Fig 2: Construction of the immune network independent of cell type by addition of globally co-expressed genes and protein-protein interacting partners. The immune network module (module 27) contained 65 genes which was expanded to 77 genes by addition of globally co-expressed genes and protein-protein interacting partners. Each gene is represented by a node and is labelled with its HUGO identifier. Genes originating from module 27 are arranged on the left-side of the diagram; genes identified as globally co-expressed or protein-protein interacting partners are arranged on the right-side of the diagram. Relationships between genes are represented as edges between nodes, either global co-expression (purple) or protein-protein interaction (pink). Only genes with edges reaching statistical significance are shown. CEBPD, LILRA2 and ITGB2 (blue nodes), represent a proposed blood-based biomarker; TREM2 (red node) protein measured in CSF correlates with disease duration in selected patients

Fig 3: Post-mortem tissues from NHD patients display lysosomal and lipid metabolism defects. a NanoString analysis was performed on hippocampi from four NHD patients (one TREM2 D134G; one TREM2 c.482 + 2 T > C; two TYROBP c.141delG) and 17 controls. Volcano plot of DEG between NHD and control brains (p < 0.05). Red: upregulated genes; blue: downregulated genes. b ATP6AP2, and c LAMP2 mRNA (raw counts) from NanoString analysis of NHD versus control brains. ****P < 0.0001. d Venn diagram of DEGs shared between NanoString analysis of PLOS tissues homogenates (hippocampi) versus controls (p < 0.05) and the DEGs of PLOS (n = 3) versus controls (n = 11) in the microglia cluster from snRNA-seq dataset [108] (Supplementary Table 7, online resource). e Representative confocal images of Iba-1+ microglia (red) and DAPI (blue) staining of frontal lobe (FL) regions of a healthy control, one NHD patient (NHD1+ (FL)), and one multiple sclerosis (MS) patient (active lesion in the medulla). Scale bar, 20 µm. f Microglia morphometric analysis of FL regions of three healthy controls, one NHD1+ (FL), and two MS patients. The analysis was performed using a ramification index [RI = 4p × cell area/(cell perimeter)2] that captures microglia cell shape. FL: CTRL n = 38 cells; NHD1+ n = 15 cells; MS n = 50 cells. ****P < 0.0001, analysis was performed between two groups using an Unpaired T test. g Representative fluorescence images of Iba-1+ microglia (red), TMEM119 (gray), CD68 (green) and DAPI (blue) staining of FL regions of a healthy control, one NHD patient (NHD1+ (FL)), and one MS patients (active lesion in the medulla). Scale bar, 10 µm. h Quantification of CD68+ signal per TMEM119+ Iba1+ cell. FL: CTRL n = 28 cells; NHD1+ n = 10 cells; MS n = 20 cells. *P < 0.05, ****P < 0.0001, analysis was performed between two groups using a Mann–Whitney T test. i Representative images of Iba-1+ microglia (red), PLIN2 (grey), LAMP1 (green) and DAPI (blue) staining of FL regions of a healthy control, one NHD patient (NHD1+ (FL)), and one MS patients (active lesion in the medulla). Scale bar, 20 µm. j Quantification of LAMP1 signal per Iba1+ cell. LAMP1 intensity: FL: CTRL n = 17 cells; NHD1+ n = 10 cells; MS n = 20 cells. ****P < 0.0001, analysis was performed between two groups using a Mann–Whitney T test. k White matter (WM) and grey matter (GW) regions with no pathology in one control individual (CTRL), one NHD patient, and one MS patient (normal appearing white matter, NAWM; and active lesions in the white matter, MS ACTIVE) were stained with solochrome cyanine staining (SOLOCY). Original magnification: 4X and 20X; scale bar: 250 µm and 25 µm, respectively. l Quantification of PLIN2 signal per Iba-1+ cell. PLIN2 intensity: FL: CTRL n = 17 cells; NHD1+ n = 10 cells; MS n = 20 cells. ****P < 0.0001, analysis was performed between two groups using a Mann–Whitney T test

Fig 4: Defective immune activation in NHD iMGLs. a Pathway analysis of protein coding differentially expressed genes (DEG) in NHD versus SB CTRL iMGLs (SB CTRL, NHD1, NHD2; n = 3 replicates/line; EnrichR; DEG are defined by FDR < 0.05, DEGs in Supplementary Table 2, online resource). b Heatmap of DEGs belonging to MHC I, MHC II, NF-?B and DAM pathways in iMGLs. c Representative histograms. d-f relative quantification performed by FACS showing cell surface d CD80, e CD86 and f HLA-DR MFIs in NR CTRL, SB CTRL, NHD1 and NHD2 iMGLs. g Representative dot plots showing the percentage (%) HLA-DR+ cells in SB CTRL, NHD1 and NHD2 iMGLs. h Quantification of % HLA-DR+ and i) CD14+ cells in NR CTRL, SB CTRL, NHD1 and NHD2 iMGLs. *P < 0.5, **P < 0.01, ***P < 0.001, ****P < 0.0001, One-way ANOVA with Tukey’s post hoc test. Data are pooled from four independent experiments. j Quantification of CD68 area in NR CTRL, SB CTRL, NHD1 and NHD2 iMGLs. k Representative confocal images of iMGLs stained for CD68 (cyan), TREM2 (red) and DAPI (blue). *P < 0.05, **P < 0.01, ****P < 0.0001, One-way ANOVA with Kruskal Wallis test and Dunn’s multiple comparison test. Data are pooled from two independent experiments. Data shown are mean ± SEM

Fig 5: TREM2 p.Q33X variant in two families affected by NHD. a and b NHD family pedigrees. Black symbols denote individuals clinically affected by NHD who carry the homozygous Q33X mutation in TREM2 (NHD1, NHD2, and NHD3). White symbols denote a clinically healthy, non-carrier family member (SB CTRL). Half shaded symbols denote heterozygous TREM2 p.Q33X carriers (HET1 and HET2). Analyses presented in this manuscript were performed on the three siblings belonging to the first NHD family (SB CTRL, NHD1 and NHD2) and one unrelated control (NR CTRL). For the NHD second family, analyses were performed on two parents (HET1 and HET2), their affected child (NHD3), and one unrelated control (NR CTRL2). Triangles are used to anonymize pedigree. c Schematic representation of the human TREM2 gene (top) and protein (bottom). TREM2 gene showing exonic (numbered boxes) and intronic (line) regions. The Q33X mutation is located in exon 2 (indicated as a red star). d Study design depicting the bioinformatic and cellular analyses performed in this study. iPSC-derived human microglia (iMGLs) were generated from iPSCs (all donor lines), while human macrophages were purified from peripheral blood mononuclear cells (PBMCs)-derived from the same donors (NHD1, NHD2 and SB CTRL). Fully differentiated iMGLs and macrophages were assayed for molecular and biochemical analyses