Expression Of Foxp3 In CD4⁺CD25^high Treg Cells Of Neonatal Cord Blood

Background and Objective CD4⁺CD25⁺ regulatory T cells are one of T lymphocyte subpopulations, which possess immunological regulating function. Considerable evidence has accumulated for the existence of CD4⁺CD25⁺ T regulatory(Treg) cells that regulate self and allograft tolerance and tumor immune escaption. It has been shown that only CD4⁺CD25^high Treg cells had inhibit function in adult's peripheral blood. In more recent studies it has been confirmed that the forkhead transcription factor Foxp3 is a critical regulator of CD4⁺CD25^high Treg cells development and function. Today the generation, development, maturation and functional molecular mechanism of this cell type have yet to be realized.The purposes of this study are to investigate the amount of CD4⁺CD25^high Treg cells and the expression of Foxp3 of Treg cells in neonatal cord blood, to observe the expression of Foxp3 and suppressor cytokine IL-10 in CD4⁺CD25⁺ Treg cells induced by transforming growth factor-β1(TGF-β1), to explore the role of TGF-β1 in induced Treg cells(iTregs) in neonatal cord blood , and to provide rationale for clinical immunosupressive therapy.Methods 1. After the mononuclear cells were isolated from neonatal cord blood or healthy adult's peripheral blood, the amount of CD4⁺CD25^high Treg cells and intranuclear transcription factor Foxp3 were measured by flow cytometry. 2. Cord blood mononuclear cells (CBMC) were stimulated with CD3mAb and cultured in 10% NBS-1640 medium in presence of IL-2, with or without different concentrations of TGF-β1, the expressions of Foxp3 protein and Foxp3 gene mRNA in CD4⁺CD25⁺i Treg cells were detected by flow cytometry and RT-PCR assay, respectively. 3. CBMC were labeled with CFSE, and then stimulated with 5ng/mL TGF-β1. The phenotypes of proliferated lymphocytes on day 4, day 6, day 8 after culture were determined by flow cytometry. The dynamics of proliferation of the activated T cell subpopulations were analyzed by the ModFit software. 4.Cytokine IL-10 in supernatant from cultured CBMC with different concentrations of TGF-β1 were detected by enzyme linked immunosorbent assay (ELISA).Results 1. The amount of CD4⁺CD25^high Treg cells in CD4⁺ T cells in cord blood was significantly higher than that in adult's peripheral blood (3.86±1.63% vs 0.87±0.74% P<0.01), whereas the expression of Foxp3 in CD4⁺CD25^high Treg cells in cord blood was markedly lower than that in adult's blood (23.21±8.9% vs 71.3±11.6% P<0.01). 2. After CBMC cultured for 4 days with TGF-β1 at concentrations of 1ng/mL, 5ng/mL, and 10ng/mL, the expressions of Foxp3 in CD4⁺CD25⁺ T cells, compared to control(31.64±7.59%), were(48.39±13.51%, P>0.05), (61.35±6.79%, P<0.01), and (56.34±5.11%, P<0.05), respectively, whereas the expression of Foxp3 in CD4⁺CD25^high T cells, compared to control(47.18±8.13)%, were (69.28±3.91)%, (78.20±4.73)%, and(69.96±8.48)%, respectively(all P<0.01). 3. By using RT-PCR technique, the photodensity value of Foxp3/β-action in groups of 1ng, 5ng, 10ng per ml TGF-β1 were (0.440), (0.727), and (0.584), respectively, while that in control group(0.317). 4. ModiFit software anaslysis showed different proliferation were observed between CD4⁺CD25⁺Treg and CD8⁺ T cells, in which CD4⁺CD25⁺ Treg cells had greater reproduction at the same time point. Proliferation Index(PI) on day 6 of culture in CD4⁺CD25⁺ Treg (11.52) had significantly higher than that in CD8⁺ T cells(4.23). PI on day 8 of culture in CD4⁺CD25⁺ Treg (23.04) had markedly higher than that in CD8⁺ T cells(5.43). Proportion of the generations from 5 to 6 on day 4 of culture in CD4⁺CD25⁺ Treg (27.75%) had notably higher than that in CD8⁺ T cells(4.89%), proportion of the generations from 5 to 8 on day 6 of culture in CD4⁺CD25⁺T(81.49%) had significantly higher than that in CD8⁺ T cells(44.57%), proportion of the generations from 9 to 10 on day 8 of culture in CD4⁺CD25⁺ Treg (52.74%) had strikingly higher than that in CD8⁺ T cells(13.54%). 5. ELISA analysis showed IL-10 concentration(pg/mL) in supernatant from CBMC cultured with TGF-β1 at 1ng, 5ng, and 10ng per ml were (72.00±10.15), (133.63±25.75), and(136.14±15.53), respectively, were significantly higher than that in control group (49.92±10.37, all P<0.01).Conclusions 1. The low expression of Foxp3 in CD4⁺CD25^high Treg cells in cord blood suggests Treg cells in neonates are not mature in suppression. 2. In vitro, TGF-pi can expand the expression of the CD4⁺CD25⁺Foxp3⁺ Treg cells and increase the secretion of IL-10 in cord blood.