Figure 7. Constitutive expression of TL on intestinal epithelial cells mediates selection of mature memory CD8αβ T cells.

(a,b) Naïve Ly5.1⁺ CD8⁺OT-I cells were cultured in the presence of APC (MEC.B7.SigOVA). After 2 days' culture, CD8αα^hi and CD8αα^lo/- OT-I cells were sorted and cultured for 3 more days in vitro. Then 0.5 × 10⁶ CD8αα^hi or CD8αα^lo/- cells were adoptively transferred into B6 recipients. One month after transfer, mice were orally infected with 5 × 10⁸ ActA^- Lm-OVA. Donor Ly5.1⁺ OT-I cells were tracked in the spleen and IEL 3 d (a) and 5 d (b) p.i.. Representative data from 3-4 mice in each group are shown. At least five independent experiments were performed. (c) As shown in (a), secondary OT-I memory cells were assessed in the IEL 45 d p.i.. Representative data from three to four mice in each group are shown. At least three independent experiments were performed. (d) Sorted in vitro activated Ly5.1⁺ CD8αα^lo/- OT-I cells were cultured for 3 d and 0.5 × 10⁶ primary effector cells were transferred into WT or TL^-recipients. One month after transfer, mice were orally infected with 5 × 10⁸ ActA^- Lm-OVA. 4 months p.i., memory OT-I cells were tracked in the spleens and IEL. Pooled data ± s.e.m. are shown. At least two independent experiments were performed. (e, f) 5 × 10⁴ naïve CD8⁺ OT-I cells were transferred into Ly5.1⁺ WT or Ly5.1⁺ TL^- recipient mice. 1 d after transfer, mice were orally infected with Lm-Q4OVA. Effector OT-I cells in the peripheral blood (7 d p.i.) and memory OT-I cells (2 m p.i.) in the spleen and IEL were analyzed. Pooled data ± s.e.m. are shown. (g, h) 5 × 10⁴ naïve CD8⁺ OT-I cells were transferred into Ly5.1⁺ WT or Ly5.1⁺ TL^- recipient mice. 1 d after transfer, mice were intravenously infected with Lm-Q4OVA. Effector OT-I cells in the peripheral blood (7 d p.i.) and memory OT-I cells (2 m p.i.) in the spleen and IEL were analyzed. Pooled data ± s.e.m. are shown. Data are representative of three independent experiments(e, f, g, h). (i) Ly5.1 mice adoptively transferred with 5 × 10⁴ naïve WT or ΔE8_I OT-I cells were orally immunized with 1 × 10⁹ ActA^- Lm-OVA. Two months after immunization, mice were re-challenged orally with 1 × 10¹⁰ WT Lm-OVA. Bacterial loads in the livers were assessed day 3 p.i.. Pooled data ± s.e.m. are shown (n = 6). Representative data are shown of three independent experiments. * P < 0.05; NS: not significant (unpaired t-test).