Upon resolution of influenza A virus (IAV) infection in mice, lung memory CD4 T cells persist in clusters with other immune cells. These clusters remain for 40 days. Understanding how sustained cell interactions in clusters drive the generation of protective memory CD4 T cells may improve vaccine design.

TRACE mice allow the identification of IAV specific CD4 T cells by EYFP expression trigged by TCR activation in the presence of doxycycline. Lung sections from TRACE mice were analysed by microscopy to visualise clusters over time and interactions between IAV specific T cells and other immune cells. To investigate the outcome of these interactions, MHCII tetramer and cytokine assays were used to count IAV specific T cells in mice whose lung TCR-pMHC interactions were blocked with an anti-MHCII antibody given i.n. 6- and 12-days post infection.

At day 10 post infection, IAV specific T cells and APCs infiltrate into the lung forming large clusters. By day 40, cluster area is reduced but the total number of clusters is increased. These clusters contain elevated numbers of IAV specific CD4 T cells and CD11c+ MHCII+ cells at day 40 compared to day 10.

Blocking lung cell interactions reduced the number but did not affect cytokine production of IAV polyclonal CD4 memory T cells. In contrast, IFNy production was reduced in memory CD4 T cells that specifically recognise the immunodominant IAV nucleoprotein epitope NP311-325. T cells specific for NP311-325 also express higher levels of ICOS and PD1 than total responding T cells. This suggests that immunodominant T cells may be more dependent on continued T cell: APC interactions than other IAV-polyclonal T cells.