Cutting edge: mechanisms of IL-2-dependent maintenance of functional regulatory T cells

Abstract

IL-2 controls the survival of regulatory T cells (Tregs), but it is unclear whether IL-2 also directly affects Treg suppressive capacity in vivo. We have found that eliminating Bim-dependent apoptosis in IL-2- and CD25-deficient mice restored Treg numbers but failed to cure their lethal autoimmune disease, demonstrating that IL-2-dependent survival and suppressive activity can be uncoupled in Tregs. Treatment with IL-2-anti-IL-2-Ab complexes enhanced the numbers and suppressive capacity of IL-2-deprived Tregs with striking increases in CD25, CTLA-4, and CD39/CD73 expression. Although cytokine treatment induced these suppressive mechanisms in both IL-2(-/-) and IL-2(-/-)Bim(-/-) mice, it only reversed autoimmune disease in the latter. Our results suggest that successful IL-2 therapy of established autoimmune diseases will require a threshold quantity of Tregs present at the start of treatment and show that the suppressive capacity of Tregs critically depends on IL-2 even when Treg survival is independent of this cytokine.

Figure 1. Bim deficiency restores peripheral Foxp3+ cells in IL-2-deficient mice

Peripheral lymph node cells were stained and analyzed by flow cytometry. (A) Representative CD25 and Foxp3 expression in CD4+ T cells. (B) Percentages and (C) numbers of CD4+ T cells expressing Foxp3, with and without CD25, in 2–4 wk old mice. Individual mice and means are shown.

Figure 2. Rescued Foxp3+ T cells fail to protect IL-2−/− Bim−/− and CD25−/− Bim−/− mice from autoimmune disease and are less suppressive in culture

(A) Survival of the indicated strains was monitored from weaning to 7 wk of age. WT, N=53; IL-2−/− and CD25−/−, N=57; Bim−/−, N=42; IL-2−/− Bim−/− and CD25−/− Bim−/−, N=20. (B) Anti-erythrocyte Ab titers in mice > 3 wk of age. Erythrocytes were stained with anti-IgM or anti-IgG secondary Ab, to detect bound endogenous Ab, and analyzed by flow cytometry. Anti-erythrocyte Ab levels were determined by the mean fluorescence intensity (MFI) of secondary Ab staining. (C) Anemia was tested with hematocrit readings of blood from mice > 4 wk of age. Individual mice and means are shown in (B) and (C). (D) CD25−/− Bim−/− and control mice were bred to express a Foxp3/GFP fusion reporter. Peripheral CD4+ GFP+ (Foxp3+) T cells of each genotype were purified with matching GFP levels by high speed cell sorting. In vitro suppression was compared by titrating the number of Foxp3+ T cells per well in a co-culture assay and measuring the proliferation of naïve CD4+ Foxp3- responder T cells in response to anti-CD3 Ab. Resp: Foxp3- cells cultured without Tregs. Treg: Foxp3+ cells cultured without responder T cells. Means and SD from 1 of 3 experiments with similar results are shown.

Figure 3. Treatment with IL-2/anti-IL-2-antibody complexes rescues IL-2−/− Bim−/− mice and expands Tregs

IL-2−/− and IL-2−/− Bim−/− mice were treated with IL-2-Ab complexes beginning at 12–18 d of age, at the onset of detectable anti-erythrocyte Ab. Treatment continued for 7 wk or until mice became moribund from anemia. (A) Survival of treated IL-2−/− or IL-2−/− Bim−/− mice, compared to the untreated controls from Fig. 2A. Treated IL-2−/−, N=10; treated IL-2−/− Bim−/−, N=14. (B) Anti-erythrocyte Ab and (C) hematocrit measurements of surviving treated IL-2−/− Bim−/− mice at 3 or ≥7 wk of age, compared to age-matched controls from Fig. 2B, C. Individual mice and means are shown in (B) and (C). (D) The percentages of Foxp3+ cells were determined by flow cytometry 5–8 d after start of treatment with IL-2/Ab complexes and compared to age-matched controls. Means and SD are shown.

Figure 4. Treatment with IL-2/anti-IL-2-antibody complexes increases the expression of putative suppressive molecules in Tregs

Splenocytes from Fig. 3D were analyzed by flow cytometry for expression of molecules known to participate in Treg-mediated suppression: (A) CD25, (B) CTLA-4, detected in permeabilized cells, (C) CD39 and CD73, gated on CD4+ Foxp3+ cells, and (D) IL-10, after 4h restimulation with PMA and Ionomycin. Representative dot plots, gated on CD4+ cells except in (C), show the percentages of cells in each quadrant. Bar graphs present means and SD of expression of these molecules in Foxp3+ and Foxp3- cells.