Researches On Human CD4+CD25+ Regulatory T Cells And Its Clinical Significance In Childhood Acute Leukemia

The naturally arising CD4+CD25+ regulatory cells (nTreg) develop in the thymus of humans. They are genetically controlled, influenced by antigen recognition and various signals, in particular, cytokines such as interleukin-2(IL-2) and transforming growth factor-beta1(TGF-β) controlling their activation, expansion, and suppressive effector activity. The development of human nTreg encompasses two sequential maturation steps: initiation of a regulatory phenotype and suppressive activity in the thymus; and subsequent activation within the peripheral lymphoid organs. Human nTreg cells, which comprise 5-10% of peripheral CD4+ T cells, characteristically express CD25 (IL-2 receptorαchain), cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR), and lymphnode homing receptors L-selectin (CD62L). Recent study shows 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.Treg 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-CD28 mAb, the Treg cells in coculature experiments suppress the activation, proliferation and cytokine production of CD4+CD25-responder T (T-resp) cells and CD8+ T cells. Studies also show that Treg 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. Treg cells play an important role in the maintenance of immunological self-tolerance, control of transplantation tolerance and tumor immunity.Objectives⑴To explore the survey protocol of human peripheral blood CD4+ CD25+ regulatory T cells and establish a normal reference range of various ages. (2) To explore the phenotypic characters and the roles of CD4+ CD25+ T cells in human umbilical cord blood (CB) and newborn PB. (3)In vitro, CD4+CD25+ T cells were isolated and their phenotype, proliferation and suppressive activity were analyzed. (4) To evaluate the significance of Treg cells in childhood acute lymphocyte 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, CD62L, CD152, FoxP3 et al.) by multiparameter flow cytometry. (2) CD4+CD25+ T cells were isolated from PBMCs with CD4+CD25+ Regulatory T cell Isolation Kit (MACS, Miltenyi Biotec), and their purity was assessed by FACS. (3) In vitro whether CD4+CD25+ T cells isolated by MACS 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 cocultured with stimulator cells in the presence or absence of CD4+CD25+ T cells whose suppressive activity was evaluated by colorimetric assay.Results (1) At the time points of 0,1,2,3,4,5 and 6 hours, the proportion of CD4+ CD25hi of peripheral blood CD4+ lymphocytes was (1.41±0.26)%,(1.45±0.26) %,(1.32±0.26)%,(1.27±0.28)%,(1.09±0.24)%,(0.95±0.18)%,(0.91±0.21)%. MFI was used as a guideline to set the regions of CD4+CD25hi, and over 85% CD4+CD25hi T cells expressed Foxp3. (2) The reference ranges of CD4+CD25hi T cells in the groups of age 4-6,7-14,20-59 and elder than 60 was (0.92±0.30)%,(1.21±0.45)%,(1.44±0.52)% and (1.51±0.55)% respectively. (3) CD4+CD25+ T cells from CB and newborn peri- pheral blood are a distinct population mainly expressing CD45RA. (4) After stimulation with 300U/mL IL-2 and anti-CD3 mAb plus anti-CD28 mAb, CD4+CD25+ T cells expanded readily and nearly 1300-fold for 12 days. Cultured with Treg cells CD4+ responder T cells did not proliferate. (5) Within standard-risk ALL patients the pro- portion of CD4+CD25hi was (1.04±0.33)% in the end of first course of induction treatment groups, (1.29±0.30)% in continued complete remission groups (over 3 years),(1.60±0.44)% in maintenane treatment groups respectively,while in the intermediate and high risk ALL patients during maintenane therapy the number was (2.24±0.75)%.Conclusions (1)We have established an optimal protocol of detection of human peri- pheral blood CD4+CD25+ regulatory T cells and certificated that CD4+CD25hi T cells can be used to specifically respent CD4+CD25+ regulatory T cells. (2) Our study discovered that the number of peripheral blood CD4+CD25+ regulatory T cells varied with age, and a normal reference range was established. (3)The results suggest that as a functionally mature population CD4+CD25+ regulatory T cells in CB and newborn peri- pheral blood have distinct phenotypic characters. (4)Under specific cultural conditions CD4+CD25+ regulatory T cells can expand intensively while maintaining its suppressive activity. (5) Compared with healthy individuals, the number of Treg cells in ALL is significantly higher except in the end of first course of induction treatment groups and may be related to the effect of chemical treatment, severity of ALL and disease relapse.