NKG2D is induced de novo on naive CD4+ T cells under Th1- and Th17-polarizing conditions. (A) Thy1.2+ OT-II and Thy1.2− OT-IIxKlrk1−/− naive CD4+ T cells were cocultured and polarized toward different Th lineages as described in Materials and methods. (B) Frequency of NKG2D+ Th1 cells generated from WT (α-CD3) or OT-II (OVA323–339) mice in the presence or absence of APCs. Data shown are mean ± SEM, n = 15 (pCD3), n = 6 (sCD3/APC), and n = 16 (OVA323–339/APC). (C) Frequency of NKG2D+ Th1 cells in response to increasing doses of OVA323–339. Data shown are mean ± SEM; line connects the mean (n = 5). (D) Frequency of NKG2D+ Th1 cells in response to increasing doses of IL-12. Data shown are mean ± SEM (n = 6). (E) Frequency of NKG2D+ Th1 cells derived from WT or Stat4−/− mice in response to increasing doses of α-CD3. Mean ± SEM is shown; line connects the means (n = 3). (F) Frequency of NKG2D+ CD4+ T cells after gating on CXCR3+ CD44+ population. Each symbol represents a mouse. Data shown are mean ± SEM; n = 6 (WT) and n = 5 (Stat4−/−). Mann–Whitney U test. ns, not significant. (G) Frequency of NKG2D+ Th17 polarized as indicated, at different time points of the culture. Data show mean ± SEM (n = 3–7). Asterisk (*) indicates that IL-12 was added in the second week of the culture. (H) Frequency of NKG2D+ Th17 polarized as indicated. Data show mean ± SEM (n = 7). (I) qPCR analysis of Tbx21 expression in Th1 and Th17 cells sorted as NKG2D+ or Klrk1−/− cells and in naive CD4+ T cells. Values are normalized to housekeeping genes Eef1a1 and Ube2g1. Each symbol represents an individual culture. Data show mean ± SEM; n = 10 (Th1), n = 12 (Th17; -TGF-β), and n = 3 (Th17; -TGF-β + IL-12*). (J) Representative FC analysis of CD94 expression on NKG2D+ and Klrk1−/− Th1 or Th17 (-TGF-β + IL-12*) cells on day 10 of culture (n = 6). (K) qPCR analysis of Rorc expression in Th1 and Th17 cells sorted as NKG2D+ or Klrk1−/− cells and in naive CD4+ T cells. Values are normalized to housekeeping genes Eef1a1 and Ube2g1. Each symbol represents an individual culture. Data show mean ± SEM; n = 10 (Th1), n = 11 (Th17; -TGF-β), and n = 3 (Th17; -TGF-β + IL-12*). In A–K, data are representative of at least three independent experiments.
Figure S2.

NKG2D is induced de novo on naive CD4+ T cells under Th1- and Th17-polarizing conditions. (A) Thy1.2+ OT-II and Thy1.2− OT-IIxKlrk1−/− naive CD4+ T cells were cocultured and polarized toward different Th lineages as described in Materials and methods. (B) Frequency of NKG2D+ Th1 cells generated from WT (α-CD3) or OT-II (OVA323–339) mice in the presence or absence of APCs. Data shown are mean ± SEM, n = 15 (pCD3), n = 6 (sCD3/APC), and n = 16 (OVA323–339/APC). (C) Frequency of NKG2D+ Th1 cells in response to increasing doses of OVA323–339. Data shown are mean ± SEM; line connects the mean (n = 5). (D) Frequency of NKG2D+ Th1 cells in response to increasing doses of IL-12. Data shown are mean ± SEM (n = 6). (E) Frequency of NKG2D+ Th1 cells derived from WT or Stat4−/− mice in response to increasing doses of α-CD3. Mean ± SEM is shown; line connects the means (n = 3). (F) Frequency of NKG2D+ CD4+ T cells after gating on CXCR3+ CD44+ population. Each symbol represents a mouse. Data shown are mean ± SEM; n = 6 (WT) and n = 5 (Stat4−/−). Mann–Whitney U test. ns, not significant. (G) Frequency of NKG2D+ Th17 polarized as indicated, at different time points of the culture. Data show mean ± SEM (n = 3–7). Asterisk (*) indicates that IL-12 was added in the second week of the culture. (H) Frequency of NKG2D+ Th17 polarized as indicated. Data show mean ± SEM (n = 7). (I) qPCR analysis of Tbx21 expression in Th1 and Th17 cells sorted as NKG2D+ or Klrk1−/− cells and in naive CD4+ T cells. Values are normalized to housekeeping genes Eef1a1 and Ube2g1. Each symbol represents an individual culture. Data show mean ± SEM; n = 10 (Th1), n = 12 (Th17; -TGF-β), and n = 3 (Th17; -TGF-β + IL-12*). (J) Representative FC analysis of CD94 expression on NKG2D+ and Klrk1−/− Th1 or Th17 (-TGF-β + IL-12*) cells on day 10 of culture (n = 6). (K) qPCR analysis of Rorc expression in Th1 and Th17 cells sorted as NKG2D+ or Klrk1−/− cells and in naive CD4+ T cells. Values are normalized to housekeeping genes Eef1a1 and Ube2g1. Each symbol represents an individual culture. Data show mean ± SEM; n = 10 (Th1), n = 11 (Th17; -TGF-β), and n = 3 (Th17; -TGF-β + IL-12*). In A–K, data are representative of at least three independent experiments.

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