Fig 2: Relationship of In Vitro Activated Monocyte-Derived Cells(A and B) PCA (21,250 present probes); displayed principal components (PCs): (A) 1 versus 2 and (B) 1 versus 3.(C) Heatmap of 1,000 most variable genes in dataset. Log2-expression values, z-transformed, scaled (-2 [blue] to 2 [red]).(D) Heatmap, Pearson correlation values (PCV) calculated pairwise between all cell types (top 1,000 most variable genes).(E) PCA (23,952 present probes).(F) Relative fractions of MO, BDCA1+ DC, infM, and infDC gene signatures in CD14+ MOs, different MO-derived cells, DCs.(G and H) Heatmaps of genes specifically expressed in (G) infDCs compared to infMs, BDCA1+ DCs, MOs (dataset 1), and MOs-GM-CSFIL-4(0-72h) versus MO-derived cells, CD14+ MOs, and DCs (dataset 2), or in (H) infMs compared to infDCs, BDCA1+ DCs, and MOs (dataset 1), and in MOs-GM-CSF and MOs-M-CSF versus MOs-GM-CSFIL-4(0-72h), CD14+ MOs, and DCs (dataset 2). PCVs between indicated group patterns of dataset 1 versus dataset 2, barplot next to heatmaps (correlation cutoff > 0.4). Genes analyzed in (J) and (K) highlighted in red. Log2-expression values, z-transformed, scaled (-2 [blue] to 2 [red]).(I) Histograms (flow cytometry analysis), relative expression CD226, MARCO, VSIG4, and CCR7 (representative data, n = 4).(J) Analysis of cell culture supernatants of MOs-M-CSF, MOs-GM-CSF, and MOs-GM-CSFIL-4(0-72h) for CCL22 and CCL2 using ELISA (n = 3, 2 technical replicates each, mean + SEM, one-way RM [repeated-measures] ANOVA, Tukey’s method for multiple test correction, with *p < 0.05, **p < 0.01, and ***p < 0.001; n.d., not detected).(K) MMP12 quantification (relative) in CD14+ MOs, MOs-M-CSF, MOs-GM-CSF, and MOs-GM-CSFIL-4(0-72h) by immunoblot (n = 3, mean + SEM, one-way ANOVA and Tukey’s method for multiple test correction, with *p < 0.05).Please also see Figure S1.

Fig 3: Manual gating analysis of myeloid and NK cell subsets reveals pediatric AITD patients show a tendency of reduced frequencies of total NK and the cytotoxic CD56lo NK subset compared to pediatric controls(A) Frequencies of dendritic cell (DC) subsets are similar among AITD and control subjects. Conventional type 1 DCs (cDC1) were gated as CD3-CD19-HLA-DR + CD14-CD16-CD11c + CD1c-, conventional type 2 DCs (cDC2) were gated as CD3-CD19-HLA-DR + CD14-CD16-CD11c + CD1c+, and plasmacytoid DC (pDC) were gated as CD3-CD19-HLA-DR + CD45RA + CD123 + CD11c-.(B) The frequency of classical monocytes (CD3-CD19-HLA-DR + CD14+) is increased in pediatric AITD subjects compared to pediatric controls, whereas the frequency of intermediate monocytes (CD3-CD19-HLA-DR + CD14 + CD16+) and non-classical monocytes (CD3-CD19-HLA-DR + CD14-CD16+) are similar among AITD and control subjects.(C) Frequencies of cytokine producing monocytes at baseline demonstrates adult AITD patients have reduced frequencies of IL-23p19+ and IL-6+ monocytes compared to adult controls.(D) The frequency of natural killer (NK; CD3-CD19-HLA-DR-CD16+) cells is reduced, but not significantly, in pediatric AITD and adult AITD patients compared to controls. The reduced frequency of NK cells appears relegated to the cytotoxic CD56lo NK cell subset, as opposed to the regulatory CD56hi NK subset. PA: pediatric AITD, PH: pediatric healthy control, AA: adult AITD, AH: adult healthy control. Data are represented as mean ± SEM. Samples were processed as depicted in Figure S1. Frequency of myeloid and NK cell subsets, and cytokine production were determined by manual gating, as depicted in Figures S3 and S6. Statistical significance determined by One-way ANOVA with Sidak multiple comparisons post-test. *p < 0.05, **p < 0.01.

Fig 4: Unsupervised clustering and dimension reduction analysis of myeloid and NK cells reveals similar frequencies of subpopulations in AITD patients and controls(A) Non-T/non-B cells which encompass myeloid and natural killer (NK) cells were manually gated as CD45 + CD3-CD19-and then analyzed using the unsupervised clustering and dimension reduction programs PhenoGraph and X-shift to generate distinct clusters based on cell surface marker expression (CD15, CD1c, CD11b, HLA-DR, CD56, CD11c, CD14, and CD16). A UMAP plot was created using total non-B/non-T cells from concatenated adult AITD, adult healthy control, pediatric AITD, and pediatric healthy control samples with a minimum of 17,000 cell events. Seven unique cell subpopulations were identified. UMAP plots of each surface marker are displayed to demonstrate varying expression levels among the myeloid and NK cell subpopulations.(B) Heatmap of cell surface marker expression for identification of the 5 myeloid and 2 NK cell subsets identified using PhenoGraph/X-Shift. cDC1: conventional type 1 dendritic cell; cDC2: conventional type 2 dendritic cell.(C) Frequencies of myeloid and NK cell subsets in adult AITD (AA), adult healthy control (AH), pediatric AITD (PA), and pediatric healthy control (PH) subjects demonstrate normal frequencies in AITD patients compared to controls. Data are represented as mean ± SEM. Samples were processed as depicted in Figure S1. Statistical significance determined by Mann-Whitney non-parametric unpaired Student's t tests.

Fig 5: IRF8hi and IRF8lo Pathways Are Differentially Compromised in IRF8 Deficiency(A) PB flow analysis of monocytes and DCs in subjects carrying heterozygous IRF8R83C or IRF8R291Q mutation (Het), their child carrying IRF8R83C/R291Q (Bi), and a carrier of dominant-negative heterozygous mutation IRF8V426fs (Dom) compared with HC (Cont).(B) Trucount quantification of PB DCs and monocytes in subjects carrying IRF8 mutations (gating shown in Figure S6A; Hambleton et al., 2011; Bigley et al., 2018)). Cont, n = 25; Het, n = 4 (IRF8R83C, IRF8R291Q, and two subjects carrying IRF8K108E); Dom, n = 3 (IRF8V426fs); Bi, n = 2 (IRF8R83C/R291Q and IRF8K108E/K108E).(C) Flow cytometry phenotyping of CD1c+ DC subsets derived from the CD1c+CD2+ gate (gray) in (A) to identify CD14+ DC3s (orange), CD14-BTLA- DC3s (light orange), CD5-BTLA+ DC2s (light red), and CD5+BTLA+ DC2s (red).(D) Proportion of CD1c+ DC subsets (gated as in C, from the individuals represented in B). C, control; H, heterozygous parents; D, dominant-negative heterozygotes (IRF8V426fs).(E) Flow analysis of DC and monocyte precursors in PB of subjects carrying IRF8 mutations as shown, gated as in Figure 4C.(F) Proportion of DC and monocyte precursors out of all pre-DCs in PB of subjects carrying IRF8 mutations, gated as in (E). C, control; H, heterozygous; D, IRF8V426fs(G and H) Intracellular flow analysis of in vitro cytokine elaboration (percentage of positive cells) by CD14+ monocytes (black), CD14+ DC3s (orange), CD14-CD5-CD1c+ DCs (gray), and CD5+ DC2s (red) (G) and CD2+ pre-pDCs and pDCs from HC (n = 8) and subjects carrying heterozygous IRF8R83C, IRF8R291Q (mean of technical duplicates) or IRF8V426fs (IRF8, red-outlined bars) (H). See also STAR Methods and Figure 1H.Bars show mean ± SEM, and circles represent individual subjects. *p < 0.05; **p < 0.01; ***p < 0.001; ˆp = 0.053, Mann-Whitney U. See also Figure S6.