Figure 2. Tcf7^-/- memory CD8⁺ T cells are impaired in secondary expansion and in differentiation to a Tcm phenotype.

(A) Frequency of early memory OT-I cells in PBLs. B6.SJL recipients of either WT or Tcf7^-/- OT-I cells were infected with actA^-LM-Ova, and the percentage of CD45.2⁺ OT-I in CD8⁺ T cells was determined on days 34-44 after infection. Data are pooled results from 2 independent experiments.

(B) Secondary expansion of memory CD8⁺ T cells in the spleens. Sorted WT or Tcf7^-/- memory OT-I T cells (6 × 10³) were transferred into naïve B6.SJL hosts, followed by infection with 2 × 10⁵ CFU of virulent LM-Ova. The numbers of CD45.2⁺ OT-I cells in the spleens were determined 6 days later. Data are means ± s.d. (n = 3). Similar results were obtained when total splenocytes containing equivalent numbers of WT or Tcf7^-/- memory OT-I cells were transferred without sorting separation (not shown).

(C) In vivo killing capacity of memory CD8⁺ T cells. CD45.2⁺ splenocytes were differentially labeled with CFSE. Ova peptide-pulsed CFSE^lo and non-peptide pulsed CFSE^hi cells were injected at 1:1 ratio into naïve or immune chimeras (35 days after infection). The spleens were harvested 4 hrs later, and percentages of CFSE^lo and CFSE^hi cells in CD45.2⁺ splenocytes were determined. Data are representative of 2 independent experiments with similar results.

(D) Surface staining for CD62L, CCR7, IL-7Rβ, and KLRG1 on antigen-specific memory CD8⁺ T cells. During days 75-85 after infection with actA^-LM-Ova, splenocytes from WT or Tcf7^-/- OT-I recipients were stained. Percentages of CD62L^hi and CCR7⁺ subsets were shown in histograms, with shaded histogram denoting isotype control. Data are representative of 2-3 independent experiments (n ≥ 4).

(E) Intracellular detection of IFN-γ, IL-2, TNF-α, and granzyme B in memory CD8⁺ T cells. Splenocytes were stimulated with Ova peptide for 6 hrs, followed by surface and intracellular staining. Gating of positive populations was based on respective isotype controls. All data are representative of 3 independent experiments (n ≥ 5).