Immunization with live influenza virus induces greater polyfunctional CD4+ T cell response compared to inactivated virus

Previous work has shown that infection of dendritic cells with influenza virus results in more diverse and efficient antigen presentation on MHC-II compared to treatment with excess doses of killed influenza virus. Accordingly, immunization of mice with live influenza virus elicits a greater frequency and breadth of CD4+ T cells than killed virus, as determined by interferon (IFN)-gamma ELISpot. Here, we sought to more fully characterize the differences in CD4+ T cell functionality induced by live vs. killed virus vaccines. C57Bl/6 mice were immunized with live influenza virus A/Puerto Rico/8/1934 (PR8) or a large excess dose of inactivated PR8. Efforts were made to control for the antigen load and adjuvant effects. Influenza-specific CD4+ T cells were evaluated by peptide/MHC-II tetramer staining, intracellular cytokine staining after peptide restimulation, and flow cytometry. Greater frequencies of PR8-specific CD4+ T cells were elicited by live vs. killed virus, as measured by tetramer staining or expression of the cytokines IFN-gamma, TNF, or IL-2. Of particular note, live virus induced a significantly higher frequency of polyfunctional CD4+ T cells, producing two or more cytokines when stimulated with peptide. This result was evident when polyfunctional CD4+ T cells were expressed as a fraction of all CD4+ T cells or PR8 epitope-specific CD4+ T cells. The difference in polyfunctional CD4+ T cells was maintained when antigenically distinct influenza virus B/Lee/1940 was mixed with killed PR8 as an adjuvant or when killed PR8 was administered in a prime/boost series. The data are consistent with a model whereby endogenous antigen presentation on MHC-II transmits more potent activating signals to CD4+ T cells than exogenous presentation, leading to a greater level of type 1 helper cytokine production and potentially greater antiviral effects.