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 relevance and changes of mucosal immunity in asthma rats’lung, nose and intestine. Methods Twenty Wistar rats were randomly divided into a normal group and an asthma group. Asthma rat model was established by sensitization and challenge with ovalbumin. CD4 + ,CD8 + , eotaxin protein and its mRNA in rats’lung tissues, rhinal and intestinal mucosa were measured by immunohistochemical methods and situ hybridization. The content of sIgA in bronchoalveolar lavage fluid ( BALF) , nasopharyngeal washings and intestinal mucus supernatant were detected by enzyme-linked immunosorbent assay. Results Compared with the normal group, the levels of CD4 + , CD8 + in rats’lung tissues, rhinal and intestinal mucosa, the expression of eotaxin protein and mRNA in rats’lung tissues, the content of sIgA in nasopharyngeal washing, and the expression of eotaxin protein in intestinal mucosa were significantly higher in the asthma group( P lt; 0. 05) . There were no significant differences of other indices between the two groups. In the normal group, the eotaxin protein expression had a negative correlationbetween lung tissue and rhinal mucosa( r = - 0. 572, P = 0. 008) , and a positive correlation between intestinal and rhinal mucosa( r=0. 638, P =0. 002) . The eotaxin mRNA expression had a positive correlation between lung tissue and rhinal mucosa( r= 0. 502, P = 0. 024) , and a positive correlation between intestinaland rhinal mucosa( r=0. 594, P =0. 006) . In the asthma group, such a correlation was not found except the eotaxin protein expression which had a negative correlation between lung tissue and intestinal mucosa( r =- 0. 448, P = 0. 048) . Conclusions Mucosal immunity in lung, nose and intestine remains a dynamic balance. The balance of mucosal immunity is destroyed in asthma.
Objective
To examine the influence of retinal pigment epithelium(RPE) cells on antigen-specific activatedlymphocytes in vitro,and to explore the role of RPE cells in the immune privilege of the eye.
Methods
Co-culture systems of RPE cells with antigen-specific T lymphocyte lines and resting T lymphocytes were established in vitro．Induction of apoptosis was detected by genomic DNA electrophoresis,DNA in situ end-labelling and flow cytometry.
Results
RPE cells induced apoptosis in antigen-specific activated T lymphocytes. 24 hours after culture,the signs of apoptosis appeared in lymphocytes co-incubated with RPE cells．As time of co-culture went on,the number of apoptosic cells increased．Quantitative analysis of apoptosic cells showed that apoptosic cells accounted for 5.95% after 24 hours, 9.38% after 48 hours,and 17.95% after 72 hours．In contrast,RPE cells induced few apoptosis in resting T lymphocytes.
Conclusions
These results suggest that RPE cells possess the ability to induce the apoptosis of invading lymphocytes. This phenomenon serves as a restrain mechanism of immune response and may be of vital importance in the maintenance of immune privilege in posterior segment of eye and in the protection of eye from the damage of immunogenic inflammation.
(Chin J Ocul Fundus Dis, 1999, 15: 241-244)
Objective To observe the proportion changes of CD4+CD25+FOXP3+ T cells in peripheral blood of patients with VogtKoyanagiHarada disease (VKH) before and after one month of treatment. Methods he peripheral blood samples from 15 patients with VKH disease before and after one month of treatment by glucocorticoid, and from 15 healthy volunteers were collected，and lymphocytes were separated from them. CD4+CD25+ regulatory T cells were labeled by antibodies of cell surface marker CD4、CD25 and transcription factor FOXP3. The proportion of CD4+CD25+FOXP3+ T cells were detected by flow cytometry. Results Before the treatment, the percentage of CD4+CD25+FOXP3+ T cells in periphery blood was（0.30plusmn;0.19）% of CD4+ cell in VKH patients, and（1.41plusmn;0.52）% in control group, the difference was statistically significant（t=7.665,Plt;0.01）; after one month of treatment, the VKH patients group was（1.28plusmn;0.54）% which close to the control group. However there were two patients whose CD4+CD25+ T cells increased extraordinarily after one month of treatment. Conclusions The proportion of CD4+CD25+ FOCP3+ T cells in periphery blood in VKH patients were lower than control group obviously before treatment, but were close to control group after treatment. Those results indicated that VKH diseases may be associated with the decreased proportion of CD4+CD25+ regulatory T cells.
ObjectiveTo study the immunological properties of osteogenically differentiated umbilical cord blood derived mesenchymal stem cells (UCB-MSCs).
MethodsUCB-MSCs were isolated from the umbilical cord vein, and were expanded; the cells at passage 3 were osteogenically induced for 2 weeks in vitro. The expressions of human leukocyte antigen I (HLA-I) and HLA-Ⅱ molecules were observed by flow cytometry analysis before and after osteogenic induction. Peripheral blood T lymphocytes were isolated and cultured with osteoblastic induced or non-osteoblastic induced UCB-MSCs in different cell concentrations of 1×102, 1×103, 1×104, and 1×105 cells/well. The intake value of 3H-thymidine was calculated with luminescence counter. Then T lymphocytes were pretreated with PHA, and co-cultured with osteoblastic induced and non-osteoblastic induced UCB-MSCs as described above. IL-2 was further added to test the reversed effect of T lymphocytes proliferation stimulated by UCB-MSCs. Finally, to investigate whether the immunomodulatory effects on T lymphocytes proliferation depend on direct or indirect cell contact, the Transwell chamber culture system of UCB-MSCs and T lymphocytes was established.
ResultsFlow cytometry analysis showed that non-osteoblastic induced UCB-MSCs expressed HLA-I but did not express HLA-Ⅱ; the expression of HLA-Ⅱ increased in osteoblastic induced UCB-MSCs. No T lymphocyte response was stimulated by non-osteoblastic induced UCB-MSCs, but osteoblastic induced UCB-MSCs could stimulate the proliferation of allogeneic T lymphocytes, especially after IFN-γ treatment. Non-osteoblastic induced UCB-MSCs of 1×104 and 1×105 cells/well could suppress the proliferation of T lymphocytes evoked by PHA, and this suppression could be reversed by the addition of IL-2. While osteoblastic induced UCB-MSCs did not have such suppressive effect. The results of the Transwell culture system also showed that non-osteoblastic induced UCB-MSCs could obviously inhibit the proliferation of T lymphocytes, but the osteoblastic induced UCB-MSCs could not.
ConclusionThe immunological properties of UCB-MSCs will change accordingly after osteogenic induction, so UCB-MSCs might not be suitable for the seed cells of bone tissue engineering.
ObjectiveTo observe the changes of peripheral blood lymphocyte subsets of patients with primary hepatocellular carcinoma (PHCC) treated with radiofrequency ablation (RFA).
MethodsThe data of 70 cases of hospitalized patients with PHCC that voluntary accepted RFA diagnosed by clinical and pathological in our hospital between July 2011 and December 2014 were collected. According to the numbers of HCC lesions, 70 cases were divided into single focus group (n=41) and multifocal group (n=29). The changes of their immune parameters before and after RFA were analyzed.
Results①The ratioes of peripheral blood CD3+/CD19-, CD3+/CD4+, CD4+/CD8+, and NK cells on 7 days and 14 days after RFA treatment of 70 cases were significantly higher than those on 1 day before RFA treatment (P < 0.05). The ratio of CD3+/CD8+ T cells reduced from 1 day before RFA treatment to 14 days after RFA treatment, but the difference was no statistically significant (P > 0.05).②The changing trend of peripheral blood lymphocyte subsets before and after RFA treatment in single focus group and multifocal group were similar to the above.③Compared with single focus group, the ratioes of peripheral blood CD3+/CD19-, CD3+/CD4+, CD4+/CD8+, and NK cells before and after RFA treatment in multifocal group were lower, and the ratio of CD3+/CD8+ T cells was higher, but the difference were not statistically significant (P > 0.05).
ConclusionRFA can not only destroy small PHCC foci, but also to significantly improve immune function and enhance the anti-tumor effect.
Objective To investigate the effect of pre-infusion with allogeneic lymphocytes treated by 5-FU on inducting immune tolerance of liver transplantation in rats. Methods Wistar and SD rats were used as liver transplantation donors and recipients, respectively. They were divided into 4 groups as following: control group: liver was transplanted from Wistar to SD rats without any other treatment; lymphocytes group: recipient was pre-infused lymphocytes （5×106 cell/ml, 1 ml） from Wistar rat 7 d and 4 d separately before transplantation; low concentration of 5-FU with lymphocytes group: lymphocytes were treated by 5-FU (7.5 μg) before pre-infusion; high concentration of 5-FU with lymphocytes group: lymphocytes were treated by 5-FU (15 μg) before pre-infusion. Pathological changes were observed on day 7 after liver transplantation. Results Acute slight rejection was observed in low concentration of 5-FU with lymphocytes group: liver cell cords were well-arranged basically, hepatic lobules structures could be observed, a few inflammatory cells infiltrated around central veins, and a few lymphocytes infiltrated around portal area. Acute severe rejection was observed in control group, and acute moderate rejection was observed in high concentration of 5-FU with lymphocytes group and lymphocytes group. Conclusion Pre-infusion of lymphocytes treated with low level 5-FU can induce immune tolerance better in recipients after liver transplantation.
PURPOSE: To investigate the activation and immune respones of lymphocytes in
epiretinal membranes (ERMs)and subretinaI membranes (SRMs).
METHODS: A panel of morioclonal antibodies against CD23 (activated B cell), CD25 (activated T cell), CD68(macrophages) and HLA-DR
(human leukocyte antigen II antigen)were used for the study of 20 specimens of ERMs from 20 patients with proliferative vitreoretinopathy (PVR),traumatic PVR and secondary traction retinal detachment,and 2 SRMs from PVR and traumatic PVR, with positive and negative reaction specimens as controls.
RESULTS:Four cases of ERMs were found to be CD23 and CD25 positive respectively,and one case of SRMs to be CD23 and CD25 positive respectively. All the specimens of ERMs and SRMs revealed
CD68 and HLA-DR positive in this series.
CONCLUSIONS :There might be an aberrant immunoreaction
mediated by T and B cells in the ERMs and the SRMs,and they might play an important role in the patbogenesis of PVR,traumatic PVR and secondary traction retinal detachments.
（Chin J Ocul Fundus Dis,1996,12: 147-150）
ObjectiveTo describe clinical significance of eosinopenia in patients with coronavirus disease 2019 (COVID-19).MethodsThis was a retrospective study conducted in three tertiary hospitals from Anhui province, China. A total of 59 patients with COVID-19 were consecutively reviewed from January 23, 2020 to March 10, 2020.ResultsThe median age of patients with COVID-19 was 39 years old, and 32 were male, 30 with eosinopenia. Cough, sputum and fatigue were more common symptoms in eosinopenia patients compared with non-eosinopenia patients. The counts of blood lymphocytes (median: 101 cells/μL) in eosinopenia patients were significantly less than those of non-eosinopenia patients (median: 167 cells/μL, P<0.001). COVID-19 patients with eosinopenia had a higher proportion of corticosteroids therapy than patients with non-eosinopenia (50.0% vs. 13.8%, respectively, P=0.005). Decreased blood lymphocytes count was an independent risk factor for eosinopenia in COVID-19 patients (odds ratio 6.566, 95%CI 1.101 - 39.173, P=0.039).ConclusionsBlood eosinopenia is frequent in COVID-19 patients. Patients with eosinopenia have different clinical features compared to patients with non-eosinopenia. Decreased lymphocyte count is an independent risk factor for eosinopenia in COVID-19 patients.
ObjectiveTo investigate the expression of costimulatory molecules( B7，CD28, and CTLA-4) of peripheral blood lymphocytes in patients with Behcet′s disease(BD).MethodsLymphocytes were obtained in 24 patients with BD and 20 healthy individuals, and the expression of CD80(B7-1), CD86(B7-2), CD28 and CTLA-4 on T and B cells were detected by direct three-color immunofluorescence flow cytometry.ResultsSignificantly increased expression of CTLA-4 on CD 4+ T cells[(3.18±1.18)%]was found in BD patients compared with that in controls[(1.73±0.66) %](t=－3.722,P＜0.01). The expression of CD86 on CD19+B cells was also significantly increased in BD patients[(4.49±1.73)%]compared with that in controls[(2.40±1.49) %] (t=-2.071,P＜0.05). No significant difference in the expression of the other costimulatory molecules was found.ConclusionsInteraction of B7 and CD28 on peripheral lymphocytes promote the occurrence of uveitis in BD patients. Intervention with these costimulatory signals may lead to a new prevention or treatment for uveitis patients.(Chin J Ocul Fundus Dis, 2003,19:357-359)
