Figure 4. IRE1α-XBP1 supports NK homeostatic proliferation.

(a) Schematic of lymphopenia-induced homeostatic proliferation experiments in b, c. (b) Relative percentage of transferred WT and IRE1^NK cells in the spleen of Rag2^−/−Il2rg^−/− recipients at specified time points after transfer. (c) Representative flow cytometric plots and quantification of CTV dilution of transferred NK cells in the spleen of recipient mice at day 4 after transfer. (d) Relative percentages and absolute number of WT and IRE1^NK cells after co-incubation with IL-2 and IL-15. (e) Representative flow cytometric plots of FSC and SSC of cells in d. (f, g) IRE1^NK and IRE1^f/f cells from littermates were pre-labeled with CTV and cultured with IL-2 and IL-15. Flow cytomemetic assays showing (f) CTV dilution and (g) Ki-67 levels at specified time points. (h) Human primary NK cells were cultured with IL-2 and IL-15, in the presence or absence of the IRE1 inhibitor 4μ8C. (left) Quantitative real-time PCR analysis of XBP1 splicing activity after 16 hr. β-ACTIN was used as reference. (middle) Flow cytometric analysis of CTV dilution at specified time points. Plots showed representative data derived from three PBMC donors. (right) Quantification of percentages of divided cells at day 6. (i) Flow cytometric analysis of CTV dilution as in h, except CD56^bright and CD56^dim cells were plotted separately. (j) Representative flow cytometric plots and quantification of XBP1s protein levels in CD56^bright and CD56^dim NK cells before and after 16 hr of cytokine stimulation. n = 2 mice/group in b. n = 4 mice/group in c, f, g. n = 3 mice/group (and 2 wells/mice for ex vivo culture) in d, e. n = 6, 5 and 5 PBMC donors in h, i and j. All error bars represent mean with s.d.. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data were analyzed by two-tailed unpaired Student’s t-test performed (c), two-way analysis of variance (ANOVA) with the Sidak post-test (d), one-way analysis of variance (ANOVA) with the Tukey post-test (f), or two-tailed paired Student’s t-test (h-j). Data are representative of 3 (b, c) or 2 (d-g) independent experiments, or pooled from 2 independent experiments (h-j).