The Role Of Human CD4~+CD25~+ Regulatory T Cells In Immunosenscence And Tumor

Human T regulatory cells include many subsets, such as CD4⁺CD25⁺ regulatory cells (CD4⁺CD25⁺Treg), Tr1 and Th3. CD4⁺CD25⁺ regulatory cells have been the object of intense study because their function appears critical in maintaining self tolerance. These cells still express CD45RB^low, CD5^high, CD44^high, ICAM-1^high and LAF-1^high. But none of them is unique for CD4⁺CD25⁺ regulatory cells. Recent experimental studies indicate that Foxp3, which encodes a forkhead-winged helix transcription factor, is a key regulatory gene for the development and suppressive activity of regulatory T cells, and represents a more specific marker for Treg. CD127, the chain of the interleukin-7 receptor, has recently been identified as a specific marker for CD4⁺CD25⁺ regulatory cells. Human CD4⁺CD25⁺ regulatory cells express lower level of CD 127 than the other CD4⁺T cells. Expression of Foxp3 protein and the CD127^low phenotype were highly correlated within the CD4⁺CD25⁺ population. These findings will help us to research the relation between CD4⁺CD25⁺ regulatory cells and human immune function.CD4⁺CD25⁺ regulatory cells fail to proliferate or secrete cytokines (IL-2 or IFN-γ) in response to polyclonal or antigen-sepcific stimulation via their T cell receptor, but their anergic state can partially be reversed by high doses of interleukin (IL)-2. Upon stimulation with anti-CD3 plus anti-CD28mAb, the CD4⁺CD25⁺ regulatory cells in coculture experiments suppress the activation, proliferation and cytokine production of CD4⁺CD25^-responder T (T-resp) cells and CD8⁺ T cells. Studies also show that CD4⁺CD25⁺ regulatory cells can suppress anti-tumor immunity and tumor immunosurveillance which is mediated by tumor-specific CD8⁺ cytotoxic T lymphocytes (CTLs), T-resp and NK cells. Immunosenescence refers to the decline in the immune response associated with aging. In humans, the major causes of death in the elderly are infectious diseases, malignancy and uncontrolled infections that can be linked to immunosenescence. At the cellular level, evidence suggests that aging is associated with increases in the numbers of circulating CD4⁺CD25⁺ regulatory cells in peripheral blood. Therefore, CD4⁺CD25⁺ regulatory cells play important roles during immune reponses, as in immnnosenscence, tumors, infection, transplants, and graft versus host disease.Objectives (1) To explore the unique marker to discriminate CD4⁺CD25⁺ Treg cells, the percentage of CD4⁺CD25⁺ Treg cells in peripheral blood were determined with the multicolor detection and multiparameter flow cytometry. To study the correlation of CD4⁺CD25^high, CD4⁺CD25⁺FoxP3⁺ and CD4⁺CD25⁺CD127^low, and analyze the feasibility and dominance of using CD4⁺CD25⁺CD127^low to discriminate CD4⁺CD25⁺ Treg cells in peripheral blood. (2) To detect the relationship between CD4⁺CD25⁺ Treg cells and the higher prevalence of tumor in old people. (3) In vitro, CD4⁺CD25⁺ Treg cells in old tumor patients were isolated and their phenotype, proliferation and suppressive activity were analyzed. (4) To evaluate the significance of CD4⁺CD25⁺ Treg cells in human acute myeloid leukemia during different therapeutic stages.Methods (1) Two or three color monoclonal antibodies directly labeled with immunofluorescence were used to analyze the surface and cytoplasma antigens (such as CD4, CD25, CD127, FoxP3 et al.) by multiparameter flow cytometry. (2) CD4⁺CD25⁺ Treg cells were isolated from PBMCs by flow sorting (FACS), and their purity was assessed by multiparameter flow cytometry. (3) In vitro whether CD4⁺CD25⁺ Treg cells isolated by FACS undergo clonal expansion in response to stimulation with anti-CD3/ anti-CD28 mAb plus high doses of IL-2 was observed. (4) CD4⁺ CD25^- T responder cells were co-cultured with stimulator cells in the presence or absence of CD4⁺CD25⁺ Treg cells whose suppressive activity was evaluated by colorimetric assay. CD4⁺CD25⁺FoxP3⁺T and CD4⁺CD25⁺CD127^lwocells in the groups of health people and cancer patients were (1.769±0.682) % and (2.958±1.392) %, (2.905±1.772) %and (5.128±2.227) %, (5.396±1.306) % and (7.175±2.565) % respectively. There was the same change rule in CD4⁺CD25^high, CD4⁺CD25⁺FoxP3⁺ and CD4⁺CD25⁺CD127^low T cells of two groups. There was statistically different among CD4⁺CD25^high, CD4⁺CD25⁺FoxP3⁺and CD4⁺CD25⁺CD127^low T cells in three groups. (2) The reference ranges of CD4⁺CD25^high T cells among CD4+ lymphocytes in groups of health adults, healthy old people and elderly cancer patients were (1.390±0.412)%, (1.729±0.247)% and (2.150±0.769)% respectively , while CD4⁺CD25⁺FoxP3⁺T cells were (1.180±0.343)%, (2.477±0.400) % and (4.980±2.177)% respectively, and CD4⁺CD25⁺CD127^low T cells were (5.213±0.942)%,(6.186±1.196)% and (7.194±1.538)% respectively. Each marker has the same change rule. Elderly cancer patients was highest, then was healthy old people, and the last was health adults. Three markers were positive correlation in three groups (P<0.05). There was a statistically different in three groups of CD4⁺CD25^high, CD4⁺CD25⁺FoxP3⁺and CD4⁺CD25⁺CD127^low T cells. (3) After stimulation with 300U/mL IL-2 and anti-CD3 mAb plus anti-CD28 mAb, CD4⁺CD25⁺CD127^low T cells in elderly cancer patients expanded readily. Cultured with freshly isolated Treg cells CD4⁺ responder T cells did not proliferate. (4) Compare with the control group, the group which in complete remission over three years was lower, and the other groups were higher.Conclusions (1) CD4⁺CD25⁺CD127^low can be used to discriminate CD4⁺CD25⁺ Treg cells and some actived CD4⁺ T cells.This marker can improve the level of detected CD4⁺CD25⁺ Treg cells and the cytoactive does not influented. CD4⁺CD25⁺CD127^low may be an ideal marker to discriminate CD4⁺CD25⁺T reg cells. (2) The freshly isolated Treg and expanded Treg were anergic and suppressed the proliferation of autologous effective T cells. The percentages of CD4⁺CD25⁺T reg cells were increased with age. Its excessive accumulation may contribute to the development of immunosenescence and the tumor. This finding suggested that the deletion of the CD4⁺CD25⁺T reg cells may be an effective strategy to improve immunity of old people and treat cancer patients. (3) In addition to the group which in complete remission over three years, the other groups were higher than control group. These results suggest that CD4⁺CD25⁺Treg cells play an important role in acute myeloid leukemia.