Figure 9.

Bcl11b expression is critical for the magnitude and functionality of memory CD8 T cells after respiratory infection with VacV. WT (Bcl11b^flox/flox/dLck-iCre⁻; n = 9) or Bcl11b-conditional knockout (Bcl11b^flox/flox/dLck-iCre⁺; n = 9) mice were infected i.n. with VacV-WR (1.5 × 10⁴ PFU/mouse). Fourty-two days postinfection, splenocytes and lung cells were harvested and stained for CD8α, CD44, and B8R_20–27/k^b-tetramer (A,B) or stimulated with B8R peptide for 8 h and subsequently stained for CD8α, CD107a, and intracellular IFN-γ, TNF, and IL-2 (C–H). (A,B) Left, representative plots of CD8⁺CD44^hi B8R_20–27/k^b-tetramer staining after gating on live cells. Percentages of B8R_20–27/k^b tetramer + CD8 T cells within each gate are indicated. Right, Frequencies and total numbers (bar graphs) of B8R_20–27/k^b tetramer + cells per spleen (A) and lung (B). Representative dot plots (Top Panels) for CD107a/IFN-γ (C), CD107α/TNF (D), and CD107α/IL-2 (E) staining in gated CD8⁺CD44⁺ T cells. Bar graphs of the percentages (Bottom Panels) of each cytokine within CD8⁺CD44⁺CD107a⁺ fraction are shown. Representative histogram plots (Top Panels) for IFN-γ (F), TNF (G), and IL-2 (H) gated on CD8⁺CD44⁺CD107a⁺ cells recovered from the spleen and lung as indicated. Numbers indicate percentages of positive cells or mean fluorescence intensity (MFI in red) within the gated population. Gates were based on controls (filled gray), after gating on CD44^lo cells in the same host. Percentages that stained positive for each marker are indicated. Bar graphs of the MFI (Bottom Panels) of each cytokine within CD8⁺CD44⁺CD107a⁺ fraction are shown. The results shown are representative of two separate experiments each with three to four mice per group. Asterisks indicate statistical significance. The p-values are < 0.05 by two-tailed Student t-test (WT vs. knockout).