Figure S2.
Transcriptional control of SLEC CD8+ T cell differentiation and S1pr5 expression via T-bet and Zeb2.(A) Mice were adoptively transferred with naive GFP+ OT-I (OT-I WT) and CD45.1+ OT-I Tbx21−/− (OT-I Tbx21−/−) and infected with HSV-OVA. Expression of indicated genes quantified by qPCR on OT-I T cells from the spleen 8 dpi and shown normalized to a housekeeping gene. (B) Effector gBT-I T cells were transduced with control (Ctrl-RV) or T-bet (Tbet-RV) GFP-expressing RVs and cotransferred into mice infected with HSV. Expression of indicated genes quantified by qPCR on sort-purified gBT-I T cells from the spleen 8 dpi and shown normalized to a housekeeping gene. (C) Effector gBT-I T cells were nucleofected with control-nontargeting (CD45.1+CD45.2+ gBT-I sgCtrl) or Zeb2-targeting (CD45.1+ gBT-I sgZeb2) sgRNA/Cas9 RNPs and cotransferred into HSV-infected mice. Expression of indicated genes quantified by qPCR on sort-purified transgenic T cells from the spleen 8 dpi and shown normalized to a housekeeping gene. (D) Effector gBT-I T cells were transduced with control or ZEB2 (ZEB2-RV) RVs and maintained in culture with IL-15 for 3 d. Expression of indicated genes was quantified by qPCR in GFP+ gBT-I T cells and shown normalized to a housekeeping gene. (E) Ctrl-RV– or Tbet-RV–transduced gBT-I T cells were nucleofected with control nontargeting (sgCtrl) or Zeb2 targeting (sgZeb2) sgRNA/Cas9 RNPs and cotransferred into HSV-infected mice. gBT-I T cells were sort-purified from the spleen 8 d after transfer. Tbx21 expression was quantified by qPCR and shown normalized to a housekeeping gene (left), and frequencies of SLECs were analyzed by flow cytometry (right). (F) Effector OT-I WT of Tbx21−/− were transduced with Ctrl-RV or ZEB2-RV and cultured with IL-15 for 3 d. Expression of the indicated genes in transduced cells was quantified by qPCR. In A–E, data are representative of two independent experiments, with n = 4–5 mice per group per experiment. In F, data are pooled from three independent experiments. *, P < 0.05; **, P < 0.01 by Mann–Whitney test. Graph shows mean ± SD. A.U., arbitrary units; rel., relative.

Transcriptional control of SLEC CD8+ T cell differentiation and S1pr5 expression via T-bet and Zeb2. (A) Mice were adoptively transferred with naive GFP+ OT-I (OT-I WT) and CD45.1+ OT-I Tbx21−/− (OT-I Tbx21−/−) and infected with HSV-OVA. Expression of indicated genes quantified by qPCR on OT-I T cells from the spleen 8 dpi and shown normalized to a housekeeping gene. (B) Effector gBT-I T cells were transduced with control (Ctrl-RV) or T-bet (Tbet-RV) GFP-expressing RVs and cotransferred into mice infected with HSV. Expression of indicated genes quantified by qPCR on sort-purified gBT-I T cells from the spleen 8 dpi and shown normalized to a housekeeping gene. (C) Effector gBT-I T cells were nucleofected with control-nontargeting (CD45.1+CD45.2+ gBT-I sgCtrl) or Zeb2-targeting (CD45.1+ gBT-I sgZeb2) sgRNA/Cas9 RNPs and cotransferred into HSV-infected mice. Expression of indicated genes quantified by qPCR on sort-purified transgenic T cells from the spleen 8 dpi and shown normalized to a housekeeping gene. (D) Effector gBT-I T cells were transduced with control or ZEB2 (ZEB2-RV) RVs and maintained in culture with IL-15 for 3 d. Expression of indicated genes was quantified by qPCR in GFP+ gBT-I T cells and shown normalized to a housekeeping gene. (E) Ctrl-RV– or Tbet-RV–transduced gBT-I T cells were nucleofected with control nontargeting (sgCtrl) or Zeb2 targeting (sgZeb2) sgRNA/Cas9 RNPs and cotransferred into HSV-infected mice. gBT-I T cells were sort-purified from the spleen 8 d after transfer. Tbx21 expression was quantified by qPCR and shown normalized to a housekeeping gene (left), and frequencies of SLECs were analyzed by flow cytometry (right). (F) Effector OT-I WT of Tbx21−/− were transduced with Ctrl-RV or ZEB2-RV and cultured with IL-15 for 3 d. Expression of the indicated genes in transduced cells was quantified by qPCR. In A–E, data are representative of two independent experiments, with n = 4–5 mice per group per experiment. In F, data are pooled from three independent experiments. *, P < 0.05; **, P < 0.01 by Mann–Whitney test. Graph shows mean ± SD. A.U., arbitrary units; rel., relative.

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