Journal: The Journal of Biological Chemistry

Article Title: Single-cell RNA-Seq analysis identifies a noncoding interleukin 4 ( IL-4 ) RNA that post-transcriptionally up-regulates IL-4 production in T helper cells

doi: 10.1074/jbc.RA118.004111

Figure Lengend Snippet: Single-cell RNA-Seq reveals transcriptome heterogeneity of in vitro differentiated Th2 cells. Naïve CD4+ T cells (CD4+CD62LhiCD44lowCD25−) isolated from C57BL/6 mice were stimulated with anti-CD3 and anti-CD28 in the presence of IL-4 and anti-IFN-γ. Five days after stimulation, the cells were restimulated with plate-bound anti-CD3 and anti-CD28 for 6 h, and single cells were captured with a Fluidigm C1 system. A total of 56 cells were collected and sequenced with an Illumina Hiseq system, and 10,000 pooled cells were sequenced using a standard protocol (Pooled cells). A, correlations of gene expression between the pooled cells and average of the 56 single cells (left panel), between two randomly selected single cells (center panel), and between the pooled cells and a single cell (right panel). 10,565 genes were detected. Gene expression was determined by TPM, and Pearson correlation coefficient (r) is depicted. B, heatmap depicting Pearson correlation coefficients between global gene expression profiles of each pair of the 56 individual cells. C, cytokine genes were abundantly expressed and variable among Th2 single cells. Average expression (μ, x axis) and standard deviation (σ, y axis) of each gene were calculated and plotted. The blue dashed lines represent the threshold of CV (highly variable, >0.28; less variable, <0.2). The black dashed line represents the threshold of abundantly expressed genes (μ > 8). Red represents cytokine genes, green denotes housekeeping genes, and gray represents other genes. D, gene ontology (GO) analysis of the most variably expressed genes (μ > 8 and CV > 0.28, left panel) and most consistently expressed genes (μ > 8 and CV < 0.2, right panel). The top five most significantly enriched GO terms (biological processes and molecular functions) are shown. The p values were Bonferroni-corrected.

Article Snippet: Five days after stimulation, the cells were restimulated with plate-bound anti-CD3 and anti-CD28 for 6 h, and single cells were captured with a Fluidigm C1 system.

Techniques: RNA Sequencing Assay, In Vitro, Isolation, Expressing, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Single-cell RNA-Seq analysis identifies a noncoding interleukin 4 ( IL-4 ) RNA that post-transcriptionally up-regulates IL-4 production in T helper cells

doi: 10.1074/jbc.RA118.004111

Figure Lengend Snippet: IL4nc is constitutively expressed in Th2 cells. A, IL4nc was specifically expressed in Th2 and Th0 cells. Naïve CD4+ T cells were activated with anti-CD3 and anti-CD28 and differentiated into various lineages under the respective conditions. Five days after differentiation, expression of IL4nc was analyzed by real-time PCR either under steady state or after being activated by anti-CD3 and anti-CD28 or PAM and ionomycin for 5 h. B, IL4nc is primarily located in the cytoplasm. D10.G4.1 cells were fractionated into the nuclear (N) fraction and cytoplasmic (C) fraction, and the presence of the IL4nc isoform was determined by real-time PCR (left panel). The protein lysates from both fractions were further analyzed by immunoblotting for the cytoplasmic marker (B-actin) and nucleus marker (histone H3) (right panel). C, expression of either IL4nc or IL4fl in resting in vitro differentiated Th2 cells was determined by real-time PCR. D, induction of IL4nc RNA precedes IL4fl following TCR stimulation. Th2 cells were differentiated as described and restimulated with anti-CD3/anti-CD28 for the indicated time. Expression of either IL4nc or IL4fl was determined by real-time PCR. Data are representative (B) or the sum (A–D) of at least three independent experiments. Error bars stand for the standard deviation of the mean. **, p < 0.01 in paired Student's t test. ns, not significantly changed.

Article Snippet: Five days after stimulation, the cells were restimulated with plate-bound anti-CD3 and anti-CD28 for 6 h, and single cells were captured with a Fluidigm C1 system.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Marker, In Vitro, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Single-cell RNA-Seq analysis identifies a noncoding interleukin 4 ( IL-4 ) RNA that post-transcriptionally up-regulates IL-4 production in T helper cells

doi: 10.1074/jbc.RA118.004111

Figure Lengend Snippet: IL4nc RNA promotes IL-4 production in Th2 cells. A, a diagram showing the gene structure of IL4fl (top panel) or IL4nc RNAs (bottom panel). The gray boxes depict noncoding region of transcripts, and the black boxes depict coding regions. B and C, overexpression of IL4nc promotes IL-4 production in Th2 cells. Th2 cells were differentiated in vitro as described in Fig. 1 and transduced with a retrovirus containing either IL4nc RNA or empty vector (Mock). 5 days after stimulation, the cells were restimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml) for 5 h. Expression of IL-4 was determined by intracellular staining. Data shown are representative (B) or summary (C) of IL-4+% or IL-13+% cells from three independent experiments. D, Th2 cells were differentiated and stimulated as in B, and secreted IL-4 in the supernatant was determined by ELISA. Data are summary of three independent experiments. E–G, exon 3 of IL4nc is required for promoting IL-4 production post-transcriptionally. As above, full-length IL4nc (FL) or its mutants depleted the indicated exons (Δexon1, Δexon2, and Δexon3, respectively) and were retrovirally expressed in Th2 cells. Production of IL-4 was analyzed by intracellular staining (E and F) or ELISA (G). H, as in E. Expression of IL-4 and GATA3 mRNAs 2 h after restimulation was analyzed by real-time quantitative PCR. I, as in B. Th2 cells were transduced with a retrovirus overexpressing IL4nc RNA, and the cells were selected with puromycin for 3 days (3 μg/ml). 5 days after differentiation, expression of IL4fl was analyzed by real-time PCR after activation by anti-CD3 (5 μg/ml) and anti-CD28 (1 μg/ml) (left panel) or PMA (50 ng/ml) and ionomycin (500 ng/ml) (right panel) for the indicated time. Data are representative (B and E) or the sum (C, D, and F–I) of at least three independent experiments. Error bars stand for the standard deviation of the mean. *, p < 0.05; **, p < 0.01; paired Student's t test. ns, not significantly changed.

Article Snippet: Five days after stimulation, the cells were restimulated with plate-bound anti-CD3 and anti-CD28 for 6 h, and single cells were captured with a Fluidigm C1 system.

Techniques: Over Expression, In Vitro, Transduction, Plasmid Preparation, Expressing, Staining, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Activation Assay, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Single-cell RNA-Seq analysis identifies a noncoding interleukin 4 ( IL-4 ) RNA that post-transcriptionally up-regulates IL-4 production in T helper cells

doi: 10.1074/jbc.RA118.004111

Figure Lengend Snippet: Knockdown of IL4nc RNA represses IL-4 production. Th2 cells were differentiated in vitro as described in Fig. 1 and transduced with a retrovirus containing a scramble sequence (sh-Ctl) or two independent shRNAs against IL4nc RNA. Five days after differentiation, the cells were restimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml) for 5 h. A–C, production of IL-4 was determined by intracellular staining (A and B) and ELISA (C). D, as in A. The cells were restimulated with anti-CD3 and anti-CD28, and expression of IL4fl (left panel) and IL4nc (right panel) RNA was analyzed by real-time PCR at the indicated time after restimulation. E, as in A. 5 days after differentiation, the cells were restimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml) for 3.5 h. The cells were then treated with cycloheximide (100 μg/ml), lysed, and fractionated on a 10–50% sucrose gradient. Distribution of IL4 mRNA at each fraction was analyzed by real-time PCR. Distribution of ribosomal RNA is shown in Fig. S6. F, as in E. In vitro differentiated Th2 cells were transduced with a retrovirus overexpressing IL4nc (IL4nc) or an empty vector (Mock). Distribution of IL4 mRNA at each fraction after sucrose density gradient centrifugation was determined by real-time PCR. Data are representative (A) or the sum (B–F) of at least three independent experiments. Error bars stand for the standard deviation of the mean. *, p < 0.05; **, p < 0.01; paired Student's t test.

Article Snippet: Five days after stimulation, the cells were restimulated with plate-bound anti-CD3 and anti-CD28 for 6 h, and single cells were captured with a Fluidigm C1 system.

Techniques: In Vitro, Transduction, Sequencing, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Gradient Centrifugation, Standard Deviation

Journal: STAR Protocols

Article Title: Protocol to characterize immune cell subpopulations in cerebrospinal fluid of patients with neuroinflammatory diseases using mass cytometry

doi: 10.1016/j.xpro.2024.103038

Figure Lengend Snippet:

Article Snippet: Anti-biotin-(1D4-C5)-170Er (1:100) , Standard BioTools , Cat#3170003B.

Techniques: Recombinant, Labeling, Blocking Assay, Software, Cytometry, Flow Cytometry, Single-cell Analysis, Gene Expression