Synergy of IL-21 and IL-15 in regulating CD8+ T cell expansion and function

Abstract

Interleukin (IL)-21 is the most recently recognized of the cytokines that share the common cytokine receptor gamma chain (gamma(c)), which is mutated in humans with X-linked severe combined immunodeficiency. We now report that IL-21 synergistically acts with IL-15 to potently promote the proliferation of both memory (CD44high) and naive (CD44low) phenotype CD8+ T cells and augment interferon-gamma production in vitro. IL-21 also cooperated, albeit more weakly, with IL-7, but not with IL-2. Correspondingly, the expansion and cytotoxicity of CD8+ T cells were impaired in IL-21R-/- mice. Moreover, in vivo administration of IL-21 in combination with IL-15 boosted antigen-specific CD8+ T cell numbers and resulted in a cooperative effect on tumor regression, with apparent cures of large, established B16 melanomas. Thus, our studies reveal that IL-21 potently regulates CD8+ T cell expansion and effector function, primarily in a synergistic context with IL-15.

Figure 1.

IL-21 acts synergistically with IL-15 to expand CD8 ⁺ T cells. 5 × 10⁶ splenocytes were pooled from two to three wild-type mice and cultured for 7 d in medium containing IL-15, IL-21, or combinations of these cytokines, as indicated. The number of NK cells (A), T cells (B), CD8⁺ T cells (C), and CD4⁺ T cells (D) were determined as NK1.1⁺TCRβ⁻, NK1.1⁻TCRβ⁺, CD8⁺CD4⁻, and CD8⁻CD4⁺ cells, respectively. Results shown are means ± SD from three experiments. (E and F) Cells cultured in medium for 0 d (a) or 7 d (b), or in 100 ng/ml of IL-15, IL-21, or both cytokines (c, d, and e, respectively) were analyzed by flow cytometry for 7 d. The percentages of NK1.1⁻TCRβ⁺ and NK1.1⁺TCRβ⁻ cells (E) and CD8⁻CD4⁺ and CD8⁺CD4⁻ cells (F) are indicated in the quadrant corners.

Figure 2.

IL-21 acts in concert with IL-15 or IL-7 but not with IL-2 to expand CD8 ⁺ T cells. (A and B) 5 × 10⁶ splenocytes pooled from three wild-type mice were cultured for 3, 5, and 7 d in medium containing 100 U/ml IL-2, 100 ng/ml IL-7, 100 ng/ml IL-15, 100 ng/ml IL-21, or combinations of these cytokines, as indicated. CD8⁺ T (A) and CD4⁺ T (B) cell subsets were identified as CD8⁺CD4⁻ and CD8⁻CD4⁺, respectively. Results shown are means ± SD from three experiments. (C) Representative flow cytometric analysis of cells cultured for 7 d as described in A and B. Percentages of selected cell populations are indicated in the quadrant corners. (D) Cells from A were also stained with annexin V and propidium iodide (PI). Percentages of double negative subpopulations corresponding to viable cells are indicated in the quadrant corners. Data representative of three separate experiments are shown.

Figure 3.

IL-15 and IL-21 cooperatively enhance effector function of memory-phenotype CD8 ⁺ T cells. (A and B) 5 × 10⁶ splenocytes pooled from two to three wild-type mice were cultured for 7 d in medium containing 100 ng/ml of IL-15, IL-21, or combinations of these cytokines, as indicated (b–e). The naive cells (A, subpanel a) were splenocytes from a littermate that were directly analyzed without culture. Cells were analyzed by flow cytometry after staining with (A) CD8-allophycocyanin, CD4-FITC, CD44-CyChrome, and IL-2Rβ-PE or (B) CD8-allophycocyanin, IL-2Rβ-FITC, CD44-CyChrome, and CD62L-PE, respectively. Data representative of three separate experiments are shown. The absolute numbers of gated CD8⁺ T cells and the percentages of CD44^high, CD44^intermediate, and CD44^low subpopulations are indicated in A. The percentages of CD62L^low and CD62L^high subpopulations are indicated in B. (C and D) Freshly isolated splenocytes or cells cultured for 7 d with the indicated cytokines were stimulated with anti-CD3 and anti-CD28 for 0, 1, 2, or 4 h. (C) Representative flow cytometric profiles. (D) The absolute number of IFN-γ–producing CD8⁺ T cells. Mean values for three similar experiments are shown. The increase in IFN-γ⁺CD8⁺ T cells with IL-15 and IL-21 was highly significant.

Figure 4.

IL-15 and IL-21 cooperatively increase cell cycle progression of both CD44^high and CD44^low CD8 ⁺ T cells. (A) All cell subpopulations were isolated and stained with CFSE as described in Materials and Methods. The “basal” CFSE profile on day 0 is shown as the dashed line. Cells were cultured in complete medium at 2–5 × 10⁵ cells/ml without cytokine or with 100 ng/ml of IL-15 and/or IL-21 for 4 or 7 d, as indicated, and analyzed by flow cytometry. The indicated cell numbers (×10⁻⁶) are an average of cell numbers from three independent experiments. In each experiment, the starting cell numbers were normalized to 3.75 × 10⁶ (CD8⁺ T cells), 0.75 × 10⁶ (CD44^highCD8⁺ T cells), and 2.5 × 10⁶ (CD44^lowCD8⁺ T cells), respectively, which allowed comparison of the different experiments. Flow cytometric results shown are representative of three experiments. (B) Cells in A were also stained with annexin V and PI. The percentages of double negative subpopulations corresponding to viable cells are indicated in the quadrant corners. Data representative of three separate experiments are shown.

Figure 5.

Antigen-specific CD8 ⁺ T cell responses are impaired in IL-21R ^−/− mice. Five mice in each group were immunized i.p. with 5 × 10⁶ PFU of vPE16. (A and B) On day 5, splenocytes of immunized WT and IL-21R^−/− (KO) mice were restimulated with 1.0 μM (A) or 0.001 μM (B) P18-I10 for 1 wk, and lytic activity was measured by a 5-h ⁵¹Cr release assay. P815 cells pulsed with 1.0 μM (A) or 0.001 μM (B) P18-I10 were used as target cells. Means ± SEM are shown. (C) The frequency of P18-I10–specific splenic CD8⁺ T cells was measured by H-2D^d-P18-I10 tetramer staining ex vivo without restimulation in vitro (left) or after 1 wk of restimulation with 1.0 μM P18-I10 (right). Shown is the mean percent ± SEM of tetramer-positive CD8⁺ T cells in total CD8⁺ T cells for five mice per group. The difference in the two groups was statistically significant (P < 0.05). (D) The frequency of IFN-γ⁺CD8⁺ T cells in spleens was measured by intracellular staining ex vivo without restimulation in vitro (left) or after 1 wk of restimulation with 1.0 μM P18-I10 (right). Cells were stimulated with 1.0 μM P18-I10 for 10 h in the presence of 1 μg/ml brefeldin A. Shown is the mean percent ± SEM of IFN-γ–positive (IFN-γ⁺) CD8⁺ T cells in total CD8⁺ T cells for five mice per group. The difference in the two groups was statistically significant (P < 0.05).

Figure 6.

In vivo administration of IL-21 in combination with IL-15 yields tumor regression with cures of large, established B16 melanomas. (A) Sublethally irradiated female C57BL/6 mice were implanted subcutaneously with B16 melanomas. 8 d later, mice were treated by adoptively transferring cultured pmel-1 splenocytes (0.5 × 10⁶ Vβ13⁺ CD8⁺ T cells) as indicated and, where indicated, vaccinating with rFPVhgp100. In some conditions, IL-15 and IL-21 were administered twice daily for six doses (5 μg each/dose; five to seven mice in each group). (B) Sublethally irradiated female C57BL/6 mice were implanted subcutaneously with B16 melanomas. 10 d later, mice were treated by adoptively transferring cultured pmel-1 splenocytes (10⁶ Vβ13⁺ CD8⁺ T cells) and vaccinating with rFPVhgp100. IL-15, IL-21, or both cytokines was administered twice daily for six doses (10 μg each /dose; five to seven mice in each group). A repeat experiment showed similar results. Shown are mean tumor sizes ± SEM. (C) Shown are Vβ13⁺CD8⁺ T cell numbers from mice from two experiments (days 21 and 28 from Experiment 1 in B and day 20 from Experiment 2, which was terminated at day 23). *, Mice not receiving cytokine had all died from their tumors, so samples were not available for analysis.

Figure 7.

Several genes are synergistically regulated by IL-21 and IL-15 in CD8 ⁺ T cells. Naive CD8⁺ T cells were isolated, treated without cytokine or with 100 ng/ml of IL-15 and/or IL-21 for 4 h, and subjected to RNA isolation and Affymetrix Gene Chip analysis as described in Materials and Methods. Results shown are from five independent experiments. Shown in A are the overall expression patterns of genes that are induced or repressed more than twofold by IL-15, IL-21, or both cytokines. Also shown are selected genes that are preferentially regulated by IL-15 alone (B), IL-21 alone (C), or IL-15 plus IL-21 (D).