The influence of IL-2 family cytokines on activation and function of naturally occurring regulatory T cells

Abstract

IL-2 is essential for CD4+CD25+forkhead box P3+ (FoxP3+) naturally occurring regulatory T cell (Treg) homeostasis and activation. Binding of IL-2 to its receptor leads to phosphorylation of STAT5, and binding of phosphorylated STAT5 to the foxp3 promoter increases foxp3 transcription, resulting in elevated levels of FoxP3 protein in Tregs. Transcriptional regulation by the elevated levels of FoxP3 is thought to be essential for the strong suppressor function seen in activated Tregs. IL-2 belongs to a family cytokines, which all depend on the common gamma-receptor chain (gammac). Given the well-documented effects of IL-2 on Treg function, the effect of other IL-2 family cytokines (IL-7, -15, and -21) on Tregs was examined. We observed that IL-7 and IL-15 induce STAT5 phosphorylation and up-regulation of FoxP3 in Tregs. STAT5 activation correlated with enhanced viability. However, only in the presence of IL-2 did Tregs acquire potent suppressor function. This finding is surprising, as IL-15 as well as IL-2 use the same IL-2R betac and gammac for signaling. In contrast, IL-21 activated STAT3 but did not activate STAT5 and had no effect on Treg viability, activation, or function. We therefore conclude that phosphorylation of STAT5, mediated through the IL-2Rgamma, promotes Treg survival in a resting and activated state. However, activation of STAT5 alone in conjunction with TCR signaling is not sufficient for the induction of potent suppressor function in Tregs, as IL-7 and IL-15 are not capable of inducing potent Treg suppressor function.

Fig. 1.

Treg expression of receptors for IL-2 family cytokines. Tregs and Th cells were prepared as described and stained for receptor expression. Alternatively, isolated cells were preactivated for 48 h with immobilized anti-CD3 antibody and IL-2 prior to staining for receptor expression. Gray filled, Isotype control; gray line, Th cells; black line, Tregs. Cytofluorimetric analysis for each receptor was performed at least three times.

Fig. 2.

Influence of IL-2 family cytokines on Treg proliferation. Purified Tregs were incubated with IL-2 family cytokines and immobilized anti-CD3 for 72 h, and proliferation was determined by pulsing the cultures with ³H-thymidine for the final 18 h. The mean ± sd of triplicates of one of three similar experiments is shown.

Fig. 3.

Influence of IL-2 family cytokines on Treg viability. Purified, naïve Tregs (2×10⁴) were cultured with plate-bound anti-CD3 antibody and the indicated cytokines. After 72 h, the number of total live cells (A) and the viability of the cultures, as determined by Trypan blue staining (B), were enumerated. White bars, Freshly isolated Tregs; shaded bars, freshly isolated Tregs with immobilized anti-CD3 antibody; black bars, 3-day-preactivated Tregs. Significance was tested using a Student’s t-test by comparing the cytokine-treated culture with the same population of untreated cells [without cytokine (w/o)]: ***, P < 0.001; **, P < 0.01; *, P < 0.05. The mean ± sd of triplicates of one of two similar experiments is shown. ns, Not significant.

Fig. 4.

Influence of IL-2 family cytokines on Treg suppressive function. Purified Tregs were activated with immobilized anti-CD3 and the indicated cytokines for 3 days, washed, and used in an inhibition assay together with TCR-II T cells at the indicated ratio. Cultures were supplemented with syngeneic splenocytes and antigen as described in Materials and Methods. Proliferation was determined after 72 h by pulsing the cultures with ³H-thymidine for the final 18 h. Statistical significance was tested using a Student’s t-test by comparing the cytokine-stimulated culture with the same population of untreated cells: ***, P < 0.001; **, P < 0.01; *, P < 0.05. The mean ± sd of triplicates of one of three similar experiments is shown. Treg:Eff, Treg:effector ratio.

Fig. 5.

Inhibitory activity of Treg in the presence of IL-2 family cytokines. Purified Tregs were incubated with Th cells at the indicated ratios, together with anti-CD3, APC, and the indicated cytokines. Proliferation was determined by pulsing the cultures with ³H-thymidine for the final 18 h. Statistical significance was tested using a Student’s t-test by comparing the cytokine-stimulated culture with the same population of untreated cells: ***, P < 0.001; **, P < 0.01; *, P < 0.05. The mean ± sd of triplicates of one of three similar experiments is shown.

Fig. 6.

STAT3/STAT5 phosphorylation after stimulation with IL-2 family cytokines. Freshly purified and preactivated Treg and Th cells were stimulated with the indicated cytokines as described. STAT activation was determined by intracellular staining with p-STAT3- and p-STAT5-specific antibodies and analyzed by flow cytometry. Th cells were gated on CD4⁺FoxP3⁻ and Treg on CD4⁺FoxP3⁻. The mean ± sd of triplicates of one of three similar experiments is shown.

Fig. 7.

FoxP3 expression after stimulation with IL-2 family cytokines. FACS-sorted Tregs from Foxp3-gfp knock-in mice were stimulated with anti-CD3 and the indicated cytokines. After 48 h cells, Tregs were analyzed for FoxP3 expression by determining GFP. Mean fluorescence intensity (MFI) is indicated in parentheses. Similar results were obtained using antibodies directed against FoxP3. Results presented are representative of five similar experiments.