Clonotypic analysis of protective influenza M2e-specific lung resident Th17 memory cells reveals extensive functional diversity

The fate of tissue-resident memory CD4 T cells (Trm) has been incompletely investigated. Here we show that intranasal, but not parenteral, immunization with CTA1-3M2e-DD stimulated M2e-specific Th17 Trm cells, which conferred strong protection against influenza virus infection in the lung. These cells rapidly expanded upon infection and effectively restricted virus replication as determined by CD4 T cell depletion studies. Single-cell RNAseq transcriptomic and TCR VDJ-analysis of M2e-tetramer-sorted CD4 T cells on day 3 and 8 post infection revealed complete Th17-lineage dominance (no Th1 or Tregs) with extensive functional diversity and expression of gene markers signifying mature resident Trm cells (Cd69, Nfkbid, Brd2, FosB). Unexpectedly, the same TCR clonotype hosted cells with different Th17 subcluster functions (IL-17, IL-22), regulatory and cytotoxic cells, suggesting a tissue and context-dependent differentiation of reactivated Th17 Trm cells. A gene set enrichment analysis demonstrated up-regulation of regulatory genes (Lag3, Tigit, Ctla4, Pdcd1) in M2e-specific Trm cells on day 8, indicating a tissue damage preventing function. Thus, contrary to current thinking, lung M2e-specific Th17 Trm cells are sufficient for controlling infection and for protecting against tissue injury. These findings will have strong implications for vaccine development against respiratory virus infections and influenza virus infections, in particular.