The Mechanism Of CD4~+NKG2D~+T Cells In Progress Of T1D In NOD

Type 1 diabetes (type 1 diabetes mellitus, TID) is a kind of self reactive T cells to attack islet beta cells damaged an autoimmune disease.The process, mainly by the effect of CD4+ T cells,CD8+ T cells and other immune cells to participate in.Sexual receptor NKG2D to NK cell activation, recent studies have found that can be expressed by NKT,γδT, activated macrophages, CD4+ T cell subgroup, CD8+ T cells in humans.NKT, γδ T cells, activate CD8+ T cells, CD4+ T cells subgroup in mouse.Studies have shown that NOD islet beta cells express NKG2D ligands Rae-1, so there is a large number of infiltration with the expression of NKG2D receptor reactivity of CD8+ T, CD4+ T cells in the pancreas.In order to further explore the CD4+NKG2D+ T cells involved in the pathogenesis of NOD mice,this research to study peripheral blood of table characteristics, function and activation condition in the period of recent-onset,T1D and immune intervention of NOD.The research content is divided into three parts:Part Ⅰ:The relationships between CD4+NKG2D+ T cells and type 1 diabetes disease process in NODObjective:Observe the frequence of CD4+NKG2D+T cells in NOD and explore the relationship between CD4+NKG2D+T cells and CD8+T cells.Methods:Dynamic monitoring the frequence of NOD,Balb/c peripheral blood CD4+NKG2D+T and CD8+NKG2D+T cells in different weeks of age.Use of IGRP206-214-SAP NOD which can effectively reduce the blood sugar and the incidence of T1D, the CD4+NKG2D+/CD4+T and CD8+NKG2D+/CD8+T cells frequency is analyzed.Proceeds to each weeks CD4+NKG2D+/CD4+T and CD8+NKG2D+/CD8+T cell frequency linear regression analysis.Results:The NOD mice peripheral blood CD4+NKG2D+/CD4+T and CD8+NKG2D+/CD8+T frequence with TID disease process development increase.But the frequence of Balb/c peripheral b1ood CD4+NKG2D+/CD4+T and CD8+NKG2D+/CD8+T cells almost no change.Use our previous successful preparation of specific CTL IGRP206-214 clear NOD mice model (the model NOD were significantly lower incidence of blood glucose than blank group), the results show with the model group NOD mice peripheral blood CD8+NKG2D+/CD8+T cells reduce the frequency of peripheral blood CD4+NKG2D+/CD4+T cells also lower,and positive linear correlation between them.Conclusion:The increased frequency of CD4+NKG2D+T cells in NOD associated with the progress of type 1 diabetes.The frequence of CD4+NKG2D+T cells and CD8+T cells in the body between a positive linear correlation.Part Ⅱ:The biological characteristics of CD4+NKG2D+T cells in NODObjective:Analyze and compare the features,phenotypic characteristics, active state of CD4+NKG2D+T cells in NOD in recent-onset and T1D.A preliminary probe into the group of cells in vitro and the interaction relationship between the CD8+T cells.Methods:By flow cytometry in pathogenesis of recent-onset NOD and stage was detected in NOD mice peripheral blood CD4+NKG2D+T cell function (IFN-γ, ROR γt, IL-17, CD107A, Perforin and granzyme B, FasL, TGF-β, IL-10), phenotypic characteristics (CD44, CD62L) and activation status (CD69, CD25).This group of cells and were compared under two different stages of disease, the differences between the phenotypic characteristics and the active state.With the method of magnetic bead separation, from the onset of recent stage NOD mice spleen for CD4+NKG2D+ T cells respectively with the CFSE tags CD4+NKG2D- T cells and CD8+T cells develop.Four days after detection system of CD4+NKG2D- T cells and CD8+T cells CFSE fluorescence intensity changes.Results:Recent-onset NOD peripheral blood CD4+NKG2D+T cells (IFN-γ secretes a recent-onset stage disease no change.RORyt express a marked increase in the incidence prodromal stage, the secretion of IL-17A level also rises.On behalf of the cytotoxic activity of CD107A,granzyme B and Perforin had no obvious change.Onset of NOD mice peripheral blood CD4+NKG2D+T cells express FasL more frequently than the onset of prodromal stage increased slightly but the change is not obvious. TGF-β and IL-10 is also slightly higher, but the cause of the individual difference is bigger makes no statistical significance between them. Onset of NOD mice of the group of cells in the peripheral blood CD4+NKG2D-T cells, the frequency of the effect of memory T cells, and the central memory T cells and CD4+NKG2D-T cells are slightly ascending, compared between the two initial T cell frequency is increased slightly but also no statistical difference. Onset of NOD mice of the group of cells in the peripheral blood CD4+NKG2D-T cells, the expression of CD69 frequency increases, CD25 expression frequency also increases significantly. The magnetic bead separation results of CD4+NKG2D+ CD4+NKG2D-T cell co-culture four days, compared with the separate CD4+NKG2D-T cells, join CD4+NKG2D+T cell culture system, the CD4+NKG2D-T cell proliferation.And the proliferation of CD8+T, no change.Conclusion:NOD mice from the onset of type 1 diabetes prodromal stage between the period, the peripheral blood CD4+NKG2D+T cells in the frequency increased not only, and the group of cells to enhance the degree of activation have some margin, when reached the period of time, the group of initial cells dominated by the initial type to the effect of memory cells dominate shift, and the biggest change is on the function, the RORγt express and the secretion of IL-17 improved significantly.And the group of cells is directly based on CD4+NKG2D-T cell regulation of indirect effects on CD8+T cells.Part Ⅲ:The biological characteristics of CD4+NKG2D+T cells in over expression of mInsB15-23H-2kd NODObjective:Dynamic testing mInsB15-23H-2Kd-dtsct eukaryotic expression vector after the intervention of the NOD mice had the frequency group of cells, and the functions and characteristics of the cells of the group, phenotypic characteristics, active state for further research.In vitro to explore the immune intervention after the group of cells and CD8+T cells and CD4+NKG2D" effect relationship between T cells.Methods:In NOD mice 3 weeks of subcutaneous inoculation mInsB1s-23H-2Kd-dtsct eukaryotic expression vector, in 4 or 5 weeks in the same position to strengthen a injections.After the third immunization of 0,1,12,24,48,72 H, and 1,3,6,9 w take mice peripheral blood test DC surface expression of H-2 kd.The dynamic monitoring of immune NOD mice peripheral blood CD4+NKG2D+/CD4+T and CD8+NKG2D+/CD8+T cells frequency.At 20 w, immune NOD mice peripheral blood, the CD4+NKG2D+T cell function (IFN-gamma, ROR gamma T, IL-17, CD107A, Perforin and granzyme B, FasL, TGF-beta, IL-10), phenotypic characteristics (CD44, CD62L) and activation status (CD69, CD25) is analyzed.Time NOD mice with prodromal stage, the group of cells in the body of the function, the phenotypic characteristics and activation status.Using the method of magnetic bead separation from the NOD mice spleen immune CD4+NKG2D+T cells respectively with the CFSE tags NKG2DT cells and CD8+T cells develop.Four days after detection system of CD4+NKG2D"T cells and CD8+T cells CFSE fluorescence intensity changes.Results:Compared with empty plasmid, after the third immunization within 0 to 72 hours and 1 week, H-2kd DC surface expression were significantly elevated and significantly higher than the empty plasmid group.However, after the third immune 3 week, DC surface expression of H-2kd plummeted, and continuously below the empty plasmid group.At 6,8,10 week, plasmid group had a significantly higher CD4+NKG2D+T the empty plasmid group,12 week almost flat, after 14 week has been below the empty plasmid group.And CD8+NKG2D+/CD8+T cells in 6,8 week is significantly higher than the empty plasmid group,10 week when both is almost flat, after 12 week has been below the empty plasmid group.NOD mice immune intervention in the peripheral blood CD4+NKG2D+T cell secretion of IFN-y change is not obvious, is higher than the control group in 8,10 week,12 weeks almost flat with the control group, after 16 weeks has remained at the levels of slightly lower than the control group.20 weeks immune intervention group than untreated prodromal stage of slightly lower, but the variance due to individual differences, resulting in no statistical significance compared with the period.NOD immune intervention in the peripheral blood CD4+NKG2D+T cells RORyt is higher than the control group in 8,10 weeks,12 weeks almost flat with the control group, after 16 weeks has remained at the levels of slightly lower than the control group.The 20 weeks data and pathogenesis of prodromal stage and the corresponding weeks data contrast, found that the recent-onset of immune intervention group compared with untreated prodromal stage slightly lower, compared with the period decreased significantly.NOD mice immune intervention in the peripheral blood CD4+NKG2D+T cells, the secretion of IL-17A in 8,10,12 weeks is higher than the control group, after 16 weeks has remained at the levels of slightly lower than the control group.Immune intervention group of 20 weeks relatively unprocessed prodromal stage, the period were significantly reduced.NOD mice immune intervention in the peripheral blood CD4+ NKG2D+T cells expressing CD 107A more frequently than prodromal stage, disease stage had no obvious change.And intracy to plasmic particles enzyme granzyme B a prodromal stage, the period were significantly reduced.Perforin a prodromal stage, the period also significantly reduced.NOD mice immune intervention in the peripheral blood CD4+NKG2D+ T cells express FASL more frequently than prodromal stage, disease stage had no obvious change.And TGF-βa prodromal stage, the period were significantly increased.IL-10 a prodromal stage, the period also increased significantly.The CD4+NKG2D+T cells, effect of memory T cells frequency is more, the central memory T cells and CD4+NKG2D" compared to T cells are slightly increases. While the initial T cell frequency more CD4+NKG2D-T cells decreased significantly.NOD mice immune intervention of the group of cells in the peripheral blood a prodromal stage, come on stage, the expression of CD69 frequency are higher, a marked increase in the incidence prodromal stage, expression of CD25 frequency and the period almost flat.Compared with single CD8+ T cells, join CD4+NKG2D+T cell culture system, the CD8+ T cell proliferation.And CD4+NKG2D’T cell proliferation, no obvious change.Conclusion:NOD mice immune intervention of CD4+NKG2D+T cells in peripheral blood not only frequency decreases in the empty plasmid group, the group of cells activation degree also has certain enhancement, and mainly effect the memory cells in the majority.The biggest change is and the function, the RORyt express and the secretion of IL-17 significantly reduced.And, the group of cells is to bypass regulation of CD4+NKG2D-T cells directly impact on CD8+T cells.