Secondary influenza challenge triggers lung resident memory B cell mobilization and rapid relocation for antibody secretion at infected loci

Resident memory B (BRM) cells develop and persist in the lungs of influenza infected mice and humans but their contribution to recall responses following rechallenge has not been defined. We used scRNA-seq to identify early changes in lung immune cell composition and gene expression following secondary influenza infection and tested the effect of alveolar macrophage depletion (using intranasally delivered CLL). We found an alveolar macrophage dependent upregulation of IFNG-associated pathways after influenza rechallenge, that included increased expression of CXCL9, CXCL10 and CCL5 in myeloid cells. B6 mice were infected with X31 influenza and were allowed to reach a memory phase (day 42 post primary infection). The mice were divided into 3 groups (n=3). One group was assessed without rechallenge. The second group was pre-treated with PBS intranasally on days 6 and 3 before being rechallenged with X31 influenza. The third group was pre-treated with CLL 3- and 6-days before rechallenge with X31 influenza. One day after rechallenge, the mice were in vivo labelled with anti-CD45 for 4min prior to tissue collection. Cells were sorted as (live, IV CD45-, ex vivo CD45+) and stained with a CITESEQ antibody panel to facilitate population identification. Samples were multiplexed in three pools using barcoded antibodies with one replicate of each condition present in each pool. There was a different assignment of HTO id to condition in pool 2. Each pool was captured on a seperate 10X channel.