Interleukin-4 Supports the Suppressive Immune Responses Elicited by Regulatory T Cells

Abstract

Interleukin-4 (IL-4) has been considered as one of the tolerogenic cytokines in many autoimmune animal models and clinical settings. Despite its role in antagonizing pathogenic Th1 responses, little is known about whether IL-4 possesses functions that affect regulatory T cells (Tregs). Tregs are specialized cells responsible for the maintenance of peripheral tolerance through their immune modulatory capabilities. Interestingly, it has been suggested that IL-4 supplement at a high concentration protects responder T cells (Tresps) from Treg-mediated immune suppression. In addition, such supplement also impedes TGF-β-induced Treg differentiation in vitro. However, these phenomena may contradict the tolerogenic role of IL-4, and the effects of IL-4 on Tregs are therefore needed to be further elucidated. In this study, we utilized IL-4 knockout (KO) mice to validate the role of IL-4 on Treg-mediated immune suppression. Although IL-4 KO and control animals harbor similar frequencies of Tregs, Tregs from IL-4 KO mice weakly suppressed autologous Tresp activation. In addition, IL-4 deprivation impaired the ability of Tregs to modulate immune response, whereas IL-4 supplementation reinforced IL-4 KO Tregs in their function in suppressing Tresps. Finally, the presence of IL-4 was associated with increased cell survival and granzyme expression of Tregs. These results suggest the essential role of IL-4 in supporting Treg-mediated immune suppression, which may benefit the development of therapeutic strategies for autoimmune diseases.

Keywords: cell survival; granzyme; immunosuppression; interleukin-4; regulatory T cell.

Figure 1

Incompetence in exerting in vitro immune suppression of interleukin-4 (IL-4)-deficient regulatory T cells (Tregs). (A) A representative dot plot demonstrating the strategy for analyzing the presence of CD25⁺Foxp3⁺ Tregs in the splenic T cell population (gate on CD4⁺ T cells). (B) The percentages of Tregs (Foxp3⁺) among CD4⁺CD25⁺ T cells and (C) the intensity of Foxp3 expression in CD4⁺CD25⁺Foxp3⁺ T cells in healthy IL-4 knockout (KO) (n = 6) and WT (n = 6) mice were analyzed by flow cytometry. Results are given as the mean ± SEM. (D) Tregs and responder T cells (Tresps) were sorted from CD4⁺ T cells from IL-4 KO and WT mice for the in vitro suppression assay. (E) The suppressive capability of IL-4 KO and WT Tregs was compared in suppressing their autologous Tresps. Data are representative of three independent experiments. The proliferation of WT Tresps alone was used as the reference for cell proliferation. Results are presented as the relative percentage of cell proliferation to corresponding Tresps alone. ○, WT Treg:WT Tresp; ●, IL-4 KO Treg:IL-4 KO Tresp (*p < 0.05; **p < 0.01; and ***p < 0.001).

Figure 2

Loss of interleukin-4 (IL-4) deteriorated the immune suppressive responses elicited by regulatory T cells (Tregs). Tregs and responder T cells (Tresps) were sorted from IL-4 knockout (KO) or WT mice, as described in Figure 1D, for the suppression assay. (A) The effect of IL-4 removal on the suppressive capability of WT Tregs. (B) The effect of IL-4 supplement on the suppressive capability of IL-4 KO Tregs. Data are representative of three (A) and four (B) independent experiments. The proliferation of WT Tresps alone (A) and IL-4 KO Tresps alone (B) were used as the reference for cell proliferation. Results are presented as the relative percentage of cell proliferation to corresponding Tresps alone (*p < 0.05; **p < 0.01; and ***p < 0.001).

Figure 3

Insufficiency in mediating in vitro antigen-specific immune suppression of interleukin-4 (IL-4)-deficient regulatory T cells (Tregs). (A) Schematic diagram illustrating the protocol for enriching OVA-specific Tregs from WT and IL-4 knockout (KO) mice. (B) The percentages of Foxp3 expressing cells among CD4⁺CD25⁺ T cells in OVA-immunized WT (n = 13) and IL-4 KO (n = 12) mice were analyzed by flow cytometry. (C) The suppressive capability of IL-4 KO and WT Tregs in exerting antigen-specific suppression. (D) The suppressive capability of IL-4 KO Tregs in suppressing WT responder T cells (Tresps). (E) The suppressive capability of WT Tregs in suppressing IL-4 KO Tresps. Results are presented as the relative percentage of cell proliferation to OT2 Tresps alone (C), WT Tresps alone (D), and IL-4 KO Tresps alone (E). Data are representative of two (B,C,E) and three (D) independent experiments (*p < 0.05; **p < 0.01; and ***p < 0.001).

Figure 4

Interleukin-4 (IL-4) deficiency resulted in increased cell death and decreased granzyme expression in regulatory T cells (Tregs). (A) The schematic diagram shows the sorting strategy after culture by labeling Tregs with eFluor 670 cell proliferation dye. Tregs and responder T cells (Tresps) from IL-4 knockout (KO) and WT mice were prepared and cocultured 1:1 for 72 h. The IL-4 neutralization antibody or recombinant IL-4 was supplied as indicated. (B) The effect of anti-IL-4 on the cell death of Tregs and Tresps was determined by PI staining. (C–F) The differences of granzyme A and granzyme B expression levels were compared by real-time RT-PCR in Tregs from IL-4 KO or WT mice with supplementation of IL-4 neutralization antibody or recombinant IL-4. Data are representative of two independent experiments. Results are presented as the mean ± SEM (*p < 0.05, **p < 0.01; and ***p < 0.001).