Co-expression of TNFR2 and CD25 identifies more of the functional CD4+FOXP3+ regulatory T cells in human peripheral blood

Abstract

Previously, we found that co-expression of CD25 and TNFR2 identified the most suppressive subset of mouse Treg. In this study, we report that human peripheral blood (PB) FOXP3(+) cells present in CD25(high), CD25(low) and even CD25(-) subsets of CD4(+) cells expressed high levels of TNFR2. Consequently, TNFR2-expressing CD4(+)CD25(+) Treg included all of the FOXP3(+) cells present in the CD4(+)CD25(high) subset as well as a substantial proportion of the FOXP3(+) cells present in the CD4(+)CD25(low) subset. Flow cytometric analysis of PB identified five-fold more Treg, determined by FOXP3 expression, in the CD4(+)CD25(+)TNFR2(+) subset than in the CD4(+)CD25(high) subset. In addition, similar levels of FOXP3(+) cells were identified in both the CD4(+)CD25(+)TNFR2(+) and CD4(+)CD25(+)CD127(low/-) subsets. Furthermore, the CD4(+)CD25(+)TNFR2(+) subset expressed high levels of CTLA-4, CD45RO, CCR4 and low levels of CD45RA and CD127, a phenotype characteristic of Treg. Upon TCR stimulation, human PB CD4(+)CD25(+)TNFR2(+) cells were anergic and markedly inhibited the proliferation and cytokine production of co-cultured T-responder cells. In contrast, CD4(+)CD25(+)TNFR2(-) and CD4(+)CD25(-)TNFR2(+) T cells did not show inhibitory activity. As some non-Treg express TNFR2, the combination of CD25 and TNFR2 must be used to identify a larger population of human Treg, a population that may prove to be of diagnostic and therapeutic benefit in cancer and autoimmune diseases.

Figure 1. Human PB FoxP3⁺ Tregs express high levels of TNFR2

Freshly isolated human PBMCs were stained for CD4, CD25 and TNFR2 and then were fixed and stained intracellularly for FoxP3. CD4 cells were analyzed by FACS, gating on lymphocytes via their forward and side scatter properties and CD4 staining. (A) FoxP3 and TNFR2 expression by CD25^hi, CD25^lo and CD25⁻ subsets of CD4 cells. (B) Percentage of TNFR2-expressing cells in CD4 subsets. (C) FoxP3 and TNFR2 expression by total CD4 cells, CD4⁺CD25^hi cells, CD4⁺CD25^lo cells and CD4⁺CD25⁻ cells. Date shown in (A) and (C) are representative of at least three separate experiments on different donors with similar results. Data shown in (B) are summarized from three different donors (mean ± SEM, N=3). The numbers in the quadrants indicate the percentage of positive cells. The numbers in histograms are mean fluorescence intensity (MFI) and percentage of positive cells (%). Dashed line histogram shows isotype control. Comparison of TNFR2 expression on CD25^loFoxP3⁺ and CD25^loFoxP3⁻ or CD25⁻FoxP3⁻ and CD25⁻FoxP3⁺ cells: ** P<0.01.

Figure 2. FoxP3 expression by human PB CD4⁺CD25⁺TNFR2⁺ cells

Freshly isolated human PBMCs were stained for CD4, CD25 and TNFR2. The cells then were fixed and stained intracellularly for FoxP3. For FACS analysis, the PBMCs were gated on CD4⁺ lymphocytes based on forward and side light scatter and CD4 staining. (A) Expression of CD25 and TNFR2 on PB CD4 cells. Data from three different donors are shown. The numbers in the quadrants indicate the percentage of positive cells. (B) Expression of FoxP3 by CD4⁺CD25^hi cells (upper panel) and CD4⁺CD25⁺TNFR2⁺ cells (lower panel). Data shown are representatives of at least three separate experiments on different donors with similar results. The numbers in the dot plots are the number of gated cells. The numbers in histograms are the percentage of positive cells (%). (C) Expression of FoxP3 by different subsets of PB CD4 cells. The data shown are summarized from eight different donors (mean ± SEM, N=8). Comparison of percentage of FoxP3⁺ cells in CD25⁺TNFR2⁺ subset and CD25⁺TNFR2⁻ subset or CD25⁻TNFR2⁺ subset and CD25⁻TNFR2⁻ subset: ** P<0.001.

Figure 3. Phenotype of human PB CD4 subsets

Freshly isolated human PBMCs were stained for CD4, CD25, TNFR2 and additional phenotypic markers. Intracellular expression of CTLA-4 and surface expression of CD45RO, CD45RA, CCR4, CCR7, CD127 and HLA-DR by different subsets of PB CD4 cells, gating on indicated populations were analyzed by FACS. Black filled histogram: antibody staining; grey histogram: isotype control. Numbers in the figures indicate the percentage of gated cells expressing relevant marker. Data shown are representative of at least three separate experiments on different donors with similar results.

Figure 4. Comparison of functional capacities of human PB CD4 T cell subsets

CD4⁺CD25⁺TNFR2⁺ and CD4⁺CD25⁻TNFR2⁻ T cells were FACS-sorted from freshly isolated PBMCs. 2.5×10⁴ cells/well of different CD4 subsets were cultured alone. The cells were stimulated with APCs and anti CD3 Ab for 72 h. Proliferation was determined by [³H] thymidine incorporation assay (A). IFNγ level in the supernatants were determined (B). Data shown are two separate experiments on different donors.

Figure 5. Suppressive activities of human PB CD4 T cell subsets

CD4⁺CD25⁺TNFR2⁺, CD4⁺CD25⁺TNFR2⁻, CD4⁺CD25⁻TNFR2⁺ and CD4⁺CD25⁻TNFR2⁻ T cells were FACS-sorted from freshly isolated PBMCs. In some experiment MACS-purified CD4 cells were used as responder cells. (A) 2.5×10⁴ cells/well of CD4⁺CD25⁻TNFR2⁻ cells, used as responder cells, were cultured alone or co-cultured with indicated CD4 subsets at ratio of 1:1. Background proliferation (APC alone, cpm: 1888.7) was subtracted. (B) 2.5×10⁴ cells/well of CD4⁺CD25⁻TNFR2⁻ cells were cultured alone or co-cultured with CD4⁺CD25⁺TNFR2⁺ cells at indicated ratios. Proliferation was determined by [³H] thymidine incorporation assay. (C) 5×10⁴ cells/well of CD4 cells were labeled with CFSE, cultured alone or co-cultured with indicated CD4 subsets at 1:1 ratio. (D) 5×10⁴ cells/well of MACS-purified CD4 cells were labeled with CFSE, cultured alone or co-cultured with indicated ratio with FACS-purified CD4⁺CD25⁺TNFR2⁺ cells. After 72 h incubation, dilution of CFSE by responder CD4 cells was determined by FACS. The cultured cells were stimulated with APCs and anti CD3 Ab. (A~D) Representative data from 3 separate experiments on different donors with similar results were shown. (E) Percent inhibition of proliferation of CD4⁺CD25⁻TNFR2⁻ T cells by CD4⁺CD25⁺TNFR2⁻ cells and CD4⁺CD25⁺TNFR2⁺ cells [(CPM (responder cells alone)-CPM (responder cells co-cultured with Treg cells))/CPM (responder cells alone)×100%]. Data shown were summarized from three separate experiments on different donors. (F) MACS-purified CD4 cells and FACS-purified CD4⁺CD25⁺TNFR2⁺ cells were cultured alone or co-cultured at ratio of 1:1. The cells were stimulated with APCs and anti CD3 Ab for 72 h. IFNγ level in the supernatants were determined. Data shown are representative of three separate experiments on different donors with similar results. Data in (E–F) are presented as Mean and SEM.