Divergent Role for STAT5 in the Adaptive Responses of Natural Killer Cells

Abstract

Natural killer (NK) cells are innate lymphocytes with the capacity to elicit adaptive features, including clonal expansion and immunological memory. Because signal transducer and activator of transcription 5 (STAT5) is essential for NK cell development, the roles of this transcription factor and its upstream cytokines interleukin-2 (IL-2) and IL-15 during infection have not been carefully investigated. In this study, we investigate how STAT5 regulates transcription during viral infection. We demonstrate that STAT5 is induced in NK cells by IL-12 and STAT4 early after infection and that partial STAT5 deficiency results in a defective capacity of NK cells to generate long-lived memory cells. Furthermore, we find a functional dichotomy of IL-2 and IL-15 signaling outputs during viral infection, whereby both cytokines drive clonal expansion, but only IL-15 is required for memory NK cell survival. We thus highlight a role for STAT5 signaling in promoting an optimal anti-viral NK cell response.

Keywords: IL-15; IL-2; MCMV; NK cell; STAT5; clonal expansion; memory.

Figure 1.. IL-12- and STAT4-Dependent Induction of STAT5 in NK Cells during MCMV Infection

(A) RNA-seq was performed on Ly49H⁺ NK cells on days 0, 2, 4. 7, 14, and 35 of MCMV infection. Normalized counts of Stat5a and Stat5b are displayed. (B) RNA-seq on WT versus Il12rb2^−/− or Stat4^−/− from mixed BMC mice on day 2 PI. Normalized counts of Stat5a and Stat5b are displayed. (C) ChIP-seq was performed for STAT4 and H3K4me3 on WT and Stat4^−/− NK cells cultured in media alone (unstim) or with IL-12 and IL-18. Representative gene tracks of mapped STAT4 ChIP (blue tracks) or H3K4me3 ChIP (black tracks) are displayed. (D) NK cells were cultured in media (unstim) or with IL-12 + IL-18, and RNA-seq was performed after 3 h of culture. Normalized counts of Stat5a and Stat5b are displayed. Error bars indicate SEM.

Figure 2.. STAT5-Dependent Anti-viral NK Cell Response

(A) Stat5^fl/fl, NK^Cre × Stat5^fl/+, and NK^Cre × Stat5^fl/fl mice were infected with high-dose MCMV. Viral titers in the serum were measured on day 4 PI. Data are representative of 2 independent experiments (n = 4–6). (B and C) Mixed WT:NK^Cre × Stat5^fl/+ BMC mice were infected with MCMV. Ly49H⁺ WT or NK^Cre × Stat5^fl/+ NK cells from spleen were analyzed on day 2 PI for granzyme A and B expression (B) or indicated activation/maturation markers (C). Data are representative of at least 2 independent experiments (n = 4). (D) RNA-seq was performed on Ly49H⁺ WT or NK^Cre × Stat5^fl/+ NK cells at day 2 PI (n = 3). ChIP-seq was performed on NK cells stimulated with IL-2 and IL-15 for 3 h. Donut graph displays the proportion of DE genes (adjusted p value [p_adj] < 0.05) that are STAT5 bound (purple). Pie graph displays STAT5-bound DE genes (p_adj < 0.05) categorized by direction of gene expression. (E) Heatmap shows Z scores of all DE and STAT5-bound genes described in (D). (F) Representative gene tracks of mapped STAT5 ChIP. (G and H) Splenocytes from mixed WT:NK^Cre × Stat5^fl/+ BMC mice were adoptively transferred into Rag2^−/− × Ly49h^−/− mice and infected with MCMV. (G) Graph shows the relative WT to NK^Cre × Stat5^fl/+ NK cell percentages that were measured over the course of infection. (H) Bar graphs show percentage of memory Ly49H⁺ WT and NK^Cre × Stat5^fl/+ NK cells in spleen and liver and KLRG1 surface expression (liver) on day 30 PI. Data are representative of 2 independent experiments (n = 3–4). All error bars indicate SEM.

Figure 3.. Both IL-2 and IL-15 Drive NK Cell Expansion In Vivo

(A) Equal numbers of WT and Ubc^Cre-ERT2 × Il2rb^fl/fl or NK^Cre × CD25^fl/fl NK cells were transferred into Ly49h^−/− mice, followed by infection with MCMV. Donor Ubc^Cre-ERT2 × Il2rb^fl/fl mice were treated with tamoxifen on days −3, −2, and −1 prior to adoptive transfer. Following MCMV infection, relative percentages of Ly49H⁺ WT and KO NK cells are displayed (n = 4–5). (B) NK cells from WT mice, NK^Cre × CD25^fl/fl mice, or Ubc^Cre-ERT2 × Il2rb^fl/fl mice treated with tamoxifen on days −3, −2, and −1 were labeled with CTV and transferred into Ly49h^−/− mice, followed by infection with MCMV. Representative flow plots and graphs show amounts of undivided WT and KO NK cells from the liver at day 3 PI. (C) WT Ly49H⁺ NK cells were transferred into Ly49h^−/− mice treated followed by infection with MCMV and treatment with PBS, anti-IL-2, or anti-IL-15 on days −1 to 4 PI. Flow plots and graph show amount of Ki67 and granzyme A on transferred Ly49H⁺ NK cells at day 4 PI. Data are pooled from 2 independent experiments (n = 4–5). All error bars indicate SEM.

Figure 4.. IL-15 and STAT5 Are Required for the Survival of Memory NK Cells

(A–C) Splenic NK cells from WT:Ubc^Cre-ERT2 × Stat5^fl/fl mBMC mice were transferred into Rag2^−/− × Ly49h^−/− mice. Tamoxifen or oil (as a control) was administered on days 8 to 10 following MCMV infection. (B) Graphs show relative percentages of Ly49H⁺ WT and KO NK cells in the peripheral blood at indicated time points and in spleen and liver at day 32 PI. Data are representative of 2 independent experiments (n = 2–4). (C) Graph shows percentage of NK cells from spleen staining positive for FLICA on day 13 PI. Data are pooled from 2 independent experiments (n = 4–5). (D) WT Ly49H⁺ NK cells were transferred into Ly49h^−/− mice infected with MCMV, and recipient mice were treated with PBS, anti-IL-2, or anti-IL-15 from days 8 to 18 PI. Graph shows the number of NK cells in the spleen on day 30 PI. Data are representative of 2 independent experiments (n = 4–5). All error bars indicate SEM.