Expression of costimulatory TNFR2 induces resistance of CD4+FoxP3- conventional T cells to suppression by CD4+FoxP3+ regulatory T cells

Abstract

Our previous study showed that TNFR2 is preferentially expressed by CD4(+)FoxP3(+) regulatory T cells (Tregs), and expression of this receptor identified maximally suppressive Tregs. TNFR2 is also expressed by a small fraction of CD4(+)FoxP3(-) conventional T cells (Tconvs) in normal mice, and its expression is upregulated by T cell activation. This raises questions about the role of TNFR2 signaling in the function of Tconv cells. In this study, by using FoxP3/gfp knock-in mice, we showed that TNFR2 signaling did not induce FoxP3(-) CD4 cells to become suppressive. Ki-67, a marker of proliferation, was concomitantly expressed with TNFR2 by CD4 cells, independent of forkhead box P3 expression, in normal mice and Lewis lung carcinoma-bearing mice. TNFR2 is associated with greater suppressive functions when expressed by Tregs and is associated with greater resistance to suppression when expressed by Tconv cells. In mice bearing 4T1 breast tumor or Lewis lung carcinoma, intratumoral Tconv cells expressing elevated levels of TNFR2 acquired the capacity to resist suppression by lymph node-derived Tregs. However, they remained susceptible to inhibition by more suppressive tumor-infiltrating Tregs, which expressed higher levels of TNFR2. Our data indicate that TNFR2 also costimulates Tconv cells. However, intratumoral Tregs expressing more TNFR2 are able to overcome the greater resistance to suppression of intratumoral Tconv cells, resulting in a dominant immunosuppressive tumor environment.

Conflict of interest statement

Disclosures

The authors have no financial conflicts of interest.

Figures

FIGURE 1.

Concomitant expression of TNFR2 and Ki-67 by CD4 subsets from normal mice. A, Spleen cells from normal C57BL/6 mice were stained with anti-CD4, TNFR2, and FoxP3 Abs. The expression of TNFR2 by CD4+FoxP3+ and CD4+FoxP3− cells was analyzed by FACS. B, Spleen cells and axillary and inguinal LN cells from normal C57BL/6 mice were stained with anti-CD4, Ki-67, and FoxP3 Abs. The expression of Ki-67 and FoxP3 was analyzed with FACS by gating on CD4. C, Spleen cells from normal C57BL/6 mice were stained with anti-CD4, TNFR2, FoxP3, and Ki-67 Abs. The expression of Ki-67 by FoxP3−TNFR2+, FoxP3− TNFR2−, FoxP3+TNFR2−, and FoxP3+TNFR2+ subsets was analyzed with FACS by gating on CD4 cells. Numbers indicate the percentage of cells in the respective quadrant. Data shown are representative of at least three separate experiments with similar results.

FIGURE 2.

Suppressive activity of CD4 subsets from FoxP3/gfp KI mice. Flow-sorted CD4+FoxP3/gfp−TNFR2− cells (Tconv cells), 5 × 104 cells/well, were cultured alone or cocultured with flow-sorted CD4+FoxP3/gfp+ TNFR2+ or CD4+FoxP3/gfp+TNFR2− cells (A) or CD4+FoxP3/gfp+TNFR2+ or CD4+FoxP3/gfp−TNFR2+ cells (B) from FoxP3/gfp KI mice at a ratio of 10:2, 10:5, and 10:10 (Tconv cells/Tregs). C, In other experiments, 2.5 × 104 cells/well of CD4+FoxP3/gfp−TNFR2− cells (Tconv) were cultured alone or cocultured at ratio of 1:1 with CD4+FoxP3/gfp+TNFR2+ (TNFR2+ Tregs), CD4+FoxP3/gfp+TNFR2− cells (TNFR2− Tregs), or unfractionated CD4+ FoxP3/gfp+ cells (total Tregs). The cells were stimulated with APCs (2 × 105 cells/well) and anti-CD3 (0.5 μg/ml) for 72 h. Proliferation was measured by [3H]thymidine incorporation. Data (mean ± SD, n = 3) shown are representative of at least three separate experiments with similar results. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 3.

TNFR2+ Tconv cells are resistant to Treg-mediated inhibition. A, TNFR2− Tconv cells (CD4+FoxP3/gfp−TNFR2−) (left panel) or TNFR2+ Tconv cells (CD4+FoxP3/gfp−TNFR2+) (right panel), at 5 × 104 cells/well, were cultured alone or cocultured with TNFR2+ Tregs (CD4+FoxP3/gfp+ TNFR2+) or TNFR2− Tregs (CD4+FoxP3/gfp+TNFR2−) at a ratio of 1:1. The cells were stimulated with APCs (2 × 105 cells/well) and anti-CD3 (0.5 μg/ml) for 72 h. Proliferation was measured by [3H]thymidine incorporation. B, IFN-γ levels in TNFR2− and TNFR2+ Tconv cells that were cultured alone with the same conditions as in A. C, Flow-sorted CD4+FoxP3/gfp+TNFR2− Tconv cells, freshly isolated (left panel) or cultured with APCs (2 × 105 cells/well) and anti-CD3 (0.5 μg/ml) for 24 h (right panel) were stained with anti-CD4 and TNFR2 Abs. Expression of TNFR2 was analyzed by gating on CD4 cells. D, Flow-sorted CD4+FoxP3/gfp−TNFR2− Tconv cells, freshly isolated (left panel) or cultured with APCs (2 × 105 cells/well) and anti-CD3 (0.5 μg/ml) for 24 h (right panel), were cultured alone or cocultured at a 1:1 ratio with flow-sorted Tregs. The cells were stimulated with APCs and anti-CD3 Ab for 72 h. Proliferation was measured by [3H]thymidine incorporation. E, Flow-sorted CD4+CD25−TNFR2− Tconv cells from normal BALB/c mice were cultured with 2-fold greater number of APCs and anti-CD3 Ab (0.5 μg/ml). After 72 h of incubation, the cells were flow sorted into TNFR2+ and TNFR2− fractions. The resorted cells (2.5 × 104 cells/well) were cultured alone or cocultured with freshly isolated CD4+CD25+ Tregs at a ratio of 2:1 (Tconv cells Tregs). Freshly flow-sorted CD4+CD25−TNFR2− Tconv cells were used as a comparison. The cells were stimulated with APCs (2 × 105 cells/well) and anti-CD3 Ab (0.5 μg/ml) for 72 h, and proliferation was determined by [3H]thymidine incorporation assay. Percentage inhibition is shown. Data in A, B, D, and E are mean ± SD (n = 3). Data are representative of at least three separate experiments with similar results. *p < 0.05; ***p < 0.001.

FIGURE 4.

Expression of Ki-67 by CD4 subsets in the lymphoid organs of tumor-bearing mice. Spleen cells, nondLNs (left side inguinal and axillary LNs), or dLNs (right side inguinal and axillary LNs) from mice inoculated with LLC tumor cells for 15 d were stained with anti-CD45, CD4, FoxP3, Ki-67, and TNFR2 Abs. A, Expression of Ki-67 by FoxP3−and FoxP3+ cells were analyzed by gating on CD45+CD4+ cells. B, Expression of Ki-67 by FoxP3− TNFR2+, FoxP3+TNFR2+, FoxP3+TNFR2−, and FoxP3− TNFR2− CD4 subsets present in spleen, non-dLNs, and dLNs were analyzed by gating on respective populations. Data shown in A and B are representatives of at least three separate experiments with similar results. Numbers in the dot plots indicate the percentage of cells in the respective quadrant. C, Comparison of Ki-67 expression by CD4 subset present in non-dLNs and dLNs of tumor-bearing mice. Data (mean ± SD) shown are summarized from three separate experiments with similar results (n = 9). D, Kinetic expression of Ki-67 by FoxP3+ Tregs and TNFR2+FoxP3+ Tregs present in dLNs of mice inoculated with LLC tumor cells for 0, 1, 5, 10, or 15 d. Compared with tumor-free mice (day 0). Data (mean ± SD) shown are summarized from two separate experiments with similar results (n = 6). *p < 0.05;**p < 0.01.

FIGURE 5.

Tumor-infiltrating FoxP3+ and FoxP3− subsets of CD4 cells upregulated their TNFR2 expression and were actively replicating. Spleen cells and TILs from C57BL/6 mice inoculated with LLC tumor cells for 15 d were stained with anti-CD45, CD4, FoxP3, and Ki-67 Abs or isotype control Abs. A, The expression of TNFR2 by FoxP3− cells and FoxP3+ cells were analyzed by gating on CD45+CD4+ cells. B, The expression of Ki-67 by FoxP3− cells and FoxP3+ cells were analyzed by gating on CD45+CD4+ cells. Numbers indicate the percentage of cells in the respective quadrant. Data shown are representative of at least three separate experiments with similar results.

FIGURE 6.

The expression of Ki-67 by FoxP3+ and FoxP3− subsets of CD4 cells in the lymphoid tissues and TILs from mice with early tumor development. Spleen cells, non-dLN cells, dLN cells, and TILs from C57BL/6 mice inoculated with LLC tumor cells for 5 d were stained with anti-CD45, CD4, FoxP3, and Ki-67 Abs. The expression of Ki-67 by FoxP3− and FoxP3+ cells was analyzed by gating on CD45+CD4+ cells. A, Lymphoid tissues. B, TILs. Numbers indicate the percentage of cells in the respective quadrant. Data shown are representative of two separate experiments with similar results.

FIGURE 7.

Phenotype of CD25+ and CD25− subsets of tumor-infiltrating CD4 cells from 4T1 tumor-bearing mice. A, LN cells from normal BALB/c mice or 4T1 tumor TILs were stained with anti-CD45, CD4, CD25, and FoxP3 Abs. The expression of FoxP3 was analyzed by FACS, gating on CD4+CD25+ cells (shaded graph) or gating on CD4+CD25− cells (open graph, upper panel). The relationship of CD25 and FoxP3 expression on CD4 cells present in the control LN cells and in the TILs is shown in the dot plot (lower panel). B, Expression of FoxP3 by total CD4 cells from tumor-free BALB/c mouse LN cells (open graph) or by tumor-infiltrating CD4 cells from 4T1 tumor-bearing mice (shaded graph) was analyzed by FACS, gating on CD4+ cells (upper panel). The percentage and mean fluorescence intensity (MFI) of FoxP3+ cells in the CD4 population present in control LNs and in the TILs are shown in the lower panel. Data shown are mean 6 SD (n = 3). *p < 0.05; **p < 0.01. C, Splenocytes from tumor-free BALB/c mice (black line) and TILs from 4T1 tumor-bearing mice (shaded graph) were stained with anti-CD45, CD4, CD25 and phenotypic markers (CD45RB, CD62L, CD44, CD69, CTLA-4, and GITR). Expression of phenotypic marker was analyzed by FACS, gating on CD4+CD25+ cells (left panel) or CD4+CD25− cells (right panel). Isotype controls are represented by dashed line. Data shown are representative of three separate experiments with similar results.

FIGURE 8.

Tumor-infiltrating Tregs potently inhibit tumor-infiltrating Tconv cells. Proliferative responses of Tconv cells: FACS-sorted CD4+ CD25− cells from LNs of tumor-free or from TILs of 4T1 tumor-bearing BALB/c mice (A) or from LNs of tumor-free or from TILs of LLC-bearing C57BL/6 mice (B) were cultured at 2.5 × 104 cells/well. The cells were stimulated with APCs (2 × 105 cells/well) and anti-CD3 for 72 h. Proliferation was measured by [3H]thymidine incorporation. Proliferative responses of Tregs: FACS-sorted CD4+CD25+ cells from LNs of tumor-free or from TILs of 4T1 tumor-bearing BALB/c mice (C) or from LNs of tumor-free or LLC-bearing C57BL/6 mice (D) were cultured at2.5 × 104 cells/well. The cells were stimulated with APCs (2 × 105 cells/well) and anti-CD3 for 72 h. Proliferation was measured by [3H]thymi-dine incorporation. Suppressive activity of Tregs: flow-sorted CD4+ CD25− cells (2.5 × 104 cells/well) from TILs of 4T1 tumor-bearing BALB/c mice were cultured alone or cocultured with the same number of CD4+CD25+ cells from LNs of tumor-free or TILs of 4T1 tumor-bearing BALB/c mice (E) or CD4+CD25− cells (2.5 × 104 cells/well) from TILs of LLC tumor-bearing C57BL/6 mice were cultured alone or cocultured with the same number of CD4+CD25+ cells from LNs of tumor-free or TILs of LLC tumor-bearing C57BL/6 mice (F). The cells were stimulated with APCs (2 × 105 cells/well) and anti-CD3 for 72 h. Proliferation was measured by [3H]thymidine incorporation. Data (mean ± SD, n = 3) shown are representative of at least three separate experiments with similar results.*p < 0.05;**p < 0.01.