Systemic Lupus Erythematosus Regulatory T Cells Of Cd4 ~ + Cd25 ~-of Foxp3 ~ + T Cells And Their Associated Molecular Marker Expression Characteristics

BackgroundT regulatory cells (Tregs) play an essential role in maintaining immunologic homeostasis and preventing autoimmunity. The decreased Treg number and deficiency in Treg function could result in increased helper T cell activity or directly in enhanced B cell activity, both of which have been shown to be regulated by Tregs in normal subjects.In SLE, it is well recognized that B cells are hyperactive and produce a variety of autoantibodies. Furthermore, it has also become evident that SLE T cells participate in the attack on target cells or tissues .One possible explanation of the pathogenesis in SLE could be the abnormal frequency or deficient function of Tregs.The development and homeostasis of Tregs relate to several intracellular or membrane molecules, including Foxp3, GITR, CTLA-4, CCR4, CD127.Some studies have shown that the number of peripheral circulating Tregs may be decreased in active SLE, and/or function of Treg may be deficient.Our previous study showed a significant increased frequency of CD4⁺CD25^-Foxp3⁺ T cells in peripheral blood of active SLE.ObjectiveStudy the quantity of Treg cells in peripheral blood of SLE.Identify the phenotype of the CD4⁺CD25^-Foxp3⁺ Tcells, an increased frequency in our precious study.Investigate the relationship between Foxp3 and other molecules related with Treg cells, including Foxp3, GITR , CTLA-4, CCR4, CD127.Search for surface markers on T cells to substitute intracelluar Foxp3 to facilitate live cell sorting.Further investigate the change of membrane/intracellular markers of Tregs in SLE. MethodsFITC,PE,PEcy5,APC,PEcy7 labeled fluorescent antibodies were used for the surface or intracellular staining of PBMC isolated from peripheral blood in SLE and healthy controls. The expression of Treg cells, CD4⁺CD25^-Foxp3⁺T cells, and other Treg related molecule markers(CD127,CCR4,GITR,CTLA-4)were tested by the Flow cytometric analysis.For immunostaining, mouse PE-, FITC-, PEcy5-,PEcy7 membrane antibodys against human CD4(L3T4), CD25(IL-2R), CD127(hIL-7R-M21), GITR,CCR4(1G1), CTLA-4(CTLA4), and corresponding mouse isotype controls were used. Cells were permeabilized and stained with Foxp3-APC according to the manufacturer's instructions for fixation and permeabilization, after the cells were stained for surface expression.Results1. The percentage of CD4⁺CD25^highTregs was significantly decreased in active SLE(0.93±0.38%,P＜0.01) and inactive SLE 1.31±0.36%,P＜0.01)compared with healthy controls(2.47±0.08%), and correlated positively with SLEDAI.The percentage of CD4⁺CD25^highTregs increased significantly after treatment of prednisolone and immunosuppressive agents (P＜0.01).2. The expression of Foxp3 on CD4⁺cells was significantly decreased in active SLE(5.04±0.73%,P＜0.01) and inactive SLE (11.03±3.32%,P＜0.01) compared with healthy controls(15.81±6.25%).There was no significant difference in the expression of Foxp3 on CD4⁺CD25^high and CD4⁺CD25⁺ T cell subpopulations in SLE (P＞0.05). Furthermore, the expression of Foxp3 on CD4⁺CD25T cells in active SLE (7.32±4.58%, P＜0.01) and inactive SLE (4.63±2.01%, P＜0.01) was significantly increased compared with healthy controls (1.08±0.33%).3.The expression of GITR, CTLA-4 and CCR4 on CD4⁺CD25^-Foxp3⁺T cells had no significant difference with that on CD4⁺CD25⁺Foxp3T cells, whereas significantly lower than that on CD4⁺CD25⁺Foxp3⁺ T cells in patient with SLE. 4.There was no significant difference between the percentage of CD4⁺CD25⁺CD127^low/- T cells (4.72±0.96%) and CD4⁺CD25⁺Foxp3⁺Treg cells(4.48±0.78%) in patients with SLE. Moreover, our study showed a high expression of Foxp3 on CD4⁺CD25⁺CD127^low/- T cells (86.53±5.32%). Similarly, the expression of CD127^low/- on CD4⁺ CD25⁺Foxp3⁺T cells was also in a high level (92.1±2.38%).5. The expression of CD127^low/- on all the CD4⁺Foxp3⁺ T cells was in a high level (93. 6 + 2. 2%), no matter in CD4⁺127^low/-CD25^high,CD4⁺127^low/- CD25^low or CD4⁺127^low/- CD25^-subpopulations, whereas, the expression of Foxp3 on CD4⁺CD127^low/- T cells showed significantly difference between CD4⁺CD127^low/-CD25^high (91.38±2.57%) and CD4⁺CD127^low/- CD25^- subpopulations (9.02±2.21%).6.The frequency of CD4⁺CCR4⁺CD25^high T cells(1.10±0.17%,P＜0.01) was significantly lower in Lupus Nephritis compared with SLE without Lupus Nephritis(1.61±0.23%) and health controls(1.75±0.10%,). And the frequency of CD4⁺CCR4⁺CD25^low/- T cells(11.49±2.26%,P＜0.01)in Lupus Nephritis was significantly higher in Lupus Nephritis compared with healthy controls(8.04±0.96%).Conclusions1. The frequency of Tregs was decreased in the PBMC of SLE patients.CD4⁺CD25^high Tregs in PBMC of SLE was significantly decreased and correlated positively with SLEDAI.CD4⁺CCR4⁺CD25^high Treg was significantly decreased in Lupus Nephritis, which suggest the Tregs with CC chemokine receptor 4 might be involved in the pathogenesis of Lupus nephritis.2. The phenotype of increased CD4⁺CD25^-Foxp3⁺T cells might play a role of activated effective function in patients with SLE.3. The expression of Foxp3 on CD4⁺CD25T cells was significantly increased, which might suggest the activation of T cells instead of regulatory function.4. CD4⁺CD25⁺CD127^low/- T cells might facilitate the purification and sorting of live CD4⁺CD25⁺Foxp3⁺ regulatory T cells in SLE.5. CD127^low/- was sensitive, but not specific to Foxp3⁺ in CD4⁺T cell populationselected from PBMC in SLE patients. Whereas, CD127^low/- was not only sensitive but also specific to Foxp3⁺ in CD4⁺CD25^high T subpopulation, which might facilitate purification of Foxp3⁺ regulatory T cells in CD4⁺CD25^high T cells.