Figure 6. Phenotypic and Functional Characterization of CD8^hi and CD8^lo Daughter T Cells.

CFSE-labeled OT-I T cells were injected into congenic mice, and immunization with peptide and LPS followed. After 24 hr (Q4R7), once-divided T cells (2^nd CFSE peak), were sorted into CD8^hi (proximal daughter) and CD8^lo (distal-daughter) populations by FACS.

(A) Sorted T cells were cocultured with antigen-pulsed DCs, and conjugation was assessed by flow cytometry after 20 min incubation (n = 3 experiments). *p < 0.005 by an unpaired two-tailed Student’s t test. Results are expressed as the mean percentage of conjugated cells ± SD.

(B–D) Sorted T cells were adoptively transferred into congenic mice that had been infected with Lm-Q4R7 1 or 3 days previously (three experiments, n = 3 mice per group). *p < 0.005. (B) Total splenic OT-I T cell numbers at days 4 and 6 after T cell transfer. (C) Representative FACS plots of KLRG1, IL-7Rα, and VLA-4 6 days after T cell transfer into mice that had been infected 72 hr prior to transfer. Numbers on the axes represent the log10 of fluorescence. (D) Total splenic OT-I T cell numbers of SLEC and MPEC cells at day 6 after infection (three experiments, n = 3 mice per group). *p < 0.001 (unpaired two-tailed Student’s t test). Results in (B) and (D) are expressed as the mean number of cells from triplicate mice ± SD.

(E) Sorted proximal or distal daughters were injected into RIP-OVA mice that had been infected with Lm-Q4R7 1 day previously. Diabetes development was assessed as in Figure 1 (see also Figures S3D and S3H).