| BackgroundPig-derived xeno organs are an important source to address the shortage of transplant donors,however,controlling the immune response to xenotransplantation requires strong anti-rejection therapy.Regulatory T cells(Treg),especially graft antigen-specific Treg,can specifically inhibit the acquired immune rejection of xenotransplantation,and have stronger inhibitory function.Therefore,exploring and discovering xenoantigen-specific Treg subsets can effectively protect xenografts and reduce the number of adoptively infused cells,which is benefit to the development of xenotransplantation.ObjectiveThis project aims to screen an appropriate surface marker for sort xeno antigen-specific Treg subset,and evaluate the immune suppressive function,xeno antigen specificity,stability and possible molecular mechanisms of this subset.Methods1.Treg cells were extracted from peripheral blood mononuclear cells(PBMC)of healthy adult donor,and polyclonal(poly-)or xeno-antigens(xeno-)were used to stimulate and expand Treg cells in vitro.Evaluate the suppression of poly-Treg and xeno-Treg by xeno-mixed lymphocytes reaction(xeno-MLR).2.FACS was used to screen suitable surface marker of xeno-antigen specific Treg via compare the phenotype of poly-Treg and xeno-Treg.And evaluate the magnetic bead sorting and flow sorting by purity and yield.3.Suppression of CD27~+and CD27~-xeno-Treg compared by xeno-MLR,and the xeno-antigen specificity by poly-and allo-MLR.4.The expression of m RNA and protein of Treg function related markers was evaluated by RT-q PCR,west blotting and FACS.5.TSDR demethylation and Th17 induction with inflammatory conditioned(exogenous inflammatory factors and PBMC-secreted inflammatory factors were used to mimic inflammatory condition)were used to evaluate stability of Treg.6.Western blot used to analyze the JAK3-STAT5 pathway related protein levels.Expression of functional markers have been tested by FACS after p STAT5 inhibition.7.Use NCG mice to establish a humanized immune system xenogeneic skin transplantation mouse model,adoptively transfer CD27~+and CD27~-xeno-Treg into the mouse model to study the protective effect of CD27~+xeno-Treg on xenografts in vivo.Results1.In xeno-MLR in vitro,the suppressive percentage of xeno-Treg at the Treg:respond cells ratio of 1:4 and 1:16 was 84.48%(±2.11%)and 54.18%(±4.59%),higher than poly-Treg(57.29%±4.84%,26.40%±6.20%)(P<0.001).2.After 21 days expansion,the expression of CD27(61.08%66.98%)was significantly upregulated in xeno-Treg than poly-Treg(16.18%±3.74%,P<0.01).The yield of CD27~+xeno-Treg sorted by magnetic bead was 2.28%(±0.86%),which was significantly lower than flow sorting method(43.19%±6.26%).The purity of CD27~+and CD27~-xeno-Treg separated by flow sorting was 97.59%(±1.88%)and 93.95%(±5.6%),respectively.Viability of sorted cells is greater than 90%after overnight culture.3.In xeno-MLR,the suppressive capacity of CD27~+xeno-Treg at ratio of Treg:respond cells is 1:4(81.46%±5.78%)and 1:16(47.98%±9.42%)is stronger than CD27~-xeno-Treg(70.25%±11.75%,P<0.05 and25.01%±9.60%,P<0.0001).In allo-MLR and poly-MLR,the suppression between CD27~+and CD27~-xeno-Treg has no statistic difference.4.The m RNA and protein levels of Foxp3,Helios and CLTA4 in CD27~+xeno-Treg were statistically higher than CD27~-xeno-Treg,however,the m RNA expression of inflammatory factors IL17 and IFN-γare lower in CD27~+xeno-Treg.5.The demethylation of Treg-specific demethylation region(TSDR)in CD27~+xeno-Treg(89.41%±5.47%)was higher than CD27~-xeno-Treg(67.54%±9.20%,P<0.01).Under Th17 induction condition,the proportion of IL17~+cells in CD27~+xeno-Treg(0.45%±0.25%)was lower than CD27~-xeno-Treg(7.64%±0.58%,P<0.0001).Under PBMC inflammatory condition,the proportion of IL17~+cells in CD27~+xeno-Treg(0.34%±0.10%)was also lower than CD27~-xeno-Treg(3.89%±0.11%,P<0.0001).6.The protein level of activated JAK3(p-JAK3)and STAT5(p-STAT5)in CD27~+xeno-Treg is higher than that of CD27~-xeno-Treg.After inhibited p STAT5,the expression of Foxp3,Helios and CTLA4 were downregulated but no difference observed in CD27~-xeno-Treg.7.In immunofluorescence staining of xenogeneic skin grafts from mice model,the average number of CD4~+and CD8~+T cells per field(40×)in the CD27~+Treg group(43.75±9.54)was less than CD27~-Treg group(70.5±17.62,P<0.01).The CD27~+Treg group(12.25±1.28)had more CD4~+Foxp3~+cells per field(40×)than CD27~-Treg group(5.63±1.06,P<0.0001).Conclusions1.CD27~+xeno-Treg show xeno-antigen-specific suppressive function in vitro,which may be a xeno-antigen-specific Treg subset.The flow sorting method is suitable for the sorting of CD27~+xeno-Treg subset.2.CD27~+xeno-Treg has higher suppressive related markers,and more stable under inflammatory conditions,which may relate to the activation of JAK3-STAT5 pathway.3.CD27~+xeno-Treg can target and protect xenogeneic skin grafts. |
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