The Malat1-miR-15/16 Circuit Increase CD28 Expression and Co-stimulation induced Gene Expression
(A) Ago2 HITS-CLIP binding at the Cd28 locus. Sequencing libraries generated from CD8+ T cells isolated from spleens and cultured for 5 days (combined libraries from n = 2 for each genotype). Grey bar indicates the peak containing the TargetScan predicted miR-15/16 binding site.
(B) Schematic illustrating the assay scheme to assay acute gene expression downstream of CD28 co-stimulation
(C) Representative flow cytometry plots of CD28 expression on naive (CD62L+ CD44-) CD8+ T cells from spleens of unchallenged mice. Mean fluorescence intensity for the sample reported in the upper right of the plot.
(D) Quantification of CD28 mean fluorescence intensity normalized to the relevant control. (Malat1scr/scr compared to WT from 3 independent experiments; miR-15/16fl/fl compared to miR-15/16Δ/Δ from 2 independent experiments)
(E-H) Cumulative density plots comparing expression of CD28 responsive gene set defined as genes from (Martínez-Llordella et al., 2013) with ɑCD3ɑCD28 vs ɑCD3 log2(FC) > 1.5 and adjusted p value < 0.001.
Kolmogorov-Smirnov test used to determine significant differences in the distributions of target and non-target genes. ɑCD3 used at 1 μg/mL, and ɑ CD28 used at 1 μg/mL.
(E) Comparison of CD28 responsive genes in WT vs Malat1scr/scr cells stimulated with ɑCD3 alone
(F) Comparison of CD28 responsive genes in WT vs Malat1scr/scr cells stimulated with ɑCD3 and ɑCD28
(G) Comparison of CD28 responsive genes in miR-15/16fl/fl vs miR-15/16Δ/Δ cells stimulated with ɑCD3 alone
(H) Comparison of CD28 responsive genes in miR-15/16fl/fl vs miR-15/16Δ/Δ cells stimulated with ɑCD3 and ɑCD28
(I-J) Heatmaps of CD28 responsive gene set expression by genotype and stimulation condition. Dendrograms represent unbiased hierarchical clustering of the samples.
(I) Malat1scr/scr and WT samples compared with ɑCD3 ± ɑCD28
(J) miR-15/16fl/fl vs miR-15/16Δ/Δ samples compared with ɑCD3 ± ɑCD28 (*, p<0.05; **, p<0.01;***,p<0.001; ****, p<0.0001)