Phenotypic characterization of splenic APCs upon SE infection. A Splenocytes were gated for size, excluding debris (FSC-A vs SSC-A), singlets (FSC-A vs FSC-H) and viability (Live/Dead marker-negative) consecutively. B A t-SNE analysis was performed on spleen samples of 7 dpi uninfected (uninf, blue) and SE-infected (SE-inf, red) chickens combined. Based on the t-SNE analysis, two population (subset 1 and subset 2) were found enriched among the splenocytes of SE-infected chickens. C The populations were evaluated for expression of MRC1LB versus CD11. Subset 2 was evaluated for its FSC-A vs SSC-A scatter pattern and further subdivided into subset 2a and subset 2b. D Subset 1 (CD11+ MRC1LB+), subset 2a (CD11+ MRC1LB− FSClow) and subset 2b (CD11+ MRC1LB− FSChigh) were sorted by FACS to assess gene expression of immune markers by RT-qPCR relative to the total splenocyte population. E The presence (%) and F numbers (cells/mg spleen) of macrophages in uninfected and SE-infected chickens were assessed over time. Mean + SEM per treatment and time point is shown (n = 5), for uninfected chickens at 0 dpi n = 3 and at 7 dpi n = 4. Statistical significance is indicated as ** p < 0.01.