt-Koyanagi Harada disease (VKH) has not yet been fully defined. Current studies mainly suggest that VKH is actually an autoimmune disease, especially related to the immune response mediated by various signal transduction pathways involved in the function of T cells. In recent years, the influence of the balance imbalance of various T cell subsets in cellular immunity on the pathogenesis of VKH has been a hot research direction. Currently, T helper cell 17/T regulatory cells, balance is the focus of clinical research, meanwhile, new discoveries and potential clinical treatment schemes have been made for related cellular pathways, particularly the Janus kinase/signal transducers and activators of transcription pathway and NF-kappa B pathway. The exploration of B cells in the pathogenesis of VKH has also achieved initial results through the successful application of various targeted drugs. In the future, further screening and localization of genes or proteins that are abnormally regulated or expressed in VKH, for which early comprehensive and in-depth exploration will be helpful, thus improve the efficacy of clinical treatment programs and develop new therapeutic targets.
Objective To assess the effects of different immunosuppressive drugs on proliferation and function of regulatory T cells (Tregs). Methods We searched MEDLINE (1966 to November 2009), EMbase (from inception to September 2009), and The Cochrane Library (Issue 4, 2009) for clinical and basic research about the effects of various immunosuppressive drugs on Tregs. Data were extracted and methodological quality was assessed by two independent reviewers. Outcome measures for clinical research included blood Tregs levels, acute rejection episodes, and graft function. Outcome measures for basic research included percentage of Tregs proliferation, function, Tregs phenotype, and evidence for possible mechanisms. We analyzed data qualitatively. Results Forty-two studies, including 19 clinical trials and 23 basic studies, were included. The immunosuppressive drugs studied were calcineurin inhibitors (CNIs), Rapa, anti-metabolism drugs, IL-2 receptor-blocking antibodies, T-cell depleting antibodies, and co-stimulation blockade antibodies. Most of the studies were on Rapa and CNIs. Eight basic studies on Rapa and CNIs showed that Rapa could promote the proliferation and function of Tregs, while CNIs could not. Five clinical trials involving a total of 158 patients showed that patients taking Rapa had higher blood concentration of Tregs than those taking CNIs, but no differences were found in graft function (6-42-month follow-up). Conclusion There is substantial evidence that Rapa favors Tregs survival and function. However, the larger number of the blood Tregs in the patients treated with Rapa does not show any correlation with better graft function. Large-sample and high-quality clinical studies with longer follow-up are needed to thoroughly assess the efficacy of immunosuppressive drugs on Tregs and to reveal whether a relationship exists between Tregs and graft function.
Regulatory T cells (Treg) are critical for regulation of tolerance, control immune responses to self-antigens thereby preventing autoimmunity, and limiting responses to foreign antigens thereby minimizing T cell-mediated immunopathology. Recent data indicate that suppression of organ-specific autoimmunity is dependent on the antigen specificity of Treg. An emerging model of Treg action is that organ-specific Treg acquire suppressive activity through activation by dendritic cells expressing specific antigens. Thus, the efficacy of Treg-based therapy should be increased by using antigen-specific Treg rather than polyclonal Treg. It is necessary to identify relevant antigens and to expand antigen-specific Treg from polyclonal populations. Here, we discuss recent techniques for expansion of antigen-specific Treg, function and antigen specificity of Treg and the therapeutic potential of Treg in controlling autoimmune disease and inducing transplant tolerance.
