Supplementary Figure 4: DC immunophenotyping, HLA-DR expression in monocytes and DCs, Flt3 ligand production, and IL-12p40 secretion by whole blood.

a) Example of the gating strategy for Fig. 3a,d,g. b) Graphic representation of CD141⁺ cDC1s as the percentage of total PBMCs in healthy controls, healthy family members (heterozygous or homozygous carriers), patients with AR SPPL2a deficiency and patients with AD IRF8 deficiency from Fig. 3a. c) Levels of HLA-DR expression in monocytes, CD1c⁺ cells, cDC1s and pDCs from healthy controls and SPPL2a-deficient patients. Mean and S.E.M. are indicated. d) Flow cytometry histograms of the samples plotted in (c). Numbers indicate the MFI for HLA-DR PerCP-Cy5.5. e) CD1c⁺ cDCs (cDC2s) from controls and patients had higher levels of HLA-DR and CD11c expression than CD1c^– cDCs. We assessed the presence of cDC2s in a CD1c-independent manner, by plotting CD11c vs. HLA-DR in live cells, FSC-A vs. SSC-A, FSC-A vs. FSC-W, HLA-DR⁺CD16^– CD14^–Lin-CD11c⁺CD1c⁺ or CD1c^–. The CD1c^– population in patients and controls did not display HLA-DR^high CD11c^high expression, indicating that the smaller CD1c⁺ cDC2 population in the patients had not shifted to the CD1c^– gate due to an absence of CD1c expression. These data provide further evidence of a lower levels of (cDC2s) CD1c⁺, HLA-DR^high, and CD11c^high cDCs in the patients with AR SPPL2a deficiency or AD IRF8 deficiency. f) Flt3 ligand levels were determined in the plasma of healthy controls, patients with AR SPPL2a deficiency, patients with AD IRF8 deficiency, two heterozygous SPPL2A carriers (P1’s parents) and seven patients carrying heterozygous germline mutations of GATA2 causing monoMAC syndrome, as positive controls. g) IL-12p40 production by whole blood from healthy controls, WT and heterozygous family members and SPPL2a-deficient patients after stimulation with BCG or BCG+IFN-γ. In f and g mean and S.D. are indicated. The p values observed in this figure correspond to an unpaired two tailed t-test with a 95% confidence interval (* p < 0.05, ** p < 0.01).