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)
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.
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.
Objective To study the immunological tolerance induced by blocking the second signal of T cell with extrinsic cytotoxic T lymphocyteassociated antigen 4 immuno globlin(CTLA4-Ig). Methods Fifty-four BALB/C mice, inbred strains, were employed as recipients of bone allografts, using a model of heterotopic muscle pouch. The 54 mice were divided into 3 groups and18 for each group. The first group, in which the donor was C57BL/6 with intraperitoneal injection ofL6(as a control), was named AL group. The second group,also C57BL/6 with injection CTLA4-Ig, was named AC group. The third group,homologous BALB/C with injection of PBS buffer solution, was named AB group.The serum antibody, lymphocyte proliferation of the second stimulation by splenic cell and bone supernatant of donor, the analysis of lymphocyte subsets, a regraft experiment and histology were determined 2, 4 and 6 weeks after transplantation. The second transplantation was to regraft C57BL/6(BC group) and C3H(BHgroup) mice respectively after first 12 mice being transplantated with C57BL/6 and injected with CTLA4-Ig as to detect donor-specificity of immunological tolerance. Results Compared with AB group, AL group created more intensive immune rejection: CD4 T cell subsets(Plt;0.05), the serum antibody(Plt;0.05) and lymphocyteproliferation of the second stimulation by splenic cell and bone supernatant ofdonor (Plt;0.01 and 0.05) were significantly increased. However, the results of AC group showed that CTLA4-Ig significantly inhibited the immune rejection: CD4T cell subsets(Pgt;0.05), the serum antibody (Pgt;0.05), and lymphocyte proliferation of the second stimulation(Pgt;0.05) were similar to those of AB group. Histological observation of AC group showed that lymphocyte infiltration disappeared,cartilage and new bone formed, and bone marrow cavities emerged. A regraft experiment showed that CD4 T cell subsets (Plt;0.05) and lymphocyte proliferation of the second stimulation by splenic cell and bone supernatant of donor(Plt;0.05), BC group was significantly lower than those of BH group. So theimmunological tolerance induced by CTLA4-Ig was of donor-specificity. Conclusion The immunological tolerance induced by CTLA4-Ig was prolonged for 6 weeks. This study provides a brand-new path for bone transplantation, which can be helpful to other organ transplantation.
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.
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 expression of Human leucocyte antigen(HLA)-DP, -DQ, -DR and CD40 in human retinal pigment epithelial (RPE) cells, to determine their molecule expression in immune response process, and their abilities to stimulate T lymphocyte activation. Methods Human RPE cells were cultured with or without (IFN respectively. Expression of HLA-DP, -DQ, -DR and CD40 was measured by immunohistochemical staining. Meanwhile, peripheral blood mononuclear cells (PBMC) were cocultured with RPE cells in vitro, and then the expression of activated lymphocytes CD69 was measured by fluorescence activated cell sorter(FACS). Results Expression of HLA-DP, -DQ, -DR and CD40 antigen were enhanced by gamma;-interferon inducement. Increasing amount of CD69 positive lymphocytes were found in the co-culture system of RPE cells and PBMC. Conclusion T-lymphocytes in the peripheral blood were activated by human RPE cells which is antigen presenting cells with immunological characteristics potential.
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.
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)
ObjectiveTo analyze dynamic characteristics of peripheral blood cells in patients with different types of coronavirus disease 2019 (COVID-19), so as to investigate the predictive value of peripheral blood cells and their dynamic changes for clinical outcome of patients with COVID-19.MethodsForty-eight patients with COVID-19 were collected and analyzed from East Hospital of Renmin Hospital of Wuhan University from February 2 to March 15, 2020. These patients were divided into general group (group A, 17 cases), severe survival group (group B, 21 cases), and severe death group (group C, 10 cases). Blood routine examination was done and analyzed before and after admission and among the three groups. The changes of neutrophils and lymphocytes were compared. The predictive power of neutrophils, lymphocytes, neutrophil-to-lymphocyte ratio (NLR), and platelet-to-lymphocyte ratio (PLR) for clinical outcomes was analyzed through the receiver operating characteristic (ROC) curve.ResultsIn group B, the lymphocyte count at discharge was significantly higher than at admission (P=0.002), and the neutrophil count, NLR and PLR were significantly lower than at admission (P values were 0.012, 0.001 and 0.007, respectively). The lymphocyte counts in the A, B, and C groups were ranked from high to low upon admission, and the differences among the three groups were statistically significant (P values were 0.020, <0.001 and 0.006 for the contrasts between groups A and B, groups A and C, groups B and C, respectively), the NLR were ranked from low to high, and the differences among the three groups were statistically significant (P values were 0.001, <0.001 and 0.026 for the contrasts between groups A and B, groups A and C, groups B and C, respectively). Before discharge or death, there was no significant difference in lymphocyte counts and NLR between A and B groups (P>0.05), and there were statistically significant differences between group C and groups A and B (all P values were<0.001). The proportions of “Neutrophils Lymphocytes Convergence” in groups A and B were 64.7% and 76.2%, respectively, which were significantly higher than that in group C (10.0%). The proportions of “Neutrophils Lymphocytes Separation” in group C was 70.0%, which was significantly higher than those in groups A (0) and B (4.8%). The area under the curve of NLR predicting patients with severe disease (excluding death) was 0.843, with the sensitivity and specificity of ≥3.55 be 0.810 and 0.882; The area under the curve of lymphocyte count predicting death in severe patients was 0.845, with the sensitivity and specificity be 0.700 and 0.905, respectively.ConclusionsDynamic changes in the composition of peripheral blood cells are one of the clinical features of COVID-19, “Neutrophils Lymphocytes Convergence” and “Neutrophils Lymphocytes Separation” predict better and worse clinical outcomes, respectively. NLR and lymphocyte counts are effective indicators for predicting the severity and death of COVID-19.
