The Method Study On Detecting GAD65-specific T Cells In Type1Diabetes By ELISPOT

The comparison of the effect of IL-2and IL-7on GAD65reactive T cell responses in patients with T1DMObjective:To explore the better cytokine between IL-2and IL-7and its most optimal concentration which can improve the signal-to-noise ratio of glutamic acid decarboxylase65(GAD65) maximally in enzyme-linked spot (ELISPOT) assay in type1diabetic (T1DM) patients.Subjects and Methods:Fourty-one T1DM patients and28healthy controls matched with age and sex were enrolled in our study and their Peripheral blood mononuclear cells(PBMCs) were isolated by Ficoll. GAD65, internal control and Pediacel were selected as the stimulating antigen in the IFN-gamma secretion CD4+T cell responses which were determined by ELISPOT. The T cell spots in GAD65and internal control were compared in different concentrations of IL-2including OU/mL,0.5U/mL,2.5U/mL and12.5U/mL which were added to the culture system. Next, the best optimal concentration of IL-2was further compared with IL-7(0.5ng/mL). The spots number in GAD65(noise), internal control (background), net values and stimulating index (SI) were compared among the different cytokine group and between T1DM and healthy controls.Results:1) The number of GAD65reactive T cells increased gradually with the increasing of the concentration of IL-2, meanwhile the number of spots (background) also increase accordingly. There was not significant difference for net value (signal-noise) between the concentration of2.5U/mL and12.5U/mL. As for the comparison of stimulation index (SI, ratio of signal-to-noise) among the four IL-2subgroups,2.5U/mL was proved to be the highest one which can maximize the signal-to-noise ratio; and only in this concentration, the SI of T1DM patients was significantly higher than that in healthy controls (P<0.05).2) The number of GAD65spots in IL-7group was although slightly higher than that of the IL-2’s, but there was also a much higher increase in the background of IL-7group accordingly. In T1DM patients, the net value spot and SI in IL-2group were both significantly higher than that in IL-7group (both P<0.05).Conclusion:1) The concentration of2.5U/mL for IL-2is proved to be the best optimal concentration for GAD65-specific T-cell responses in ELISPOT in patients with T1DM.2) IL-2is much better than IL-7that can improve the signal-to-noise ratio in the ELISPOT in T1DM patients. Effect of cryopreservation method on islet antigen reactive T cell responses in type1diabetic patientsObjective:To investigate the better freezing protocol to preserve PBMCs, islet antigen reactive T cell responses compared with freshly isolated PBMCs samples in T1DM patients.Subjects and Methods:Nine T1DM patients and9age and sex matched healthy controls freshly isolated were enrolled and PBMCs were isolated and were further divided into3parts. One part was analyzed for cell subset composition by FACS, the other two parts were freezed by cold and warm freezing protocols recommended by IDS-TCW. One month later, the freezing PMBCs were thawed and further received FACS and ELISPOT assay. Human GAD65, control, Pediacel vaccine and PMA were selected as four antigens in ELISPOT. Cell recovery, viability, cell subset constitution and T cell responses in ELISPOT were compared in two freezing methods and the fresh cells.Results:1) Although warm frozen-thawed (W) samples seemed yielded a slightly higher recovery rate and viability as compared to the cold frozen ones (C), the differences were not significant.2) Both freezing protocols led to a relative loss in monocytes as compared with the fresh cells (F)[3.2±1.1%(C) and3.0±0.9%(W) vs7.0±1.l%(F), both P<0.05], while other PBMCs subsets including CD4+T, CD8+T, B cells, NK cells and NKT cells didn’t.3) Freezing and fresh samples showed similar IFN-y secretion responses to polystimuli in ELISPOT. Irrespective of freezing protocol, recall antigen Pediacel and islet antigen-reactive responses were both lower in frozen cells compared to fresh PBMCs. The stimuli index (SI) of GAD-specific T cell response in fresh samples from TID patients was5.3, which were both higher than that of frozen samples with either cold protocol (1.1) or warm one (1.5)(both P<0.05). Moreover, only fresh cells from TID patients showed significantly higher GAD-specific T cell responses than the healthy controls no matter in spot forming cells (8.6vs0.3, P<0.05) and SI (5.3vs1.1, both P<0.05), whereas the two frozen samples couldn’t produce such difference.Conclusion:More researches are needed to verify a freezing method that will produce comparable islet antigen specific T cell responses in TID patients to fresh PBMCs.