Fig. 3.

ADAM10 deficiency results in the selective absence of splenic ESAM^hi cDC2A that is compensated for by an emerging Clec12a⁺ cDC2B subset. (A) Absolute cell counts of splenic ESAM^hi and ESAM^lo SIRP-α⁺ cDC2 in control and ADAM10^ΔCD11c mice. (B) Phenotypic comparison of splenic CD11c⁺MHCII⁺SIRP-α⁺ cDC2 isolated from control (Top) and ADAM10^ΔCD11c (Bottom) mice, assessed by flow cytometry. (C) Frequency of splenic ESAM^hi cDC2 in control, ADAM10^ΔXCR1, and ADAM10^ΔCX3CR1 mice. (D) t-distributed stochastic neighbor embedding (t-SNE) analysis of SC-seq-WTA profiled control and ADAM10-deficient cDC2 (gated for cells expressing Itgax, H2ab1, and Sirpa) showing automated clustering into an ESAM^hi cDC2 subpopulation (subpopulation I, purple) and two ESAM^lo cDC2 subpopulations (subpopulations II and III, green). Each dot represents an individual cell. (E) Comparison of the expression of CX₃CR1 versus ESAM and Ly6C on SIRP-α⁺ cDC2 in spleen (Top) and pLN (Bottom) from control and ADAM10^ΔCD11c mice assessed by flow cytometry. Filled gray histogram: isotype control. (F) Flt3 RNA expression (Reads Per Kilobase Million) in splenic CD4⁺ and CD4^– cDC2 subsets from control and ADAM10^ΔCD11c mice. (G) Flow cytometry plots show the frequency of Clec12a⁺SIRP-α⁺ splenic cDC2 in control and ADAM10^ΔCD11c mice, while bar graphs represent the absolute numbers of ESAM^hi and Clec12a⁺ cDC2. T-bet and Ror-γT expression, either as protein expression on control and ADAM10-deficient SIRP-α⁺ cDC2 determined by flow cytometry (H) or as RNA counts (Reads Per Kilobase Million) in bulk-sorted splenic CD11c⁺MHCII⁺CD4⁺ cDC2 (I). (J, Left) Violin plots show the expression of 69 Tbet⁺ cDC2A-associated key signature genes (purple) and 155 Tbet^– cDC2B-associated key signature genes (green) across bulk-sorted CD4⁺ cDC2 subsets. Expression is log₂ fold change between ADAM10 deficient/control. Gene sets are retrieved from published database and accessible as GEO GSE130201 (16). (Right) Unbiased t-SNE representation of SC-seq-WTA depicting cDC2 subpopulations I through III (Left, as in C), indicating the expression levels of the cDC2A-associated gene signature (Top) and the cDC2B-associated gene signature (Bottom) in these cDC2 subpopulations. Each dot represents an individual cell. (K) Unbiased t-SNE representation of SC-seq-WTA depicting cDC2 subpopulations I through III (Left, as in C), indicating the expression levels of splenic cDC2-associated gene signature (comprised of 464 genes, Top) and the pLN-resident cDC2-associated gene signature (comprised of 184 genes, Bottom) in the indicated cDC2 subpopulations. Each dot represents an individual cell. *P < 0.05, **P < 0.01, and ***P < 0.001 (Student’s t test), FACS plots show one representative mouse/group. Data are mean frequencies or mean fluorescent intensities ± SEM of more than five pooled experiments (n = 3 to 5 mice/experiment). RNA expression data are from three Cre-negative and three Cre-positive ADAM10^ΔCD11c mice.