FIG. 4.

Protection mediated by polyclonal influenza virus-specific memory CD4 T cells occurs in lymphocyte-deficient hosts. C57BL/6 mice were infected with 500 TCID₅₀ of PR8 influenza virus and monitored for infection, and CD4 T cells were purified from the spleens and lungs of these “flu-memory” mice at 4 to 8 weeks postinfection. (A) Frequencies of influenza virus NP-specific CD4 T cells 8 weeks after infection, as determined by intracellular cytokine staining following stimulation with 1 μg/ml anti-CD28 (medium alone), 1 μg/ml NP311-325 plus 1 μg/ml anti-CD28 (NP_311-325), or phorbol myristate acetate-ionomycin (PMA/Iono). These results are representative of two experiments, each with five mice per group. (B) CD4 T cells purified from the spleens and lungs of naive C57BL/6 mice or C57BL/6 flu-memory mice were transferred (8 × 10⁶ total cells, comprising 4 × 10⁶ cells from the spleen and 4 × 10⁶ cells from the lungs) intravenously into RAG2^−/− recipients, which were subsequently infected with 500 TCID₅₀ of PR8. The graph shows weight loss morbidities of the influenza virus-infected RAG2^−/−, RAG2+Naive, and RAG2+Memory groups up until day 7 postinfection. Weight loss differences between naive and memory groups were not significant. (C) Lung viral titers at day 7 postinfection in RAG2^−/−, RAG2+Naive, and RAG2+Memory mice, expressed as the average for 10 mice per group, compiled from two independent experiments with 5 mice per group (representative of three independent experiments). The significance of differences in viral titers between the RAG+Memory, RAG+Naïve, and RAG^−/− groups was determined by one-way ANOVA (P = 0.002).