CCR2 enhances CD25 expression by FoxP3+ regulatory T cells and regulates their abundance independently of chemotaxis and CCR2+ myeloid cells

Abstract

A wide array of chemokine receptors, including CCR2, are known to control Treg migration. Here, we report that CCR2 regulates Tregs beyond chemotaxis. We found that CCR2 deficiency reduced CD25 expression by FoxP3⁺ Treg cells. Such a change was also consistently present in irradiation chimeras reconstituted with mixed bone marrow from wild-type (WT) and CCR2-/- strains. Thus, CCR2 deficiency resulted in profound loss of CD25^hi FoxP3⁺ Tregs in secondary lymphoid organs as well as in peripheral tissues. CCR2-/- Treg cells were also functionally inferior to WT cells. Interestingly, these changes to Treg cells did not depend on CCR2⁺ monocytes/moDCs (the cells where CCR2 receptors are most abundant). Rather, we demonstrated that CCR2 was required for TLR-stimulated, but not TCR- or IL-2-stimulated, CD25 upregulation on Treg cells. Thus, we propose that CCR2 signaling can increase the fitness of FoxP3⁺ Treg cells and provide negative feedback to counter the proinflammatory effects of CCR2 on myeloid cells.

Fig. 1

CCR2 deficiency leads to reduced CD25 expression and loss of CD25⁺ FoxP3⁺ Tregs. Tissues were harvested from WT and CCR2−/− mice (five each; 9-week-old male mice) for analysis. a Representative plots showing the expression of FoxP3 and CD25 on gated CD4⁺ T cells from different tissues. b Representative plots showing the expression of FoxP3 and CD25 on gated FoxP3⁺CD4⁺ T cells from different tissues. c Bar graphs showing the levels of CD25 expression (as the mean fluorescence index (MFI)) by gated FoxP3⁺ T cells. d Bar graphs showing the percentages of cells with high CD25 expression among FoxP3⁺ T cells. e, f. Bar graphs showing the percentages and numbers of FoxP3⁺ cells. The data shown are the mean ± SEM of four individual animals. All data were analyzed using two-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P<0.0001. Five repeated experiments showed similar results

Fig. 2

The defect in CCR2−/− Tregs is cell-intrinsic. Lymphoid organs were harvested from five irradiation chimeric mice (Ly5.1/Ly5.2) reconstituted with mixed bone marrow from Ly5.1 WT and Ly5.2 CCR2−/− mice. Single-cell suspensions were analyzed for cell composition. a Representative plots showing CD4⁺ T cells and CD8⁺ T cells. The gated CD4⁺ T cells show expression of FoxP3 and CD25. b Representative histograms showing CD25 expression by gated FoxP3⁺ T cells (left). The levels of CD25 expression by FoxP3⁺ T cells are shown (right). **P < 0.01 and ****P < 0.0001 by paired Student’s t tests. c−e The bars show the percentages and numbers of the indicated T-cell populations. *P < 0.05, **P < 0.01, and ***P < 0.0001 by paired Student’s t tests. f, g CD25 expression by tissue Tregs was examined in five chimeric mice.  The scatter plots in (f) show the levels of CD25 expression (as the mean fluorescence index (MFI)) by gated FoxP3⁺ T cells. The scatter plots in (g) show the percentage of cells with high CD25 expression among FoxP3⁺ T cells. *P < 0.05, **P < 0.01, and ***P < 0.001 by paired Student’s t tests. Five independent experiments were performed in mixed chimeric mice with similar results

Fig. 3

Deletion of CCR2 monocytes/moDCs has little impact on Tregs. a, b CCR2 expression by T cells and Ly6C⁺CD11b⁺ monocytes. Spleen cells from WT mice and CCR2−/− mice were analyzed for CCR2 expression with an anti-CCR2 Ab and an isotype control. a Plots showing CCR2 expression by Tregs and non-Treg CD4⁺ T cells from WT mice and CCR2−/− mice. b Plots showing CCR2 expression by gated Ly6C⁺CD11b⁺ monocytes. c−e CCR2-DTR mice were either untreated or treated with 200 ng DT/dose every second day over 7 days. Spleens were harvested 1 day after the last dose. Spleen cells were analyzed for T cells and CD11b⁺ cells. c The contour plots show the distribution of FoxP3 in gated spleen CD4⁺ T cells with or without DT treatment. The bar graphs show the percentages of the indicated T-cell populations; >4 similar experiments were performed with similar results. d Histograms showing the expression of surface CCR2 (by Ab) and CCR2 reporter (cyan fluorescent protein, CFP) by CD11b⁺ spleen cells from DT-treated and untreated mice. DT-treated mice (dotted lines) lost CCR2⁺CD11b⁺ cells. e Mixed bone marrow chimeras of WT and CCR2-DTR donors were either untreated or treated with 200 ng DT/dose every second day over 7 days. Spleen cells were analyzed for CD25 expression

Fig. 4

CCR2−/− Tregs are less suppressive in vitro. a The FACS plots shows the coexpression of GITR and FoxP3 by Tregs from WT and CCR2−/− mice. The bars show the mean fluorescence index (MFI) of the indicated molecules on Tregs from WT and CCR2−/− mice. ***P < 0.001 by t test. B&C. GITR⁺CD4⁺ Tregs were isolated from WT and CCR2−/− mice. The histograms show the levels of GITR and CD25 expression by Tregs from WT and CCR2−/− mice (b). For the functional assay (c), graded numbers of Tregs were cultured with 5×10⁴ CTV-labeled naïve CD4⁺ T cells (responders) for 5 days under stimulation with beads coated with anti-CD3 and anti-CD28 Abs. The histograms show the proliferation profiles of responders with or without Tregs (ratio of responders to Tregs: 2:1). The bar graph shows the % of suppression (mean ± SEM) from three pooled experiments. The data were analyzed using two-tailed Student’s t tests. *P<0.05

Fig. 5

CCR2 antagonism diminishes CD25 upregulation on Tregs. Spleen cells were harvested from FoxP3 reporter mice. Spleen cells (2×10⁵/well) were cultured in 96-well round-bottom plates and stimulated with CpG, IL-2 or anti-CD3/anti-CD28 Abs for 24 h. a The representative histograms show CD25 expression by FoxP3⁺CD4⁺ (left) and Foxp3^-CD4⁺ T cells (right). The shaded areas indicate unstimulated cultures. The bar graphs show the CD25 mean fluorescence index (MFI) of expression by FoxP3⁺CD4⁺ (top) and FoxP3^-CD4⁺ (bottom) T cells in 3 replicate wells with or without stimulation. b The above cell cultures with three replicate wells were incubated with a CCR2 inhibitor (RS102895, 0.5 µM). The histograms show the expression of CD25 by FoxP3⁺ cells with or without the inhibitor. The shaded areas show expression by unstimulated FoxP3⁺ cells. The bar graph (top) shows the CD25 MFI of FoxP3⁺ cells with or without the CCR2 inhibitor. The other bar graph (bottom) shows the % CD25 MFI of FoxP3⁺ cells with the CCR2 inhibitor relative to that of cultures without the inhibitor. **P < 0.01 compared to cultures without inhibitor. The data are from one of three similar experiments. c, d Spleen cells from Foxp3 reporter mice were incubated with various concentrations of anti-CCR2 Ab (rat IgG2b) or isotype control and then with anti-rat IgG as a crosslinking Ab. The cells were then stimulated with CpG for 20 h and analyzed for CD25 expression. The histogram shows the expression of CD25 by CpG-stimulated FoxP3⁺ cells (c). Line graph and bar graphs showing the MFI of CD25 (top) and FoxP3 (bottom) under different conditions. The data are from one of three similar experiments. E&G. Spleen FoxP3⁺ CD4⁺ cells from FoxP3 reporter mice were sorted into three subpopulations: CD25^loCCR2^int, CD25^loCCR2^lo, and CD25^hiCCR2^lo (e). The three types of cells (50,000 cells per well in a U-bottom 96-well plate) were cultured in the presence of CpG and 2×10⁵ spleen cells for 20 h. The CD25 expression (f) and CCR2 expression (g) by the three populations with or without CpG are shown. **P < 0.01 by t tests between the indicated groups

Fig. 6

CpG-stimulated CD25 upregulation requires CCR2 expression by FoxP3⁺ cells. a, b CD25^loFoxP3⁺ cells were purified from Foxp3 reporter mice. CD25^loFoxP3+ cells (50,000 cells per well) were cocultured with 2×10⁵ spleen cells from WT or CCR2−/− mice in the presence or absence of CpG (1 μM) in a 96-well round-bottom plate for 20 h. The expression of CD25 by FoxP3⁺ cells was evaluated. The histograms (a) show CD25 expression by FoxP3⁺ cells with WT spleen cells or CCR2−/− spleen cells. The FACS plots (b) show the differences in myeloid cells between WT and CCR2−/− spleen cells. c, d Spleen cells from five individual chimeric mice were cultured with or without CpG for 20 h. The histograms (c) show the expression of CD25 by FoxP3⁺ cells cultured with or without CpG. The MFI of CD25 expression is shown for CpG-stimulated WT and CCR2−/− FoxP3⁺ Tregs (top), and the fold changes in CD25 expression compared to that of unstimulated Tregs are also shown (bottom). The lines indicate data from the same mouse. **P < 0.01, ***P < 0.001, and ****P < 0.0001 by paired t tests. The data are from one of three similar experiments