Reduced costimulatory marker expression on myeloid cells and reduced antigen-presenting capabilities. (A and B) UMAP map displaying 50,000 randomly sampled cells from the dLN of NIK^ΔCX3CR1 (n = 5) and littermate controls (n = 5) analyzed by flow cytometry, focusing on CD11b⁺ monocyte, Mac, and DC subsets 5 dpi. (B) Heatmap with median marker expression values for each population shown in the UMAP. (C) Relative frequencies of Ly6C^high monocytes (Mono), Ly6C^int Mono, Ly6C^lo Mono, ResDC2, MigDC2, MoDCs, Macs, other, pregated on CD11b⁺ cells, and dead cells, duplicates, T and B cells, neutrophils, and eosinophils were excluded. (D) Median (arcsinh-transformed) expression of CD80, CD86, MHCII, and PD-L1 across the eight identified myeloid cell populations. (E and F) Representative flow cytometry histograms showing CTV dilution of OT-II CD4⁺ T cells cocultured with either sorted Macs (T + Macs) (E) or migratory dendritic cells (T + migDCs) from dLNs (F) from control or NIK^ΔCX3CR1 mice. Accompanying graphs show the Division Index, reflecting the average number of divisions per input cell. Cells were pulsed with the indicated concentrations of OVA_323–339 peptide for 1 h prior to coculture. Data are shown as the mean ± SEM and analyzed using two-way ANOVA with Šídák’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. dpi = days after immunization. Each dot represents one mouse. Data are from at least two independent experiments. CTV, CellTrace Violet.