Stat5b is essential for natural killer cell-mediated proliferation and cytolytic activity

Abstract

We have analyzed the immune system in Stat5-deficient mice. Although Stat5a-/- splenocytes have a partial defect in anti-CD3-induced proliferation that can be overcome by high dose interleukin (IL)-2, we now demonstrate that defective proliferation in Stat5b-/- splenocytes cannot be corrected by this treatment. Interestingly, this finding may be at least partially explained by diminished expression of the IL-2 receptor beta chain (IL-2Rbeta), which is a component of the receptors for both IL-2 and IL-15, although other defects may also exist. Similar to the defect in proliferation in activated splenocytes, freshly isolated splenocytes from Stat5b-/- mice exhibited greatly diminished proliferation in response to IL-2 and IL-15. This results from both a decrease in the number and responsiveness of natural killer (NK) cells. Corresponding to the diminished proliferation, basal as well as IL-2- and IL-15-mediated boosting of NK cytolytic activity was also greatly diminished. These data indicate an essential nonredundant role for Stat5b for potent NK cell-mediated proliferation and cytolytic activity.

Figure 1

(A) Extracts from thymuses (lanes 1–3) or spleens (lanes 4–6) were isolated from wild-type mice (lanes 1, 4), Stat5a^−/− mice (lanes 2, 5), or Stat5b^−/− mice (lanes 3, 6), and proteins were then separated by SDS-PAGE and Western blotted with antisera specific for Stat5a (top) or Stat5b (bottom). (B) Diminished thymocyte numbers in Stat5b^−/−mice (7–10-wk-old mice were analyzed). (C and D) Flow cytometric analysis of thymocytes from 8-wk-old mice. (C) CD4 versus CD8 flow cytometric profiles. (D) CD25 versus CD44 flow cytometric profiles of double negative thymocytes. (E) Diminished numbers of splenocytes in Stat5b^−/− mice (7–10-wk-old mice were analyzed). (F–H) Flow cytometric profiles (8-wk-old mice) show a decrease in the ratio of CD3⁺ to B220⁺ cells (F) and a decrease in the percentage of CD8⁺ single positive T cells (G) in Stat5b^−/− mice. The percentage of TCR-α/β and TCR-γ/δ cells was normal in Stat5b^−/− mice (H).

Figure 2

(A) Diminished IL-2–induced IL-2Rα expression in both CD4⁺ and CD8⁺ subpopulations of T cells after their preactivation with anti-CD3. Fresh splenocytes were stimulated in Falcon 3003 plates coated with 10 μg/ml anti-CD3ε mAb for 48 h, washed twice with PBS, and then cultured with or without 2 nM IL-2 for 48 h. Cells were stained with anti-CD25–FITC, anti-CD4–PE, and anti-CD8–Cy-Chrome. Shown are IL-2Rα expression and mean fluorescent intensity on CD4⁺ or CD8⁺ T cells from one representative mouse out of eight mice analyzed. (B) Northern blotting for IL-2Rα, IL-2Rβ, or control pHe7 using RNA from preactivated splenocytes not treated or treated with IL-2. Shown are representative data from three mice. (C) The relative levels of IL-2Rα and IL-2Rβ mRNA expression in B were normalized for pHe7 expression. (D) High dose IL-2 (1 nM) could not restore proliferation of Stat5b^−/− splenocytes preactivated with anti-CD3. Shown are the mean ± SEM from a compilation of two independent experiments. (E) Proliferation to PMA plus IL-4 was done in Stat5b^−/− splenocytes. Shown are the mean ± SD from a compilation of two experiments. In one experiment, we confirmed that treatment of cells with PMA alone or IL-4 alone yielded minimal proliferation even in wild type mice. (F) Equivalent expression of Stat6 in splenocytes from wild-type, Stat5a^−/− and Stat5b^−/− mice.

Figure 3

(A) Diminished proliferation of freshly isolated splenocytes from Stat5b^−/− mice to 2 nM IL-2 or 7.75 nM IL-15. Shown are the mean ± SD from a compilation of two independent experiments. (B) Flow cytometric analysis of CD3⁻DX5⁺ NK cells in splenocytes from wild type, Stat5a^−/−, and Stat5b^−/− mice. (C–E) Decreased NK cytolytic activity in Stat5b^−/− mice in the presence of no cytokine (C), IL-2 (D), or IL-15 (E). Shown are the mean ± SEM from combining two or three independent experiments, each of which had three mice of each genotype. (F) Defective IL-2Rβ expression in NK cells in Stat5b^−/− mice, as compared with levels in wild type or Stat5a^−/− mice. A total of 14 Stat5a^−/− and 17 Stat5b^−/− mice were examined.

Figure 4

(A) Northern blotting for perforin or control pHe7 using RNA from fresh splenocytes or from splenocytes treated with IL-2 or IL-15 for 90 h. (B) For each condition, the relative level of perforin mRNA expression in A was normalized for pHe7 expression.