CD4+ CD25+ FOXP3+ regulatory T cells from human thymus and cord blood suppress antigen-specific T cell responses

Abstract

Activation of self-reactive T cells in healthy adults is prevented by the presence of autoantigen-specific CD4+CD25+ regulatory T cells (CD25+ Treg). To explore the functional development of autoantigen-reactive CD25+ Treg in humans we investigated if thymic CD25+ Treg from children aged 2 months to 11 years and cord blood CD25+ Treg are able to suppress proliferation and cytokine production induced by specific antigens. While CD4+CD25- thymocytes proliferated in response to myelin oligodendrocyte glycoprotein (MOG), tetanus toxoid and beta-lactoglobulin, suppression of proliferation was not detected after the addition of thymic CD25+ Treg. However, CD25+ Treg inhibited interferon (IFN)-gamma production induced by MOG, which indicates that MOG-reactive CD25+ Treg are present in the thymus. In contrast, cord blood CD25+ Treg suppressed both proliferation and cytokine production induced by MOG. Both cord blood and thymic CD25+ Treg expressed FOXP3 mRNA. However, FOXP3 expression was lower in cord blood than in thymic CD25+ T cells. Further characterization of cord blood CD25+ T cells revealed that FOXP3 was highly expressed by CD25+CD45RA+ cells while CD25+CD45RA- cells contained twofold less FOXP3, which may explain the lower expression level of FOXP3 in cord blood CD25+ T cells compared to thymic CD25+ T cells. In conclusion, our data demonstrate that low numbers of MOG-reactive functional CD25+ Treg are present in normal thymus, but that the suppressive ability of the cells is broader in cord blood. This suggests that the CD25+ Treg may be further matured in the periphery after being exported from the thymus.

Figure 5

Flow cytometric analysis of CD25 expression and analysis of FOXP3 expression by quantitative RT-PCR in thymus and cord blood. (a) One representative histogram depicting bead-purified CD4⁺CD25⁺ T cells (thick line), CD4⁺CD25⁻ (thin line) and isotype control (broken line) is shown for thymus and cord blood. (b) Normalized ratios of FOXP3/GAPDH in the bead-purified CD25⁺ and CD25⁻ T cell fractions of thymus (n = 4) and cord blood (n = 9). (c) One representative contour plot of CD4⁺ cord blood T cells used for FACS-sorting depicting CD25⁺CD45RA⁺, CD25⁻CD45RA⁺ and CD25⁺CD45RA⁻, CD25⁻CD45RA⁻ T cells. (d) Normalized ratios of FOXP3/GAPDH in the FACS-sorted T cell fractions of cord blood (n = 4). Each dot represents one individual and horizontal bars depict median values. Statistical significance between cultures is shown as, *P < 0·05, **P < 0·01.

Figure 1

Capacity of thymic CD4⁺CD25⁺ T cells to suppress proliferation of CD4⁺CD25⁻ thymocytes after antigen-specific stimulation. (a–c) Absolute level of proliferation of thymic CD25⁻ and CD25⁺ T cells cultured alone or together in a 1:1 ratio after stimulation with MOG (a), beta-LG, (b) and TT (c). Data points from the same individual are connected by lines. (d,e) Percentage of proliferation relative to the level of CD25⁻ T cells for each individual after stimulation with MOG (D, n = 13), beta-LG (E, n = 8) and TT (F, n = 8). Bars depict mean percentage proliferation ± SEM. Statistical significance between cultures is shown as: *P < 0·05, **P < 0·01, ***P < 0·001.

Figure 2

Cytokine levels in cultures of thymic cells after stimulation with specific antigens. (a) Production of IFN-γ from thymic CD4⁺CD25⁻ T cells, CD4⁺CD25⁺ T cells and cocultures thereof stimulated with MOG (n = 6). (b) IL-10 production after stimulation with MOG (n = 7). (c) IFN-γ production after stimulation with beta-LG (n = 3). (d) IL-10 production after stimulation with beta-LG (n = 3). Lines connect data points from the same individual and horizontal bars depict median values. Statistical significance between cultures is shown as: *P < 0·05.

Figure 3

Capacity of CD4⁺CD25⁺ T cells to suppress proliferation and cytokine production of CD4⁺CD25⁻ T cells in cord blood after stimulation with MOG. (a) Absolute level of proliferation of cord blood CD25⁻ and CD25⁺ T cells cultured alone or together in a 1:1 ratio after stimulation with MOG (n = 12). (b) Percentage of proliferation relative to the level of CD25⁻ T cells for each individual after stimulation with MOG (n = 12). Bars depict mean percentage proliferation ± SEM. (c,d) Levels of IFN-γ (c, n = 12), IL-13 (d, n = 12) and IL-10 (e, n = 9) in cultures from cord blood CD25⁻ T cells, CD25⁺ T cells and cocultures thereof stimulated with MOG. Lines connect data points from the same individual and horizontal bars depict median values. Statistical significance between cultures is shown as, *P < 0·05, **P < 0·01, ***P < 0·001.

Figure 4

Capacity of CD4⁺CD25⁺ T cells from thymus and cord blood to suppress proliferation and cytokine production of CD4⁺CD25⁻ T cells after SEB stimulation. (a,e) Proliferation of thymus (a, n = 12) and cord blood (e, n = 11) CD25⁻ T cells, CD25⁺ T cells and cocultures thereof after stimulation with SEB. Data points from the same individual are connected by lines. (b–d) Levels of IFN-γ (b, n = 11), IL-13 (c, n = 11) and IL-10 (d, n = 7) in cultures of thymic CD25⁻ T cells, CD25⁺ T cells and cocultures thereof. (f–h) Production of IFN-γ (f, n = 11), IL-13 (g, n = 11) and IL-10 (h, n = 11) from cord blood CD25⁻ T cells, CD25⁺ T cells and cocultures thereof. Individual levels are shown as dots and horizontal bars depict median values. Statistical significance between cultures is shown as: *P < 0·05, **P < 0·01, ***P < 0·001.