Platelets Activated By Thrombin Enhance The Differentiation Of Tfh In CD4~+T Cells

Objective: To observe the effect of activated platelet to the differenti- ation of CD4+T cells.Methods:1 Isolation of CD4+T cells, Verify the purity of CD4+T cells, Activation of CD4+T cells: Peripheral blood was isolated from healthy male donors(n=5). Peripheral blood mononuclear cell(PBMC)was isolated by density gradient centrifugation. Magnetic activated cell sorting was used for isolating of CD4+T cells. Sorted CD4+T cells was simulated by Anti-CD3/CD28 antibody. Using Flow cytometry to detect the expression of CD69 on CD4+T cells.2 Isolation and activation of platelets: Peripheral blood was isolated from healthy male donors(n=5). Secondary centrifugation was used to isolate platelets. Isolated platelets were stimulated with thrombin(0.2 U/ml, 37°C, 5min). Using Flow cytometry to detect the expression of CD62 P on platelets.3 Activated CD4+T cells were cultured with platelets in accordance with the following groups: A Group of platelets: activated platelets+CD4+T cells; B Group of platelet supernatant: activated platelet supernatant+CD4+T cells; C Group of control: culture medium+CD4+T cells.4 When activated CD4+T cells was co-cultured with platelets after 72 hours, observation was taken for the morphological change of CD4+T cells.5 The IL-10 expressed by CD4+T cells was detected by ELISA. Co-cultured CD4+T cells was labeled with FITC-conjugated anti-CD4 antibody,Percep-conjugated anti-CXCR5 antibody, APC-conjugated anti- PD-1 antibody, and APC-Cyt-conjugated anti-Foxp3 antibody. Using Flow cytometry to detect the percentage of Tfh(defined as CD4+CXCR5+ PD-1+Foxp3-) in total CD4+T cells.Results:1 Using flow cytometry to analyze the purity of CD4+T cells and the expression of CD69. The purity of CD4+T cells isolated by MACS was(95.93±2.14)%. The CD69 expressions of CD4+T cells stimulated by anti-CD3/CD28 antibody was(13.05±2.12)%, while the unstimulated control was(1.48±0.25)%. There were statistically significant differences(P<0.01).2 Flow cytometry was used to analyze the expression of CD62 P by platelets. The CD62 P expressions of platelets activated by thrombin(0.2U/ml, 37℃, 5min) was(46.27±2.58)%, while the nonactivated control was(10.29±0.44). There were statistically significant differences(P<0.05).3 Microscopic observation of CD4+T cells(after co-cultured with activated platelets for 3 days) showed higher aggregating activity than co-cultured with platelet supernatant and culture medium.4 Detection of IL-10 by ELSA: when co-cultured for 72 hours, the expression of IL-10 in platelets group was(69.77±7.59) pg/ml,which was significantly higher than the platelet supernatant group(14.08±2.78) pg/ml, and the group of control(11.24±2.81) pg/ml, P<0.05.While there were no statistically significant differences between the platelet supernatant group and the group of control.5 Using Flow cytometry to detect the percentage of Tfh in total CD4+T cells. The percentage of Tfh in total CD4+T cells in platelets group was(10.75±2.46)%, which was significantly higher than the platelet supernatant group(6.10±3.09)% and the group of control(3.00±1.76)%, and there were statistically significant differences, P<0.05. While there were no statistically significant differences between the platelet supernatant group and the group of control.Conclusions:1 Activated platelets can promote the aggregating activity of CD4+T cells.2 Activated platelets can promote the expression of IL-10 by CD4+T cells.3 Activated platelets can promote the differentiation of CD4+T cells to Tfh.