Journal: PLoS Biology
Article Title: miR-181a/b-1 controls thymic selection of Treg cells and tunes their suppressive capacity
doi: 10.1371/journal.pbio.2006716
Figure Lengend Snippet: (A) Immunofluorescence staining of Treg cells from miR-181a/b-1 +/− or miR-181a/b-1 −/− mice with antibodies against Foxp3 and CTLA-4. Right panel, quantification of integrated density for CTLA-4 fluorescence normalized to the area of each individual cell. Data are representative of 3 independent experiments with n = 3 for each genotype (pooled). Data were analyzed using ImageJ software. DAPI, nuclear staining. Scale bar, 2 μm. (B) Immunofluorescence staining of CD4 + T cells from miR-181a/b-1 +/− or miR-181a/b-1 −/− mice with antibodies against murine CTLA-4, EEA1 (upper panel) and GM130 (lower panel). Data are representative of 3 independent experiments with n = 3 (pool). Right panels show colocalization scatter plots. (C) Immunofluorescence staining of CD4 + T cells from miR-181a/b-1 +/− or miR-181a/b-1 −/− mice with antibodies against murine CTLA-4, Rab11 (upper panel) and LAMP2 (lower panel). Data are representative of 3 independent experiments with n = 3 (pool). Right panels show colocalization scatter plots. Data were analyzed using ImageJ software. DAPI, nuclear staining. Scale bar, 2 μm. (D) Enhanced degradation of CTLA-4 in the absence of miR-181a/b-1. CD4 + T cells isolated from spls and subcutaneous LNs of miR-181a/b-1 +/− or miR-181a/b-1 −/− mice were stimulated for 2 h with αCD3/αCD28 antibodies. In order to monitor protein degradation, CHX was added and incubated with cells for an additional 3 h. Each dot represents an individual cell from 10–12 randomly chosen fields of view. Quantification of integrated density for CTLA-4 fluorescence per cell was quantified using ImageJ software. (E) CD4 + CD25 + -enriched T cells were incubated in the presence of an inhibitor of lysosomal degradation, bafilomycin, for indicated time points. Accumulation of CTLA-4 was detected using FACS, and fold increase of MFI was calculated using time point 0 as reference. Data from 3 independent experiments, n = 3. (F) Cell-intrinsic up-regulation of CTLA-4 in the absence of miR-181a/b-1. Mixed BM chimeras were generated by mixing BM cells from miR-181a/b-1 +/− (CD45.1) and miR-181a/b-1 −/− (CD45.2) mice in a 1:1 ratio and injecting them into lethally irradiated WT recipients (CD45.1/2). Chimeras were analyzed 8 weeks after reconstitution for CTLA-4 expression in TCRβ + CD4 + Foxp3 + cells from the Thy, spl, and LNs. Plots are representative of 2 independent experiments with n = 9 per experiment. Each set of paired data points represents an individual mouse. (G) RNA flow-cytometry analysis of the expression of Nr4a1 by TCRβ + CD4 + Foxp3 + Treg cells from pooled spl and LNs. Numbers indicate average MFI ± SD. Data from 2 independent experiments with n = 2–3 per experiment. Statistical analysis was performed using unpaired Student’s t test (A), two-way ANOVA (E, p -values for effect of genotype, ** p = 0.0021) and paired Student’s t test (F, p -values for effect of each genotype, ** p = 0.0021, *** p = 0.0002, and **** p < 0.0001). Numerical values are available in . AU, arbitrary unit; BM, bone marrow; CD, cluster of differentiation; CHX, cycloheximide; CTLA-4, cytotoxic T-lymphocyte–associated protein 4; EEA1, early endosome antigen 1; FACS, fluorescence-activated cell scan; Foxp3, forkhead box protein P3; GM130, cis -Golgi matrix protein 130; LAMP2, lysosome-associated membrane protein 2; LN, lymph node; MFI, mean fluorescence intensity; miR-181, microRNA-181; Nr4a , Nuclear receptor subfamily 4 group A; Rab11, Ras-related in brain protein 11; spl, spleen; TCR, T-cell receptor; Thy, thymus; Treg cell, regulatory T cell; WT, wild type.
Article Snippet: The following primary antibodies were used: purified α-mouse CTLA-4 (UC10-4F10-11, BD Biosciences) labeled with DyLight650 antibody labeling kit (Pierce, Thermo Fisher Scientific) according to the manufacturer’s protocol, AlexaFluor488-labeled rat-α-mouse Foxp3 (MF23, BD Biosciences), purified rat-α-mouse LAMP2 (Hybridoma Bank), α-mouse EEA-1 (14/EEA1, BD Biosciences), α-mouse GM130 (35/GM130, BD Biosciences), and α-mouse Rab11 (D4F5, Cell Signaling, Danvers, MA).
Techniques: Immunofluorescence, Staining, Fluorescence, Software, Isolation, Incubation, Generated, Irradiation, Expressing, Flow Cytometry, Membrane
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