The Role Of Follicular Cytotoxic T Cells In Type 1 Diabetes

Part I Exploration of the relation between Follicular Cytotoxic T Cells and the progress of immunological damage in NOD miceObjective: In the course of the pathogenesis of type 1 diabetes,autoreactive CD8+T cells are activated by interaction with antigenic peptides presented by MHC class I molecules.Autoreactive CD8+T cells directly kill islet beta in a contact-dependent manner by releasing perforin and granzymes.Such cell subsets are the main effector cells which cause type 1 diabetes.Cytotoxic CD8+T cells are the major characters in insulitis lesions and for insulitis.Recent studies have found a new subpopulation of CD8+T cells called follicular cytotoxic T cells(TFCs)which highly express the chemokine receptor 5 and have been shown to perform high cytotoxicity and pro-inflammatory effects in studies of tumors and chronic viral infections.In this part of the study,we explore the relation between TFC and type 1 diabetes to investigate the differences in the distribution of follicular cytotoxic T cells(TFCs)in different tissues of NOD mice and the proportion of TFC cells with the growth of NOD mice,observe the expression of PD-1,TIM3,CD107 a,and KLRG1 in TFC cells.Detecting the ratio of IFN-? and Gr B secreted by TFC subpopulations in NOD mice to explore the effect of TFC on immune damage in NOD mice.Methods: NOD mice were divided into high-glucose group Hi(two repeated blood glucose values >16.7 mmol/L)and according to the weekly ages divided into five groups of 4-week,10-week,15-week,20-week,and 25-week.Spleens,lymph nodes,and thymus tissues from NOD mice were used to detect the number of TFC cells.The expression levels of four surface markers of PD-1,TIM3,CD107 a,and KLRG1 on TFC cells were detected and statistically analyzed.Detecting and analyzing the level of IFN-? and Gr B in the spleen TFC cells of NOD mice before and after PMA stimulation.Statistical methods include Student's t test,one-way analysis of variance,multi-component comparison using the Kruskal-Wallis test.Results: The distribution of TFC cells in different tissues of NOD mice was different.The thymus tissue had almost no TFC,but the TFC of the spleen was slightly higher than that of the lymph nodes,but it was not statistically difference.The proportion of TFC cells increased with the age of NOD mice.The proportion of TFC cells was significantly lower than that of CXCR5-cell subpopulations in total of CD8+T cells(P<0.01),but the expression of multiple cell surface markers was significantly higher than that of CXCR5-cell subpopulations,except of KLRG1 expression levels.The expression levels of PD-1,TIM3,and CD107a(P<0.01)in TFC cells were significantly higher than those of CXCR5-cell subsets.The amount of IFN-? and Gr B released from the subset of TFC cells in the spleen of NOD mice was higher than that of CXCR5-cell subsets after stimulation of PMA(P<0.05).Conclusions: With the progressive development of immune damage in NOD mice,the proportion of TFC cells also increased.Although the proportion of TFC cells was much lower than that of CXCR5-cell subpopulations,the ratio of expression of multiple inhibitory and stimulatory molecules was significantly higher than that of CXCR5-cell subsets.It shows that although the proportion of TFC cells is low,the immune effects and capabilities exerted by them may be stronger.TFC can release higher levels of IFN-? and Gr B than CXCR5-cell subsets.In summary,TFC may be involved in some aspects of T1 D disease and play a role in the pathogenesis of type 1 diabetes.The role of TFC in type 1 diabetes still needs further exploration and research.Part II Analysis of Peripheral Follicular Cytotoxic T Cells in Type 1 and Type 2 Diabetes and ControlObjective: Series of studies have demonstrated that autoreactive CD8+ T cells are a subpopulation of cells that play an important role in the pathogenesis of T1 D.As a newly discovered subpopulation of CD8+ T cells,follicular cytotoxic T cells(TFCs)make an important impact on tumor and chronic viral infections,and have strong cytotoxicity and pro-inflammatory effects.The purpose in this part of the study was to detect and analyze the differences in the percentage of follicular cytotoxic T cells(TFCs)in peripheral blood PBMCs and the proportion of TFC-released Gr B in patients with type 1 diabetes(T1D),type 2 diabetes(T2D),and healthy controls.Detecting and analyzing the difference of the ratio of Gr B released from peripheral blood TFC in patients with different course of T1 D so that to verify that TFC plays a certain role in the pathogenesis of type 1 diabetes.Methods: A total of 43 patients with type 1 diabetes,37 patients with type 2 diabetes,and 30 healthy controls were enrolled.Peripheral blood was collected from three groups and PBMCs were isolated from peripheral blood.The ratio of follicular cytotoxic T cell(TFC)and TFC released granzyme(Gr B)were detected and analyzed from the patients and healthy controls.Statistical methods include the t-test,one-way ANOVA,and Kruskal-Wallis test.Results: The overall proportion of TFC cells in peripheral blood of T1 D patients(4.6±4.4%)was higher than that of healthy controls(2.33±1.4%),and there was a statistical difference between healthy controls and the T1 D group(P<0.05).However,there was no statistical difference in the ratio of TFC in PBMCs between patients with T1D and T2D(3.85±1.8%).There was a significant difference in the ratio of peripheral blood TFC between patients with T2 D and the healthy controls(P<0.001).There were no significant differences in the levels of Gr B secreted by follicular cytotoxic T cells in the three groups of subjects.The proportion of CXCR5-CD8+T cell subsets in the peripheral blood of the three groups was detected.The ratio of CXCR5-cell subsets between T1 D and T2 D,T2D and healthy controls was different and has statistical differences(T1D vs T2 D,P<0.05,T2 D vs Control,P <0.005).The proportion of Gr B released by CXCR5-CD8+ T cell subsets in T1 D patients(26.54±15.8%)was significantly lower than that in healthy controls(37.46±16.91%)and T2 D patients(44.74±16.92%),and there was a statistical difference between T1 D and T2 D patients(P<0.0001).The proportions of follicular cytotoxic T cells in peripheral blood PBMCs in patients with newly diagnosed type 1 diabetes(with duration <1 year)and long-term type 1 diabetes(with duration>1 year)were detected and compared.No significant difference was found between the two groups.The rate of TFC(21.61±8.67%)released granzyme B(Gr B)in peripheral blood of patients with long course of T1 D was higher than that of the short course of T1D(15.77±8.55%),and the difference was statistically significant(P<0.05).Conclusions: The ratio of peripheral blood PBMC follicular cytotoxic T cells in patients with T1 D and T2 D were both higher than that of the normal control group which indicated that TFC may make an effect on the pathogenesis of both T1 D and T2 D.Although the proportion of TFC-released Gr B between the three groups had no significant differences,we found that the proportion of Gr B released from TFC in peripheral blood of T1 D with long-course was higher than that of patients with newly diagnosed type 1 diabetes which was consistent with the results of our first part of study on NOD mice,that is,with the development of immune injury process,TFCs persist and may participate in the process of chronic immune damage.In addition,we found that the level of granzyme B released from CXCR5-CD8+T cell subsets in peripheral blood of T1 D patients were significantly lower than that in T2 D and healthy controls.The function of this subset of CXCR5-CD8+Tcell subsets remains unclear.In general,our study still needs to be further improved to increase the sample size and follow-up related functional verification.