Fig 2: 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 3: 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 4: NIB.2 binds human and cynomolgus cell-based CD1d and demonstrates high affinity for recombinant CD1d antigens. (A) Representative SPR trace showing that NIB.2 binds strongly to human CD1d and not to related human proteins CD1a, CD1b, CD1c, CD1e, HLA-B37, and MR1. (B) NIB.2 binds recombinant human and cynomolgus CD1d/β2M expressed on transfected HEK293E cells (top panels, black histograms) and to native CD1d on primary human and cynomolgus PBMCs (bottom panels, black histograms). Negative control isotype-matched antibody showed no binding (white histograms).

Fig 5: CD1c+ DC Heterogeneity Is Evident in Human BM(A) Flow phenotyping of CD1c+ DCs from HC PB mononuclear cells (PBMCs) (representative example of n = 22), distinct from SIRPA-CD141+ cDC1s, CD123+CD303/4+ pDCs, and CD88+monocytes (Mono). CD14+CD163+BTLA- (orange), CD14-CD163+BTLA- (light orange), CD163-BTLA+CD5- (light red), and CD163-BTLA+CD5+ (red) CD1c+ DC subsets are indicated.(B) 3D representation of CD14, CD5, and BTLA expression (flow cytometry) across the CD1c+ DC population. Heatmap shows expression of CD163.(C) PCA of NanoString gene expression profiling of fluorescence-activated cell sorting (FACS)-purified DC subsets from n = 3 HC PBMCs. CD1c+ DCs were purified based on their expression of CD14, CD5, and BTLA (A).(D) Intracellular flow analysis of in vitro cytokine elaboration (percentage of positive cells) by PB monocytes (black) and CD1c+DC subsets CD14+ (orange), CD14-CD5- (gray), and CD5+ (red) from n = 9 HC donors in response to 14-h stimulation with TLR agonists (CpG, poly(I:C), CL075, and lipopolysaccharide [LPS]). p values were derived from paired two-tailed t tests; *p < 0.05; **p < 0.01; ***p < 0.005. Bars show mean ± SEM, and circles represent individual donors.(E and F) Representative examples of the flow profiling of DC subsets in human spleen (n = 3), dermis (n = 3) (E) and BM (n = 13) (F), gated as in (A). Histograms show CD163 and BTLA expression on CD14+ (orange), CD5+ (red) and CD14-CD5- (gray) CD1c+ DCs.(G) tSNE visualization of the expression of TFs and surface markers across HC PB and BM lineage(lin, CD3,19,20,56,161)-HLA-DR+ cells by CyTOF analysis. Black gates indicate the CD1c+DC population distinct from CD88+monocytes, CLEC9A+cDC1 and CD303+pDC. Red and orange gates indicate expression of lymphocyte- or monocyte-associated antigens, respectively.(H) Hierarchical clustering of single-cell transcriptomes of mature DCs from BM using all protein-coding, non-cell-cycle genes. Marker genes were identified within SC3 with parameters p < 0.01, area under the receiver operating characteristic curve (AUROC) > 0.85; cluster 1, pDCs (GZMB, JCHAIN); cluster 2, monocytes (S100A8, VCAN); cluster 3, CD14+ DC3s (HLA-DPB1); cluster 5, cDC1s (CD59). The top rows show fluorescence intensity of surface antigens (“Antigens”) from index-sorted cells, and “Phenotype” denotes their classification defined by surface markers.See also Figure S1.