Extended Data Fig. 1: Havcr2 is highly expressed by microglia and induced by TGFβ.
From: Immune checkpoint TIM-3 regulates microglia and Alzheimer’s disease
a, Representative gating strategy for microglia. b, Representative gating strategy for immune cells in the spleen. c, Representative gating strategy for HEK293 cells transfected with Havcr2 constructs. d, Immune checkpoint genes were quantified by RT-qPCR in microglia in the brain (live CD45intCD11b+), and peripheral immune cells in the spleen (n = 3/condition, independent mice). e, The expression of immune checkpoint genes, including Havcr2, Lag3, and Vsir, is shown in mouse microglia and other cell populations in the central and peripheral nervous system using a public scRNAseq dataset19. f, The previous dataset19 was reanalyzed to detect a dissociation signature103 in microglia and perivascular macrophages. Unsupervised clustering divided microglia into 3 populations (MGL1, 2, and 3). Though genes induced by tissue dissociation were highly expressed in two of the three mouse microglial clusters103, Havcr2, Lag3, and Vsir showed high and specific expression in microglia (e). g, The expression of immune checkpoint genes and TGFβ pathway-related genes is shown in human microglia and other cell populations in the brain20. h, Immune checkpoint receptor expression on microglia was analyzed by flow cytometry during the early postnatal period (n = 9, 3, 5 for day 7, 13, 20, respectively, independent mice). i, Developmental alterations of MGnD and homeostasis-associated genes in microglia were analyzed using a published dataset21. MGnD and homeostasis-associated genes include the top 10 DEGs in Clec7a+ plaque-associated microglia10 and several selected genes. j, Primary microglia (live CD45intCD11b+) were cultured with either TGFβ (2, 10, or 50 ng/mL), M-CSF (10 ng/mL), GM-CSF (10 ng/mL), IL-27 (10 ng/mL), or LPS (10 ng/mL) for 24 h. RT-qPCR quantified the immune checkpoint gene expression (n = 3/condition, independent samples). k, TPM value of Havcr2 in the RNAseq analysis of sorted microglia (live CD45intCD11b+) from one-month-old Havcr2cKO mice (n = 4 (3 males, 1 female)), and Havcr2flox/flox mice (n = 5 (4 males, 1 female)). Avg Expr, average expression; Pct Expr, percent expressed; MGL, microglia; PVM, perivascular macrophage. Results are shown from one experiment, representing at least two independent experiments for h. Data are mean ± s.e.m. One-way ANOVA with Dunnett’s multiple comparisons test.
