CITE-seq Analysis of Dendritic Cells in the Gut-Draining Lymph Node at Homeostasis with LIPSTIC Labeling of Akkermansia muciniphila-Presenting Cells

Commensal-specific T cells contribute to tissue homeostasis within the intestines, and can influence disease pathology in the context of both cancer and autoimmunity. Antigen presenting cells play a critical role in influencing the differentiation of these T cells, but how the complex network of antigen presenting cells in the intestines drives commensal-specific T cells towards a variety of differentiation trajectories remains poorly understood. We examined the priming of T cells specific for the intestinal commensal bacterial species Akkermansia muciniphila, using the LIPSTIC system combined with single-cell RNA-sequencing to identify the APCs that prime A. muciniphila-specific T cells both at homeostasis when it elicits T follicular helper cells, and during inflammation, when it also elicits TH17 and TH1 cells. We found that A. muciniphila-specific T cells were primed by several transcriptionally distinct subpopulations of migratory cDC2s in both contexts. The identity of these subpopulations did not change with inflammation, but the distribution of presentation across the subpopulations shifted, with increased presentation by inflammatory cDC2s favoring TH1 and TH17 polarization. These results reveal how distinct T cell differentiation trajectories can be determined through varied interactions with multiple, functionally distinct subpopulations of APCs. Overall design: CITE-Seq: A. muciniphila-specific T cells engineered to express a CD40L-SortaseA fusion protein were adoptively transferred into CD40G5 mice, in which the CD40 gene had been modified to express an additional five N-terminal glycines. The CD40G5 mice were vertically colonized with a simplified microbiota known as Altered Schaedler's Flora plus A. muciniphila. 2.5 days after the T cell transfer, we performed LIPSTIC labeling by repeated injections of an LPETG-biotin peptide to identify APCs that were interacting with A. muciniphila-specific T cells. After 6 injections, gut-draining lymph nodes were harvested from 5 mice and enriched for dendritic cells. Enriched dendritic cells were stained with CITE-seq antibodies (ADTs and HTOs) and then dendritic cells were isolated by fluorescence-activated cell sorting before running on a 10X