Clonotypic analysis of protective influenza M2e-specific lung resident T helper memory cells reveals extensive functional plasticity

Intranasal influenza immunization with CTA1-3M2e-DD stimulated M2e-specific Th17 tissue resident memory (Trm) cells that conferred strong antibody-independent protection against infection at 6 months after priming. These cells rapidly expanded in the lung upon infection and effectively restricted virus replication. Single-cell RNAseq transcriptomic and TCR VDJ-analysis of M2e- tetramer-sorted Trm cells on day 3 and 8 post infection revealed complete Th17-lineage dominance (no Th1, Tfh or Foxp3+ Tregs) with extensive functional plasticity involving cytotoxicity (ThCTL) as well as regulatory functions. Unexpectedly, the same clonotype hosted cells with different Th17 subcluster functions (IL-17, IL-22), Tr1 cells and ThCTLs, suggesting a tissue and context-dependent differentiation of reactivated Th17 Trm cells. A gene set enrichment analysis demonstrated up- regulation of regulatory genes in the day 8 recovery phase. Contrary to present thinking lung M2e- specific Th17 Trm cells are sufficient for controlling infection and for preventing tissue injury. These findings will have strong implications for vaccine development. Overall design: Three immunizations with 5µg CTA1-3M2e-DD in sterile PBS were done with 10 days between the doses. Mice were sacrificed 6 months after the final immunization 3 or 8 days after an influenza A virus challenge infection. Influenza A virus challenge experiments were performed using a lethal dose of 4x LD50 of the mouse-adapted X47(H3N2) virus strain (a reassortant between A/Victoria/3/75 (H3N2) and A/Puerto Rico/8/34 (H1N1)) administrated i.n. to mice under light anaesthesia. Lungs were extracted and collected. Single cell suspensions of lung lymphocytes were prepared with the lung dissociation kit from Miltenyi Biotec (ref 130-095-927). Briefly, the harvested lungs were first added into the gentleMACS C tubes containing enzyme mix and broken up into smaller pieces with gentleMAC OctoDissociator (m_lung_01 program). The samples were then incubated at 37°C for 30 minutes before the complete digestion on gentleMAC OctoDissociator (m_lung_02 program), after which, the cell suspensions were washed once with PBS, passed through a mesh and resuspended in appropriate media for further processing. We assessed the CD4+ T cell response in BALB/c mice after immunizations (day 3 and day 8, post infection with influenza A virus) by flow cytometry using the PE-labeled M2e-tetramer, designed and produced by the NIH Tetramer Core Facility. Sorted lung M2e-teramer+ TCRb+ T cells (CD19-, CD8- F4/80-) from 5 mice at each time point were directly loaded onto a Chromium chip (10X Genomics) and run on the Chromium Controller for droplet generation. Reverse transcription was conducted in droplets and cDNA recovered through demulsification and bead purification. Pre-amplified cDNA further went through 10X library preparation for quantification of both mRNA and VDJ sequences {10.1038/nprot.2018.021} using 10X genomics v2 Chemistry kit. Libraries were sequenced on an Illumina Nextseq500 (High Output) targeting a minimum depth of 10,000 reads per cell across all samples.