Prolonged T–DC macro-clustering in lymph nodes initiates site-specific Th2 differentiation

T helper 2 (Th2) responses protect against pathogens while also driving allergic inflammation, yet how large-scale Th2 responses are generated in tissue context remains unclear. Here, we used quantitative imaging to investigate early Th2 differentiation within lymph nodes (LNs) following cutaneous allergen administration. Contrary to current models, we observed extensive activation and ‘macro-clustering’ of early Th2 cells with migratory type-2 dendritic cells (cDC2s) generating specialized Th2-promoting microenvironments. Macro-clustering was integrin mediated and promoted localized cytokine exchange among T cells to reinforce differentiation, which contrasted behavior during Th1 responses. Unexpectedly, formation of Th2 macro-clusters was dependent on the site of skin sensitization. Differences between sites were driven by divergent activation states of migratory cDC2 from different dermal tissues, with enhanced costimulatory molecule expression by cDC2 in Th2-generating LNs promoting prolonged T cell activation, macro-clustering, and cytokine sensing. Thus, generation of dedicated Th2 priming microenvironments through enhanced costimulatory molecule signaling initiates Th2 responses in vivo and occurs in a skin site-specific manner. To investigate transcriptional differences in dendritic cells between auricular (Au) and brachial (Br) draining lymph nodes (LNs), we immunized mice in the ear or forepaw with papain and EaGFP. LN tissues were harvested 48 hours later and mechanically disrupted and subject to digestion in PBS with 10% fetal bovine serum (FBS) with DNase I (100µg/mL; Sigma), Dispase II (800µg/mL; Sigma), and Collagenase P (200µg/mL; Sigma) at 37°C shaking at 150rpm for 30 minutes with periodic manual disruption. We then stained and FACS sorted cells from three populations of EaGFP+ MHC-II-hi CD11c-int antigen bearing cDC2 (CD301b+CD11b+, CD301b-CD11b+, and CD301b-CD11b-) from Au or Br draining LNs 48 hours post-immunization. We also sorted non-antigen bearing DCs of the same populations from naive mice. Cells were sorted from pooled dLNs from 3 individual mice for each group. 500 of each cDC2 cell type was sorted on an Aria III (BD Biosciences) directly into reaction buffer from the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara), and reverse transcription was performed followed by PCR amplification to generate full length amplified cDNA.