Human CD4~+CD62L~+CCR7~+Central Memory T Cells Can Be Converted To Functional Foxp3~+Regulatory T Cells

Regulatory T cells is a population of T cells with regulatory function that differ-ent from Thl, Th2and Th17cells, they play an important role in self-tolerance and acquired tolerance as well as immunological homeostasis. There are multiple types of immune regulatory T cells, including Trl cells, natural killer T cells, a subset of CD8+T cells, and CD4+CD25+Foxp3+cells. CD4+CD25+Foxp3+cells (commonly known as Tregs) are the predominant regulatory cells. Deficiency or dysfunction of Tregs leads to the development of various autoimmune diseases. Tregs can be divided into natural arising Treg cells (nTregs) generated in the thymus and adaptive Treg cells (iTregs) generated in the peripheral through antigen stimulation, both of which are implicated in immune regulation. It is well known that nTregs are derived from the thymus, but the exact origin of iTregs remains incompletely defined.Previous studies suggested that iTregs may be derived from various conditions outside the thymus. It is clear that iTregs are converted from T effector cells in the periphery. And further in vitro studies found that iTregs can be efficiently differen-tiated from purified CD4+CD25-T cells stimulation with TGF-β.Naive CD4+T cells and memory CD4+T cells both can formation effect T cells after stimulation, thus which effect T cells will differentiation into iTregs is unknow. Recently, other studies reported that of CD4+CD25-T cells, only the naive CD4+T cells (Tn) but not the memory CD4+T cells (Tm) were able to differentiate into iTregs in vitro both in mouse and human models. So it is generally accepted that iTregs are converted from T effector cells that originate from activated naive CD4+CD25-T cells in vivo. However, there is controversial evidence regarding this view. One report showed that human iTregs were derived from peripheral memory CD4+Foxp3-T cells in vivo by isotopes tracking experiments and another found that mouse Th2memory T cells could also be efficiently converted into Foxp3+Tregs with a differentiation protocol in vitro. Thus, the possibility that iTregs are converted from T effector cells that are derived from activated memory CD4+CD25-T cells in vivo cannot be excluded.Because iTregs play essential roles in developing immune tolerance to foreign antigens and tumor derived neo-antigens, a better understanding of their origin and differentiation would provide important information regarding their therapeutic poten-tial. Our study wanted to define the origin of effect T cells that could differentiation into Tregs in vivo. Through flow cytometer analyzing the peripheral blood of HBV infected patients and inducing iTregs from flow cytometer sorted naive CD4+T cells, we found that naive CD4+T cells might not be the source of iTregs de-rived from effect T cells. Through phenotype analysis we found a subset of memoy CD4+T cells which could be induced into iTregs, and determined the suppressive function of these cells by CFSE labeling assay. And we analyzed the TSDR sequence of iTregs that derived from memory CD4+T cells and naive CD4+T cells by bisulfite sequencing PCR. The results and conclusions are shown as follow:1. CD45RO distinguishes the different between iTregs in vivo an in vitroPurified naive CD4+T cells from human PBMCs were activation in vitro by an-ti-CD3/anti-CD28plus TGF-β to generate Foxp3+iTregs, these iTregs were rarely co-expressed memory cell phenotype CD45RO with Foxp3. PBMCs from HBV-infected pregnant (n=38) and healthy pregnant (n=22) women were analyzed by flow cytometry, we found that the proportion of CD4+Foxp3+Tregs was significantly higher in HBV-infected pregnant women than the healthy controls, further statistic analysis found that only the CD45RO+CD4+Foxp3+Treg proportion was increased, and the CD45RO-CD4+Foxp3+Treg proportion had no change.The above results collectively showed that iTregs generated in vitro had different surface CD45RO expression from in vivo chronic virus infection-induced iTregs, which suggested that iTregs induced in vivo might not be directly derived from naive CD4+T cells.2. CD4+CD62L+CCR7+central memory T cells can be induced to differentiate into iTregsPurified memory CD4+T cells are difficult to be induced into iTregs in vitro. However, a small undefined population of memory CD4+T cells could be induced to express Foxp3. Through analysis the capability of different subsets of memory CD4+t cells to be induced to differentiate into iTregs, we found that CD62L+CCR7+central memory T cells can be induced to differentiate into iTregs efficaciously.The above results suggested that although overall memory CD4+T cells were re-sistant to differentiate into iTregs, a subset of memory CD4+T cells defined as CD62L+CCR7+central memory T cells had the capability to be induced to differen-tiate into iTregs. 3. Induced iTregs from CD4+CD62L+CCR7+memory T cells have potent suppressive function in vitroInduced iTregs from CD62L+CCR7+memory CD4+T cells or naive CD4+T cells were stimulated with CFSE labeled CD4+CD25" T cells, the proliferation of CFSE labeled cells showed that both iTregs had suppressive function, and cells de-rived from CD62L-CCR7-memory CD4+T cells through induction process did not exhibit any suppressive function.These results showed that CD62L+CCR7+central memory CD4+T cells could be induced to differentiate into functional iTregs.4. iTregs derived from CD4+CD62L+CCR7+central memory T cells showed signifi-cantly more FOXP3TSDR demethylationBisulfite sequencing PCR analysis of the status of the CpG sites in FOXP3TSDR sequence among iTregs derived from CD62L+CCR7+central memory CD4+T cells and naive CD4+T cells and purified Tregs. The results showed that CD62L+CCR7+central memory CD4+T cells derived iTregs displayed more deme-thylation of TSDR sequence than naive CD4+T cells derived iTregs, but less than pu-rified Tregs. These results indicated that CD62L+Tcm cells derived iTregs might have a more stable Treg phenotype than those from naive CD4+T cells.In conclusion, we proposed for the first time that CD62L+CCR7+central memory CD4+T cells can be induced into iTregs, and iTregs might be converted from T effec-tor cells that are derived from activated CD62L+CCR7+central memory CD4+T cells in vivo. Our study provided novel information on the origin of human iTregs and might have important clinical value in adoptive Tregs therapy.