CD88 ⁺ monocytes capture antigen in the periphery, transport, and present the antigen to cognate T cells in the LLNs. (A) Top row: WT naive LLN were plotted as CD11c and CD11b to gate on myeloid cells. CD88⁺ neutrophils were excluded as CD64⁻CD26⁻ cells by plotting myeloid cells as CD64 vs. CD26. LN mononuclear phagocytes were then plotted as CD88 vs. CD26 to identify CD88⁺CD26^lo monocytes and CD88⁻CD26^hi DCs. Bottom row: Overlay of monocytes (green) and DCs (blue) to illustrate the intensity of expression of CD11c, MHCII, CD64, and Ly6C on monocytes, resident DCs, and migratory DCs. Data shown are representative of three independent experiments; n = 3–5. (B) LLN 24 h after i.n. delivery of CFSE or PBS. Top row: Control mice given PBS (no CFSE). Bottom row: Mice given CFSE. Left gate is CFSE⁺ myeloid cells. Bottom row: Overlay of CFSE⁺ migratory cells (blue) and all myeloid cells (yellow) illustrating that only migratory cDCs (MHCII high) migrate and not resident DCs (MHCII low). Data represent two independent experiments; n = 5. (C) LLN 24 h after i.n. delivery of OVA-AF488, a TLR agonist ± pertussis toxin (PT; no PT, left plots; with PT, right plots). Top row: Flow plots illustrate the myeloid gate, followed by (middle row) gated Ag⁺ myeloid cells, which were plotted as CD88 vs. CD26 to identify Ag⁺ monocytes and DCs (bottom row). Bar graphs compile the frequency and the total number of Ag⁺ myeloid cells in the LLNs; n = 4–5 mice per group. Data represent two independent experiments. (D) LLN Ag⁺ monocytes and cDCs were sorted 24 h after i.n. delivery with OVA-AF488 and a TLR agonist. Representative histograms show in vitro proliferation of CFSE-labeled antigen-specific CD8⁺ OTI and CD4⁺ OTII T cells. Data are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, mean ± SEM. One-way ordinary ANOVA, with post hoc Tukey’s multiple comparison test (C).