A chemokine trail laid down by migratory dendritic cells directs antigen-presenting monocytes into draining lymph nodes

Antigen presenting dendritic cells (DCs) and monocytes capture and transport antigens from barrier tissues for presentation to antigen-specific T cells in the draining-lymph nodes (LNs). While DCs enter LNs through afferent lymphatics in a CCR7-dependent manner, how exactly antigen-carrying monocytes reach LNs is less clear since monocytes do not express CCR7 and can also enter LNs via the bloodstream. In steady state, and following injection of several PAMPs, scRNA-seq data on LN mononuclear phagocytes identified LN resident versus migratory type 1 and type 2 conventional (c)DCs despite downregulation of DC subset-defining transcripts, such as Xcr1, Clec9a, H2-Ab1, Sirpa, and Clec10a on migratory cDCs. Migratory cDCs gained expression of transcripts controlling cellular migration such as Ccr7, Ccl17, Ccl22, and Ccl5, while migratory monocytes expressed Ccr5 without Ccr7. Using two tracking methods and a gating strategy that clearly distinguishes migratory CD88hiCD26lo monocytes from CD88-CD26hi cDCs, we found that both captured antigens in the lung and migrated to lung-draining LNs. Using global and mixed-chimeric Ccl5-, Ccr2-, Ccr5-, Ccr7-, and Batf3-deficient mice, we found that CCR5+ monocytes follow CCL5-secreting migratory cDCs to reach the draining LN via lymphatic vessels. In a model of asthma, such recruited monocytes regulated the induction of type 2 immunity. Overall, our data suggest that CCL5-secreting migratory cDCs lay down the chemokine trail for CCR5+ antigen-presenting monocytes to reach draining lymph nodes and regulate adaptive immunity. Overall design: Five groups of mice (n=4) were immunized as described before without TLR adjuvant or with individual different TLR adjuvants: Poly:IC, LPS, CpG, R848 in 40 µl sterile PBS 24 hours before harvest. The LLNs were harvested and single cell suspensions were prepared. Cells were stained with flow cytometry antibodies and different TotalSeq A antibodies (BioLegend) according to the different TLR adjuvant treatment. After the staining, single cells suspensions were washed 3 times by resuspension in 4.5 ml HBSS plus 0.5% BSA, followed by centrifugation at 300g for 5 min at 4°C. After the final wash, cells from different treatment groups were resuspended and mixed. Myeloid cells were sorted as shown in Supplementary Figure 1A with the exclusion of Ly6G neutrophils. Sorted cells were centrifugated at 300g for 5 min at 4°C. Supernatants were aspirated, and pellets were resuspended with HBSS plus 0.5% BSA at an approximate concentration of 2.5 × 105 cells/ml.