Figure 3.

CD4⁺ T helper signals are required for optimal generation and functions of memory cytotoxic T-lymphocytes (CTLs) at higher precursor frequency (PF). (a) Ninety days after ovalbumin (OVA) -pulsed dendritic cells (DC_OVA) immunization, the peripheral blood samples were analysed for interferon-γ-positive (IFN-γ⁺) memory CTL survival by intracellular staining assay. The values represent mean % ± (SD) of IFN-γ⁺ CTL in total CD8⁺ T-cell population, and are cumulative of two independent experiments with five or six mice per group. **P < 0·01, versus wild-type (WT). (b) Sixty days following immunization, the blood samples were stained for triple markers, and the expression of activation (CD69) and memory [CD62 ligand (CD62L) and interleukin 7 receptor α (IL-7Rα)] markers were analysed in tetramer⁺ CD44^hi population (histogram grey-filled overlays). Irrelevant isotype-matched antibodies were used as controls (open black lines). (c) To determine recruitment and expansion of IFN-γ⁺ memory CTLs in lungs of challenged mice, the WT B6-immunized and Ia^b−/−-immunized mice were challenged 90 days later, and the lungs were assessed for IFN-γ⁺ CTLs by intracellular IFN-γ assay after 24 days. The values represent frequencies of IFN-γ⁺ CTLs in total CD8⁺ T-cell population, and are cumulative of two independent experiments with two or three mice per group. The horizontal bars indicate means. **P < 0·01, versus WT B6 mice.