Fig. 4.

ADAM10 is required for splenic cDC2 commitment and differentiation by impacting proliferation and cell survival. (A) Absolute numbers of committed Ly6C^– precDC1 (Top) and Ly6C⁺ precDC2 (Bottom) subsets in spleens of control and ADAM10^ΔCD11c mice. PrecDC were defined as Lin^–CD11c⁺MHCII^–FLT3⁺cells. (B) Flow cytometric analysis of SIRP-α and CD24 expression on CD11c⁺MHCII⁺CD11b^–CD8-α^– cells, which represent natural immature stages of cDC2 (aubergine gate) and cDC1 (orange gate) development, respectively. Cells were isolated from control (Top) or ADAM10^ΔCD11c (Bottom) spleens. Bar graphs indicate the frequency and absolute numbers of these cells found in the spleen. Control or ADAM10^ΔCD11c mice were injected with BrdU and proliferating cells were detected 12 h later as BrdU⁺ cells among splenic CD24⁺ precDC1 and SIRP-α⁺ precDC2 (C) or CD11c⁺MHCII⁺ mature splenic XCR1⁺ cDC1 and SIRP-α⁺ cDC2 (D). (E) Flow cytometry analysis for the expression of the proliferation-associated marker KI-67 in CD8-α⁺ cDC1 and CD4⁺ or CD4^– cDC2 subsets isolated from control and ADAM10-deficient mice. (F) Violin plots showing expression of cell-cycle signature genes across bulk-sorted CD11c⁺MHCII⁺ CD8⁺ cDC1 (orange), CD4⁺ (purple), and CD4^– (green) cDC2 subsets. Expression is log₂ fold change between ADAM10 deficient/control. Gene sets are retrieved from the publication by Tirosh et al. (60). (G) The frequency of (7AAD⁺Annexin V⁺) apoptotic cells among splenic ESAM^hi and ESAM^lo cDC2 in control and ADAM10^ΔCD11c mice. (H) Flow cytometric analysis of IRF-4 expression in splenic CD11c⁺MHCII⁺ SIRP-α⁺ cDC2. Cells were isolated from control or ADAM10^ΔCD11c mice. Filled histogram: isotype control. (I) The frequency of FAS (CD95)⁺ cells among splenic cDC1 and cDC2 subsets in control and ADAM10^ΔCD11c mice. *P < 0.05, **P < 0.01, and ***P < 0.001 (Student’s t test); data are pooled from three or more experiments (n = 3 to 5 mice/experiment); FACS plots show one representative mouse/group with mean fluorescent intensity ± SEM.