TNFR2 Expression on CD25(hi)FOXP3(+) T Cells Induced upon TCR Stimulation of CD4 T Cells Identifies Maximal Cytokine-Producing Effectors

Abstract

In this study, we show that CD25(hi)TNFR2(+) cells can be rapidly generated in vitro from circulating CD4 lymphocytes by polyclonal stimuli anti-CD3 in the presence of anti-CD28. The in vitro induced CD25(hi)TNFR2(+) T cells express a conventional regulatory T cells phenotype FOXP3(+)CTLA4(+)CD127(lo/-), but produce effector and immunoregulatory cytokines including IL-2, IL-10, and IFN-g. These induced CD25(hi)TNFR2(+) T cells do not suppress target cell proliferation, but enhance it instead. Thus the CD25(hi)TNFR2(+) phenotype induced rapidly following CD3/28 cross linking of CD4 T cells identifies cells with maximal proliferative and effector cytokine-producing capability. The in vivo counterpart of this cell population may play an important role in immune response initiation.

Keywords: FOXP3; TNFR2; Th1; effector cells; regulatory T cells.

Figures

Figure 1

Phenotype of induced TNFR2+ T cells. (A) The expression of CD25 and TNFR2 on MACS sorted CD4+CD25− T cells and CD4+CD25+ cells after 72 h of CD3/28 stimulation. (B) The expression of FOXP3 on TNFR2 subsets induced from starting population CD4+CD25+ T cells. (C) Comparison of percentages of CD25hiTNFR2+ T cells induced from the different starting populations – the CD4+CD25−, CD4+CD25+, or un-fractionated CD4+ T cells. (D) Expression of regulatory molecules on TNFR2 subsets from starting population CD4+CD25+ T cells. Gray histograms represent isotype staining while clear histogram represents the indicated molecule. The numbers indicate percentage positive for the represented molecular marker. (E) The MFI of FOXP3 within the TNFR2 subsets. (F) The mRNA expression levels of FOXP3 within the TNFR2 subsets. Data shown in (A,B,D) are representative of four donors respectively while (C,E,F) are summarized from four donors. Kruskal–Wallis with Dunn’s multiple comparison test was used here and graphs represent mean ± SEM. *p < 0.05.

Figure 2

FOXP3 expression levels on ex vivo and induced TNFR2+ T cells. Flow cytometry was performed on both ex vivo PBMCs (N = 14) and in vitro induced T cells (N = 4). These cells were initially gated on CD3, CD4, CD25, and TNFR2 expression to identify the different TNFR2 populations, noting that the CD25/TNFR2 phenotype was considerably different between fresh and cultured cells. (A) FOXP3 expression was further compared between CD25−TNFR2− (tinted histogram), CD25intTNFR2int/− (thin clear histogram), and CD25hiTNFR2+ (thick clear histogram). (B) The FOXP3 expression levels were compared between ex vivo CD25hiTNFR2+ and induced CD25hiTNFR2+ T cells. Unpaired Student’s t-test was performed to compare FOXP3 levels and graphs represent mean ± SEM. ****p < 0.0001.

Figure 3

Suppressive capacity of induced TNFR2+ T cells. CD4+ T cells were stimulated with anti-CD3/28 for 72 h. On day 3, induced CD25intTNFR2int/− and CD25hiTNFR2+ were sorted by flow cytometry and added to autologous responders (CD4+ T cells) at a ratio of 1:1 and stimulated with anti-CD3/28 for 72 h in the (A) absence of APCs or (B) presence of APCs. (C) Suppression assays performed using MLRs as responders. PBMCs of three different donors were isolated and cultured together with the indicated TNFR2 subsets at a 1:1 ratio. One-way ANOVA with Tukey’s multiple comparison test was used for (A,C), and Kruskal–Wallis with Dunn’s multiple comparison test was used for (B). Error bars indicate SD for (A,B) and SEM for (C). Comparison of the proliferation of responders: *p < 0.05; ***p < 0.001.

Figure 4

Proliferative capacity, IL-2, and IFN-g production by induced CD25hiTNFR2+ T cells. (A) The proliferative capacity of sorted TNFR2 subsets (originated from the un-fractionated CD4+ cells) upon 72 h anti-CD3/28 re-stimulation. N = 6. (B) Intracellular expression of IL-2 (upper panel, N = 2) by the sorted cells. (C) IL-10 secreted into the supernatant during the re-stimulation of the sorted TNFR2 subsets (N = 4) was determined using CBA-flex kits (left panel) and IL-10 mRNA levels (right panel) was determined using qPCR (N = 4). (D) IFN-g secreted into the supernatant during the re-stimulation of the sorted TNFR2 subsets (N = 4) (left panel) and the mRNA expression levels of IFN-g and T-bet on sorted TNFR2 subsets (N = 4) (right panel). (E) Total PBMCs were stimulated using CD3/28 to obtain the induced TNFR2 populations. On day 3, cells were further stimulated with PMA/Ionomycin in the presence of Brefeldin A to determine intracellular IFN-g production. Flow cytometry was performed to identify the different induced TNFR2 populations and their IFN-g production was determined. Data is representative of four donors. Kruskal–Wallis with Dunn’s multiple comparison test was used and error bars indicate SEM. Comparison of proliferation, mRNA and cytokine levels: *p < 0.05; **p < 0.01; ***p < 0.001.

Figure A1

Phenotype of induced TNFR2+ T cells from CD4+CD25− T cell, CD4+CD25+ T cells, and un-fractionated CD4+ cells. Expression of regulatory molecules on TNFR2 subsets from starting population CD4+CD25− T cells, CD4+CD25+ T cells, and un-fractionated CD4+ T cells. Gray histograms represent isotype staining while clear histogram represents the indicated molecule. The numbers indicate percentage positive for the represented molecular marker.

Figure A2

FOXP3 expression levels on ex vivo and induced TNFR2+ T cells. The FOXP3 expression levels were compared between ex vivo CD25hiTNFR2+ and induced CD25hiTNFR2+ T cells. Unpaired Student’s t-test was performed to compare FOXP3 levels and graphs represent mean ± SEM. **p < 0.01.

Figure A3

Suppression assays performed using ex vivo Tregs from healthy donors. PBMCs from healthy donors were stained with Treg markers, CD4, CD25, and TNFR2 to sort for non-induced ex vivo Tregs. Tregs were identified as either CD4+CD25hiTNFR2+ T cells or CD4+CD25hiTNFR2− T cells. The sorted Tregs were added to autologous responders (CD4+ T cells) at a ratio of 1:1 and stimulated with anti-CD3/28 for 72 h in 96 well plates. (A) Represents a suppression assay using CD25hiTNFR2+ T cells as the Treg population while (B) Represents a suppression assay using CD25hiTNFR2− T cells as the Treg population. Data shown here represents mean ± SD. Unpaired t-tests was used to determine statistical significance. *p < 0.05.