Fig 2: Proposed diagram of the loss-of-function LILRB1 variant resulting in immune over activity and autoimmune diseases.

Fig 3: Flow cytometry and single cell RNA sequencing (scRNA-seq) results. (a) Comparison of mean fluorescent intensity (MFI) of LILRB1 expression on the surface of activated Treg cells in family members (b) High-dimensional transcriptomic scRNA-seq clustering reveals increased percentage of monocyte subsets in the three studied patients compared with the sex-, age- and ethnic-matched control. (c) Two-dimensional UMAP visualization of PBMCs of patients with the LILRB1 variant. Colors represent four clusters (cell types) of interest similar to (b) (top panel). Expression of marker genes for LILRB1 and SHP-1 (bottom panel). Red color indicates high expression level of the gene. (d) UMAP visualization of PBMCs of patients with the macrophage subset markers CD86 (left panel) and flow cytometry results of the frequency (middle panel) and MFI (right) of CD86 marker, where red and blue dots indicate samples of patients and controls concurrently investigated in scRNA-seq experiment, respectively. * indicates statistical significance. (e) LYZ (left panel) and CLEC10A (right panel) expression in cell population. Visualization of single-cell transcriptome data was done in R (v. 4.2.1; https://www.R-project.org) using RStudio (http://www.rstudio.com) and R packages tidyverse (v. 1.3.1; https://doi.org/10.21105/joss.01686) and ggpubr (v. 0.4.0; https://CRAN.R-project.org/package=ggpubr).

Fig 4: The c.479G > A (p.G160E) variant in LILRB1 decreases SHP-1 phosphorylation. (a) The protein levels of LILRB1 and SHP-1 in the Jurkat cells transfected with the wild-type (WT) LILRB1 and mutant (MT) LILRB1 in different time points as detected by Western blotting. GAPDH was used as a loading control. The grouping of blots was cropped from different parts of the same gel. Control denotes Jurkat cells without transfection and PV denotes pervanadate.(b) Representative Western blot graphs showing relative densitometric bar graphs of total proteins of LILRB1 and SHP-1 normalized to the intensity of the corresponding GAPDH bands. (c) The tyrosine phosphorylation status of SHP-1 was examined using Western blot analysis. The grouping of blots was cropped from different parts of the same gel. (d) Representative western blot graphs showing relative densitometric bar graphs of phosphorylated SHP-1 and GAPDH. (e) Representative blots of Human Phospho-Immunoreceptor Antibody Arrays treated with PV and incubated with a phosphotyrosine-specific antibody. Dots of LILRB1, SHP-1, and controls are boxed in green, blue, and orange, respectively. (f) Phosphorylation levels of the dot blots. The relative change in the phosphorylation state of LILRB1 and SHP-1 (the average signal of the pixel density of the pair of duplicate spots) to the average of signal reference spots in three corners of the array. The original Western blots are presented in Supplementary Fig. S8 and S9, respectively.