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Objective:To explore the type of cytokine (IL-2 or IL-7) and its most optimal concentration regarding the improvement of the signal-to-noise ratio of glutamic acid decarboxylase 65 (GAD65) in enzyme-linked immunospot (ELISPOT) assay in Type 1 diabetic (T1DM) patients.Methods:Twenty T1DM patients (Group A) and sixteen healthy controls matched with age and sex (Group B) were enrolled in our study,and their peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll method.GAD65,internal control and Pediacel served as "five-for-one" vaccine were selected as the stimulating antigen.Different concentrations of IL-2 [0 U/mL (Group 1),0.5 U/mL (Group 2),2.5 U/mL (Group 3) and 12.5 U/mL (Group 4)] were added to the culture system.The CD4+ T cells of secreting interferon-gamma (IFN-γ) in the above groups were determined by ELISPOT.The spots number,net values and stimulating index (SI) were compared in GAD65 (signal) and internal control (background).Next,another 21 T1DM patients (Group C) and 12 healthy controls matched with age and sex (Group D) were enrolled,and the specific T cell response to the GAD65 antigen was detected.The net values and SI were compared between the best optimal concentration of IL-2 (2.5 U/mL,Group 5) and IL-7 (0.5 ng/mL,Group 6).Results:1) After adding IL-2 into the Group A,the amount of GAD65 reactive T cells in different groups increased compared with Group A1,while the background in the internal control also increased gradually with the increased concentration of IL-2.There was no significant difference in net value (signal-noise) in the different concentration between the Group A3 and the Group A4 (P＞0.05).The SI in the Group A3 (2.8),the highest one,was significantly higher than that in the Group B3 (1.3) (P＜0.05).2) Although the number of GAD65 spots in the Group C6 and the Group D6 were slightly higher than that in the Group C5 and the Group D5,respectively,the background in the Group C6 and the Group D6 also increased,without statistical significance (P＞0.05).The mean net value spot and SI in the Group C5 (net value:5.5;SI:2.8) were both significantly higher than those in the Group C6 (net value:4.3;SI:1.8) (bothP9＜0.05).Conclusion:The concentration of 2.5 U/mL for IL-2 is proved to be the best optimal concentration for GAD65 specific T-cell responses in ELISPOT in patients with T1DM.IL-2 is much better than IL-7 in improvement of the SI in the ELISPOT.
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OBJECTIVE@#To explore the better freezing protocol to preserve peripheral blood monuclear cells (PBMCs), islets antigen-specific T cells responses compared with freshly isolated samples in type 1 diabetic (T1D) patients.@*METHODS@#The T cell Workshop Committee of the Immunology of Diabetes Society (IDS-TCW) organized the Freezing Study I and we were one of the 9 centers in the world to participate in the study. According to the two standardized T cell freezing protocols (warm and cold) to freshly isolated PBMCs in terms of recovery, viability, cell subset composition (FACS) and performance in Enzyme-linked immunospot (ELISPOT) assays, we chose 5 newly onset T1D patients and 5 age and sex matched healthy controls. Besides the protocols, all the freezing reagents and antigens were also centralized. The antigens used in ELISPOT were labeled blindedly.@*RESULTS@#1) Although warm frozen-thawed (W) samples had a slightly higher recovery rate (61.2% vs 60.1%, P>0.05) and viability (77.5% vs 74.9%, P>0.05) as compared with the cold frozen ones (C), the difference was not significant. 2) Both protocols led to a relative loss in monocytes as compared with the fresh samples (F) [3.2±1.1% (C) and 3.0±0.9% (W) vs 7.0±1.1% (F), both P<0.05], while other subsets including CD4+T, CD8+T, B cells, NK cells and NKT cells didn't. 3) Freezing and fresh samples showed similar IFN-γ secretion responses to polystimuli in ELISPOT. Irrespective of the freezing protocol, recall antigen Pediacel and islet antigen-reactive responses were both lower in the frozen cells compared with fresh PBMCs. The stimuli index (SI) of GADspecific T cell response in the fresh samples from T1D patients was 5.1, higher than that of frozen samples with either cold protocol (1.3) or warm one (1.4) (both P<0.05). Only fresh samples from T1D showed significantly higher GAD-specific T cell responses than the healthy controls no matter in SI (5.1 vs 0.9, P<0.05) or spot forming cells (8.1 vs 0.1, P<0.05), whereas the frozen samples did not show such difference.@*CONCLUSION@#More studies are needed to verify a freezing method to bring comparable islets antigen specific T cell responses in T1D patients to fresh PBMCs.