Journal: Advanced Science

Article Title: Spatial Transcriptomics Reveals a T Cell‐Mediated Microglial Activation Axis of Immune Checkpoint Inhibition‐Related Adverse Events in the Brain

doi: 10.1002/advs.77733

Figure Lengend Snippet: Depletion of T cells prevents ICI treatment‐induced microglial activation. (A) Experimental Design: Transgenic T‐DTR mice received diphtheria toxin A (DT) either 2 days before or 7 days after ICI treatment (anti‐CTLA‐4 and anti‐PD‐1). ICI treatments were given for 3 weeks (see Materials & Methods for details). WT (C57Bl/6) mice receiving either ITC or ICI treatment were used for comparison. Brains were collected at 72 h after completion of ICI treatments. (B) Representative dual‐immunofluorescence confocal z‐stacks depicting microglial activation (CD68, magenta, IBA1, green, and DAPI nuclear counterstaining) from T‐DTR and WT mouse hippocampus. (C) A 3D surface rendering of CD68‐IBA1 + activated microglia from the WT + ICI group (white arrows). (D) Volumetric quantification of CD68 and IBA1 immunoreactivity showing ICI treatment elevated microglial activation in WT + ICI group compared to WT + Vehicle. T cell depletion (T‐DTR mice) did not lead to microglial activation. Data are presented as Mean ± SEM, N = 6–8 mice/group. P ‐values were derived from ANOVA and Dunn's multiple comparisons test. Scale bar, 50 µm (A,B).

Article Snippet: Primary antibodies to characterize microglial activation include rat anti‐mouse CD68 (1:500, Bio‐Rad, Cat# MCA1957; RRID: AB_322219), and rabbit anti‐IBA1 (1:500, Wako, Cat# 019–19741; RRID: AB_839504).Secondary antibodies used for CD68 marker 1:1000; Goat anti Rat AF 647 (Abcam; Cat. ab150159), and 1:500; Goat anti Rabbit AF 488 (FisherSci; Cat. a11008) for IBA1 marker] For staining, tissue sections were first washed 3 times in phosphate‐buffered saline (1 × PBS, 100 m m , pH 7.4, FisherSci) and, when necessary, antigen retrieval was performed using citrate buffer incubation (10 m m , pH 6 with 0.05% Tween‐20, Sigma) at 70°C for 45 min. Tissues were then incubated in borate buffer (pH 8.5, 100 m m , Sigma, Cat. B0394) for 10 min, followed by three 5‐min washes in 1 × PBS.

Techniques: Activation Assay, Transgenic Assay, Comparison, Immunofluorescence, Confocal, Derivative Assay