Soluble IL-2Rα inhibits IL-2-mediated proliferation. (A) Purified CFSE-labeled MP CD8⁺ T cells were cultured with either murine IL-2 or human IL-2 at the concentration shown. As indicated, 2.5 μg/ml of either soluble murine IL-2Rα or soluble human IL-2Rα was added to the cultures. CFSE dilution was assessed on day 3. Representative data are shown. (B) Purified MP CD8⁺ T cells were cultured with titrated amounts of murine IL-2 with or without soluble mIL-2Rα (2.5 μg/ml). The data show mean levels of [³H]thymidine incorporation (±SD) for triplicate cultures on day 3.

Proliferation to IL-15 immobilized by IL-15Rα cannot be blocked by soluble IL-15Rα-Fc. (A) CFSE-labeled T cells were injected i.v. into Thy1-congenic B6 or IL-15Rα^−/− hosts. One day later, mice were injected i.p. with PBS or 500 ng of LPS. As indicated, mice were also treated i.p. with 10 μg of sIL-15Rα-Fc daily beginning the day of LPS injection. Three days after LPS injection, spleens were harvested, and CFSE dilution of MP CD8⁺ T cells was assessed. (B) Ninety-six-well plates were precoated overnight with 10 μg/ml of sIL-15Rα-Fc. Plates were then washed and incubated with 1 μg/ml IL-15 for 1 h at 37°C. Thereafter, plates were washed, and 5 × 10⁴ MP CD8⁺ T cells were added together with (i) 10 μg/ml sIL-15Rα-Fc, (ii) 10 μg/ml of polyclonal anti-IL-15 antibody, or (iii) control media; as an additional control, free IL-15 (32 ng/ml) was added to some wells. The data show mean levels of [³H]thymidine incorporation (SD) for triplicate cultures on day 3.

Soluble IL-15Rα augments IL-15-mediated host lymphocyte proliferation. (A) Normal B6 mice were injected i.v. on days 1 and 2 with PBS, sIL-15Rα-Fc alone, IL-15 alone, or sIL-15Rα-Fc/IL-15 as described for Fig. 2A. Total numbers of CD8⁺ MP T cells, CD4⁺ MP T cells, and NK cells recovered from spleen on day 3 are shown. (B) Spleens from A were photographed as indicated. (C) Mice were treated as in A, except that the mice also were given an i.v. injection of BrdU on day 1 and placed on BrdU in the drinking water until killing. Shown is BrdU staining for MP CD8⁺, naïve CD8⁺, MP CD4⁺, and NK cells. (D) IL-15Rα^–/– mice were injected i.v. on days 1, 3, 5, and 7, with PBS, IL-15 (0.6 μg), or IL-15 (0.6 μg)/sIL-15Rα-Fc (3 μg). The data show staining of spleen cells on day 9. For B–D, representative data are shown. All data are representative of at least two independent experiments.

Soluble IL-15Rα augments IL-15-mediated donor lymphocyte proliferation in vivo. (A) CFSE-labeled T cells were transferred i.v. into C57BL/6 (B6) recipients. On days 1 and 2 after transfer, the recipients were given i.p. injections of PBS, sIL-15Rα-Fc alone (7 μg), IL-15 alone (1.5 μg), or sIL-15Rα-Fc plus IL-15 (7 μg and 1.5 μg, respectively, which represents a 1:2 molar ratio). CFSE dilution of the donor cells was measured in spleen on day 4. Representative data for gated MP CD8⁺ cells are shown. (B) As in A except that the cells transferred were from lymphocytic choriomeningitis virus-immune mice (Upper) versus normal mice (Lower). (C) CFSE-labeled MP CD8⁺ T cells were transferred to normal B6 hosts; one day later, the hosts were injected with the indicated dose of IL-15 with or without sIL-15Rα-Fc; the dose of sIL-15Rα-Fc varied such that a 2:1 molar ratio of IL-15 to sIL-15Rα-Fc was injected. CFSE profiles for donor MP CD8⁺ cells in spleen at 2 days after injection are shown. (D) Compilation of data from C. For A–C, data shown are representative of two mice per group and are also representative of two independent experiments.

Soluble IL-15Rα augments IL-15-mediated lymphocyte proliferation in vitro. (A) Purified MP (CD44^hi) CD8⁺ T cells from IL-7 tg mice were labeled with CFSE and cultured at 5 × 10⁴ cells per well with 5 ng/ml of IL-15. As indicated, 1 μg/ml of either sIL-15Rα-Fc (dimers) or sIL-15Rα (monomers) was added to the cultures. CFSE dilution was assessed on day 4. Representative data are shown. (B) Purified MP CD8⁺ T cells were cultured with either titrated amounts of IL-15 plus a fixed concentration of soluble receptor (1 μg/ml) (Upper) or titrated amounts of soluble receptor plus a fixed concentration of IL-15 (10 ng/ml) (Lower). The data show mean levels of [³H]thymidine incorporation (±SD) for triplicate cultures on day 3. (C) Purified naïve (CD44^lo) CD8⁺ T cells, MP CD8⁺ T cells, NK cells, or MP CD4⁺ T cells were cultured with IL-15 as indicated. Soluble IL-15Rα-Fc was added at 1 μg/ml. CFSE dilution was assessed on day 3. (D) Same as in B except 10 μg/ml of anti-CD122 antibody was added as indicated. (E) MP CD8⁺ T cells from wild-type IL-7 tg (Ly5.2) and IL-15Rα^–/–/IL-7 tg (Ly5.1) mice were mixed together, labeled with CFSE, and cultured as indicated. CFSE dilution on Ly5.1^– (wild type) and Ly5.1⁺ (IL-15Rα^–/–) cells was measured on day 3.