Journal: Frontiers in Immunology
Article Title: Gastrointestinal Microbiome Dysbiosis in Infant Mice Alters Peripheral CD8+ T Cell Receptor Signaling
Figure Lengend Snippet: Distal T cell receptor-mediated signaling is compromised in maternal antibiotic treatment (MAT) naïve CD8 + T cells . Total T cells enriched from the spleens of day of life 15 control (CTRL) and MAT infant mice were analyzed for (A) expression of Erk2 (CTRL, n = 5; MAT, n = 4), (B) percentage of phospho-Erk-1/2 (pErk1/2) positive cells (CTRL, n = 5; MAT, n = 4), (C) expression of pErk1/2 (CTRL, n = 5; MAT, n = 4), and (D) expression of c-Rel (CTRL, n = 6; MAT, n = 3) gating on naïve (CD44 − ) CD8 + T cells by flow cytometry. To analyze pErk1/2 expression (B,C) , T cells were left unstimulated (−) or stimulated (+) with anti-CD3/anti-CD28 or phorbol 12-myristate 13-acetate (PMA)/ionomycin for 2 min at 37°C. Representative graphs (CTRL, white; MAT, black; dashed line, negative staining control) and analysis of the median fluorescence intensity (MFI) or percentage are shown. Data are representative of two independent experiments, presented as mean + SEM and were analyzed by one-way ANOVA with Holm–Sidak posttest (B,C) or unpaired two-tailed Student’s t -test (D) . * p
Article Snippet: TCR Signaling Analysis by Flow Cytometry Infant CTRL and MAT T cells (5 × 105 cells/50 μl) that were freshly isolated (unstimulated) or stimulated with anti-CD3/anti-CD28 for 24, 48, and 72 h were incubated with or without soluble anti-CD3 (10 μg/ml; clone 145-2C11; BioLegend) and soluble anti-CD28 (10 μg/ml; clone 37.51, BioLegend) in cold RPMI 1640 supplemented with 0.5% FBS in 96 well round bottom plates at 4°C for 15 min, washed with cold medium, incubated with or without soluble goat anti-hamster IgG (20 μg/ml; Jackson ImmunoResearch Laboratories) at 4°C for 15 min, washed with cold medium, then resuspended in 50 μl of medium and incubated in a 37°C water bath for 2 min. After CD3/CD28 crosslinking, cells were immediately fixed with 50 μl of pre-warmed Cytofix buffer (BD Biosciences) for Erk2 and pErk1/2 staining or IC Fixation buffer (eBioscience) for ZAP-70, pZAP-70, pTyr, and c-Rel staining, incubated at 37°C for 10 min, and washed with FACS buffer (HBSS containing 1% FBS and 0.1% sodium azide) prior to staining with antibodies for CD8α, CD44, and CD62L at 4°C for 30 min. For intracellular staining of Erk2 and pErk1/2, cells were permeabilized with pre-chilled Phosflow Perm III buffer (BD Biosciences).
Techniques: Mouse Assay, Expressing, Flow Cytometry, Cytometry, Negative Staining, Fluorescence, Two Tailed Test