Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Peripheral Circadian Clock Rhythmicity Is Retained in the Absence of Adrenergic Signaling

doi: 10.1161/atvbaha.107.152538

Figure Lengend Snippet: Figure 3. Rhythms in peripheral tissues are maintained in Dbh/ mice. A through I, Dbh/ (black) and Dbh/ (gray) mice entrained on a 12-hour light-dark regimen (Lights on at ZT0) were switched to constant darkness for 24 hours before tissue harvest (n6). Per1, Per2, dbp, E4bp4, and Adrb2 mRNA was monitored by qPCR.

Article Snippet: Protein lysates were electrophoresed, transferred to nitrocellulose and probed with anti Per1 (Chemicon), anti E4bp4 (Novus) and β Actin (Sigma).

Techniques:

Journal: bioRxiv

Article Title: Microbiota-derived inosine programs protective CD8 + T cell responses against influenza in newborns

doi: 10.1101/2024.04.09.588427

Figure Lengend Snippet: A) Lung from control and dysbiotic infant mice (n=4, 2 in each group) was obtained at 7 days post-infection (d.p.i). Lung samples were dissociated into cell suspensions, magnetically enriched (EPCAM - CD31 - CD45 + CD8 + ) and used for single-cell RNA sequencing (scRNAseq). B ) Uniform manifold approximation and projection (UMAP) embedding of all samples (n∼9000 cells) colored by cell clusters was performed on scRNAseq data. C0: terminally differentiated effector CD8 + T cells, C1: naive-like CD8 + T cells C2: CD8 + T exhausted progenitor characterized. C3: effector-memory CD8 + T cells. C4: regulatory T cells. C ) Scaled expression of genes unique to each cluster. Bubble size represents the percentage of cells expressing the gene, and the color intensity represents the log (mean expression +0.1) [Benjamini and Hochberg-adjusted p -values < 0.01, log 2 fold change > 2, Wald’s test]. D ) UMAP embedding of all samples colored by pseudo time with overlaid trajectory and E ) scatter plots showing expression of Sell , Ctla4 , Tcf7 and Il7r across pseudo time. F ) Proportion of clusters in dysbiotic and control samples showing predominance of naïve-like CD8 + T cells (C1) in dysbiotic mice and terminally differentiated effector CD8 + T cells (C0) in controls. Lines indicate 95% confidence intervals. G) Unsupervised analysis of live, single live CD8 T + cells using a self-organizing map (SOM). CD8 + T cell clusters identified by SOM were mapped to H ) uniform manifold approximation and projection (UMAP) embedding and colored by naïve, effector, memory, and exhausted progenitor clusters. I ) The proportion of memory or naïve or exhausted progenitor or effector CD8 + T cells in control and dysbiotic infant lungs at ten d.p.i. [n=6, * indicated p -values < 0.05, NS – not significant. One-way ANOVA with Tukey’s correction for multiple comparisons. Solid lines, mean; dotted lines, quartiles). J) Gene Ontology (GO) terms associated with DEG in in control and dysbiotic mice. [Benjamini-Hochberg-corrected p -values < 0.05 (one-sided Fisher’s exact test) are shown and colored by gene ratio]. K) MFI of indicated markers in live CD8 + T cells from control or dysbiotic infant lungs [n=6 per group, * indicated p -values < 0.05, one-way ANOVA with Tukey’s correction for multiple comparisons. Solid lines, mean; dotted lines, quartiles]. L ) Row-scaled regulon activity for all samples. k-means clustering was used to arrange clusters and regulons [Benjamin and Hochberg-adjusted p-values < 0.01]. Regulons related to TRM identity, cell survival, and inflammation are highlighted. M ) Network diagram showing the core regulator relationship, with the circle size indicating # of regulated genes. N ) Average Nfil3 expression (left) or NFIL3 MFI (right) in CD8 + T cells from control or dysbiotic infant lung, at seven d.p.i. [Benjamini and Hochberg-adjusted p -values < 0.01].

Article Snippet: Sheared chromatin was immunoprecipitated with an anti-NFIL3 antibody (Clone PM097, MBL Bio).

Techniques: Infection, RNA Sequencing Assay, Expressing, Activity Assay

Journal: bioRxiv

Article Title: Microbiota-derived inosine programs protective CD8 + T cell responses against influenza in newborns

doi: 10.1101/2024.04.09.588427

Figure Lengend Snippet: A) Experimental approach. Transgenic dLck cre and Nfil3 flox on C57Bl/6J background mice were crossbred. dLck ΔNfil3 and control littermate infant mice were challenged with a sublethal dose of murine-adapted influenza A H1N1 strain-PR8, expressing OVA 257–264 epitope (PR8-OVA) [10 2 TCID50] via the intranasal route on PN14. B) Unsupervised analysis of live, single live CD8 T + cells using a self-organizing map (SOM). CD8 + T cell clusters identified by SOM were mapped to uniform manifold approximation and projection (UMAP) embedding and colored by naïve, effector, memory, and exhausted progenitor clusters (on left) and by genotype (right). CD8 + T cells from dLck ΔNfil3 infant lungs are colored blue, whereas CD8 + T cells from dLck cre infant lungs are colored red. C) The proportion of memory or naïve or exhausted progenitor or effector CD8 + T cells in lungs of dLck ΔNfil3 and dLck cre CD8 + T cells [n=8, * indicated p -values < 0.05, one-way ANOVA with Tukey’s correction for multiple comparisons. Solid lines, mean; dotted lines, quartiles]. D) Mean fluorescent intensity (MFI) of key phenotypic markers in live lung CD8 + T cells from dLck ΔNfil3 or dLck cre infant mice [* indicated p -values < 0.05, one-way ANOVA with Tukey’s correction for multiple comparisons. Solid lines, mean; dotted lines, quartiles]. E) Experimental approach. Infant B6 mice were treated with anti-CD8α antibody (Ab) on PN12 to deplete endogenous CD8 + T cells and received adoptive transfer of CD8 + T cells from age-matched dLck cre or dLck ΔNfil3 infants on PN14. F ) Representative histogram of CFSE expression in CD8 + T cells and G ) computationally modeled CFSE peaks corresponding to cell divisions in CD8 + T cells. H ) Proliferation index of CD8 + T cells from lungs of dLck ΔNfil3 or dLck cre infant mice in recipient mouse, five days after transfer [Solid lines, mean; dotted lines, quartiles, n=6, *indicated p -values < 0.05, Student’s t-test]. I ) Representative biaxial plots of KLRG1 and CFSE expression in CD8 + T cells in recipient mice at indicated cell divisions or J ) Representative histogram of KLRG1 expression in CD8 + T cells in recipient mice at indicated cell divisions. K) Proportion of KLRG1 hi CD8 + T cells from lungs of dLck Δ Nfil3 or dLck cre infant mice recipient mouse at indicated cell divisions. [Solid lines, mean; dotted lines, quartiles, n=5, *indicated p -values < 0.05, one-way ANOVA with Tukey’s correction for multiple comparisons]. L ) Proportion of OVA 257–264 specific CD8 + T cells or M) IFNγ + OVA 257–264 specific CD8 + T cells or N ) OVA 257–264 specific TRM cells in lungs of dLck ΔNfil3 and dLck cre infant mice [n=5, * indicated p -values < 0.05, Student’s t-test. Solid lines, mean; dotted lines, quartiles]. O ) Chromatin Immuno-Precipitation with sequencing (ChiPseq) of CD8 + T cells isolated from lungs of control or dysbiotic infant mice at PN14 days with anti-NFIL3 antibody. Heat map of the NFIL3 ChIP-seq signals around transcription start site (TSS) compared to the random genomic control in CD8 + T cells from indicated infant mice. P ) Denovo motifs identified by HOMER. Q ) Gene Ontology (GO) terms associated with NFIL3 peaks. [Benjamini-Hochberg-corrected p -values < 0.05 (one-sided Fisher’s exact test) are shown and colored by gene ratio]. R ) NFIL3 ChIP-seq or ENCODE-identified cis-regulatory elements (CRE) in Tcf7 loci or S ) Lef1 loci in CD8 + T cells from lungs of control compared to dysbiotic mice. T ) Fold enrichment of H3K4me3 binding at ENCODE-identified CRE in Tcf7 or U) Lef1 promoter in indicated experimental groups. [n=3, * indicated p -values < 0.05, one-way ANOVA with Tukey’s correction for multiple comparisons. Solid lines, mean; dotted lines, quartiles].

Article Snippet: Sheared chromatin was immunoprecipitated with an anti-NFIL3 antibody (Clone PM097, MBL Bio).

Techniques: Transgenic Assay, Expressing, Adoptive Transfer Assay, Chromatin Immunoprecipitation, Sequencing, Isolation, ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: Microbiota-derived inosine programs protective CD8 + T cell responses against influenza in newborns

doi: 10.1101/2024.04.09.588427

Figure Lengend Snippet: A) Frequencies of HSC (identified as Lin - c-Kit + cells) and B ) CLP (identified as Lin-Flt3 + c-Kit mid Sca-1 mid − CD48 + cells) in the bone marrow of dLck ΔNfil3 or dLck cre infant mice (PN14). [n =6, NS – not significant, paired student’s t-test, violin plots, Solid lines, mean; dotted lines, quartiles]. C ) Pairwise Euclidean distances between CD8 + T cells in the lungs of dysbiotic, dLck ΔNfil3 , and dLck cre infants. D ) Photomicrographs of hematoxylin and eosin (H&E) stained sections of lungs from either dLck ΔNfil3 or dLck cre infant mice 10 d.p.i. Areas highlighted by broken white lines represent lung damage. E ) Lung injury scores as per American Thoracic Society Guidelines from dLck ΔNfil3 or control or dysbiotic infant mice at indicated times post-infection. Data for control infant mice was re-used from . [Solid lines, mean; dotted lines, quartiles, * indicated p -values < 0.05, one-way ANOVA with Tukey’s correction for multiple comparisons. Solid lines, mean; dotted lines, quartiles]. F ) Fold change in Tcf7 or G ) Lef1 mRNA in lung CD8 + T cells from dLck ΔNfil3 and dLck cre infant mice (PN14) [n=3, * indicated p -values < 0.05, Student’s t-test. Solid lines, mean; dotted lines, quartiles].

Article Snippet: Sheared chromatin was immunoprecipitated with an anti-NFIL3 antibody (Clone PM097, MBL Bio).

Techniques: Staining, Infection

Journal: bioRxiv

Article Title: Microbiota-derived inosine programs protective CD8 + T cell responses against influenza in newborns

doi: 10.1101/2024.04.09.588427

Figure Lengend Snippet: A) Experimental approach. Fecal samples from control and dysbiotic humans and intestinal contents from control or dysbiotic infant mice samples were subjected to shotgun metagenomics and targeted metabolomics to quantify differences in microbiota taxonomy and function. B) Linear discriminant analysis effect size showing the differentially abundant clades between dysbiotic and control infant mice with an abundance of Bifidobacteria and Lactobacilli genera in controls and Proteobacteria in dysbiotic infant mice. C) Linear discriminant analysis effect size showing the differentially abundant clades between dysbiotic and control infant humans with an abundance of Bifidobacterium in controls and Proteobacteria in dysbiotic infant humans. D) Volcano plot depicting the abundance of various metabolites in plasma of dysbiotic and control infant mice. E) Enriched (CD3ε + CD8 + ) single-cell suspension from control and dysbiotic infant mice were co-incubated with inosine with or without an A2AR antagonist (ZM241385). F) Representative histogram of NFIL3 expression on CD8 + T cells in indicated experimental groups. Proportion of NFIL3 + CD8 + T cells is indicated. G ) Mean fluorescent intensity (MFI) of TCF1 or G) LEF1 in CD8 + T cells in indicated experimental groups. [* indicated p -values < 0.05, n=5, Student’s t-test. Solid lines, mean; dotted lines, quartiles]. I ) Representative histogram of CFSE expression and J ) proportion of proliferating CD8 + T cells or K ) IFNγ + CD8 + T cells in indicated experimental groups [n=5, * indicated p -values < 0.05, one-way ANOVA with Tukey’s correction for multiple comparisons. Solid lines, mean; dotted lines, quartiles]. L ) Representative histogram of phosphorylated CREB (pCREB) expression. Proportion of pCREB + CD8 + T cells is indicated. L) Experimental approach. Pregnant C57/B6 dams were treated with a cocktail of antimicrobials from embryonic day (E) 15 to postnatal day (PN) 5 (dysbiosis) or with saline (control). Infant mice in each experimental group were treated with inosine (300 μg g -1 ) on PN10,12 and 14 via intraperitoneal route and subsequently challenged with a sublethal dose of murine-adapted influenza A H1N1 strain-PR8, expressing OVA 257–264 epitope (PR8-OVA) [10 2 TCID50] via the intranasal (i.n.) route on PN14. N ) Fold enrichment of H3K4me3 binding at ENCODE-identified CRE in Tcf7 promoter in lung CD8 + T cells in indicated experimental groups. [n=3, * indicated p -values < 0.05, one-way ANOVA with Tukey’s correction for multiple comparisons. Solid lines, mean; dotted lines, quartiles]. O) Fold change in Tcf7 transcripts in lung CD8 + T cells in indicated experimental groups. [n=3, * indicated p -values < 0.05, one-way ANOVA with Tukey’s correction for multiple comparisons. Solid lines, mean; dotted lines, quartiles]. P ) Proportion of OVA 257– 264 specific CD8 + T cells in lungs [n=5, * indicated p -values < 0.05, one-way ANOVA with Tukey’s correction for multiple comparisons. Solid lines, mean; dotted lines, quartiles] or Q ) weight change in indicated experimental groups (n=6, * p -values < 0.05, one-way ANOVA with Tukey’s correction for multiple comparisons. Solid lines, mean; dotted lines, quartiles).

Article Snippet: Sheared chromatin was immunoprecipitated with an anti-NFIL3 antibody (Clone PM097, MBL Bio).

Techniques: Suspension, Incubation, Expressing, Saline, Binding Assay

Journal: bioRxiv

Article Title: Microbiota-derived inosine programs protective CD8 + T cell responses against influenza in newborns

doi: 10.1101/2024.04.09.588427

Figure Lengend Snippet: A) Enriched (CD3ε + CD8 + ) single-cell suspension from control and dysbiotic infant mice were co-incubated with inosine with or without an A2AR antagonist (ZM241385). Proportion of NFIL3 + CD8 + T cells in indicated experimental groups [n=5, * indicated p -values < 0.05, one-way ANOVA with Tukey’s correction for multiple comparisons. Solid lines, mean; dotted lines, quartiles]. B) Representative biaxial plots of IFNγ in CD8 + T cells in each experimental group. The proportion of IFNγ + CD8 + T cells is indicated.

Article Snippet: Sheared chromatin was immunoprecipitated with an anti-NFIL3 antibody (Clone PM097, MBL Bio).

Techniques: Suspension, Incubation

Journal: bioRxiv

Article Title: Microbiota-derived inosine programs protective CD8 + T cell responses against influenza in newborns

doi: 10.1101/2024.04.09.588427

Figure Lengend Snippet: Influenza virus susceptibility in dysbiotic infant mice is caused by CD8 + T cell hyporesponsiveness and diminished persistence as tissue-resident memory cells. Nuclear factor interleukin 3 (NFIL3) repressed memory differentiation of CD8 + T cells in dysbiotic mice by epigenetic regulation of T cell factor 1 (TCF 1) expression. Pulmonary CD8 + T cells from dysbiotic human infants share these transcriptional signatures and functional phenotypes. Mechanistically, intestinal inosine was reduced in dysbiotic human infants and newborn mice, and inosine replacement reversed epigenetic dysregulation of Tcf7 and increased memory differentiation and responsiveness of pulmonary CD8 + T cells.

Article Snippet: Sheared chromatin was immunoprecipitated with an anti-NFIL3 antibody (Clone PM097, MBL Bio).

Techniques: Virus, Expressing, Functional Assay

Journal: The Journal of Clinical Investigation

Article Title: E4BP4-mediated inhibition of T follicular helper cell differentiation is compromised in autoimmune diseases

doi: 10.1172/JCI129018

Figure Lengend Snippet: (A) Analysis of naive CD4+ T cells from 8-week-old C57BL/6 mice stimulated in vitro under Th0, Th1, Th2, Th17, Treg, and Tfh cell–polarizing conditions for 3 days (Th0, Th1, Th17, Treg, and Tfh cells) or 5 days (Th2 cells) by flow cytometry for mean fluorescence intensity (MFI) of E4BP4 (n = 9). (B) mRNA expression of E4bp4 in T cell subsets indicated in A (n = 6). (C) Naive CD4+ T cells, anti-CD3/CD28–activated CD4+ T cells, and in vitro–polarized Tfh cells were stained by anti-E4BP4 (red) and DAPI (blue) and analyzed by confocal microscopy. Scale bar: 5 μm. (D) Statistical intensity of E4BP4 in nuclei (n = 6). (E) Flow cytometric analysis of E4BP4 expression in mice CD4+ T cells (n = 10). (F) Gating strategy of CXCR5+PD-1+(Tfh) or CXCR5–PD-1– (non-Tfh) cell phenotype in CD4+ T cells from KLH immunized C57BL/6 mice. Analysis of E4BP4 and BCL6 expression (MFI) is shown in the right panel. (G) Statistical analysis of F (n = 10). (H) Gating strategy of human tonsillar CD45RO+ memory/effector or CD45RO– naive CD4+ T cells. CD45RO+ cells were subsequently divided into CXCR5lo, CXCR5int, and CXCR5hi gates. (I and J) Representative histograms of CXCR5, BCL6, PD-1, and E4BP4 MFI expressions in subsets outlined in H (n = 10). Data are representative of 3 independent experiments. For A, B, D, and J, 1-way ANOVA with Dunnett’s post hoc test, E and G with Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The following antibodies were used: FITC anti–human CD4 (BD Biosciences, catalog 550628), APC mouse anti–human CD69 (BD Biosciences, catalog 555533), PE mouse anti–human CD154 (BD Biosciences, catalog 555700), PE anti–human IL-17A (BD Biosciences, catalog 560487), Alexa Fluor 647 anti–human IFN-γ (BioLegend, catalog 502516), PE anti–human IL-4 (BioLegend, catalog 500704), APC-Cy7 anti–human Bcl-6 (BD Biosciences, catalog 563581), PE-CY7 anti–human CD185 (BioLegend, catalog 356923), Alexa Fluor 647 anti–human FOXP3 (BD Biosciences, catalog 561184), PE anti–human CD278 (Thermo Fisher Scientific, catalog 12-9185), APC anti–human CD279 (Thermo Fisher Scientific, catalog 85-17-2799-42), human E4BP4 (NFIL3) monoclonal antibody (Thermo Fisher Scientific, catalog 12-9812-42), FITC anti–mouse CD4 (BioLegend, catalog 100406), PerCP-Cy5.5 rat anti–mouse Foxp3 (BD Biosciences, catalog 563902), APC-Cy7 mouse anti–Bcl-6 (BD Biosciences, catalog 563581), Biotin rat anti–mouse CXCR5, APC-Cy7 rat anti–mouse CD62L (BD Biosciences, catalog 560514), PE-Cy7 rat anti–mouse CD44 (BD Biosciences, catalog 560569), APC hamster anti–mouse CD279 (PD-1) (BD Biosciences, catalog 562671), PE rat anti–mouse CD278 (ICOS) (BD Biosciences, catalog 552146), PE hamster anti–mouse CD95 (BD Biosciences, catalog 554258), Alexa Fluor 647 anti–mouse T and B cell activation antigen (BD Biosciences, catalog 561529), FITC anti–mouse CD45R/B220 (BD Biosciences, catalog 553087), and mouse E4BP4 (NFIL3) monoclonal antibody (BD Biosciences, catalog 551960; Thermo Fisher Scientific, catalog 12-5927-82).

Techniques: In Vitro, Flow Cytometry, Fluorescence, Expressing, Staining, Confocal Microscopy

Journal: The Journal of Clinical Investigation

Article Title: E4BP4-mediated inhibition of T follicular helper cell differentiation is compromised in autoimmune diseases

doi: 10.1172/JCI129018

Figure Lengend Snippet: (A) Flow cytometric analysis of the CD4+ and CD8+ T cell populations in CD3+ T cells of nonimmunized WT and E4bp4-cKO mice. Statistical analysis is shown on the right. (B and C) Flow cytometric analysis of CD44+ and CD62L+ cells in CD4+ and CD8+ T cell populations (n = 10). (D and E) Flow cytometric analysis of Treg, Tfh, and Tfr cells. (F) Statistical analysis of D and E (n = 6). Data are representative of 4 independent experiments. LN, lymph node; EM, effector memory. Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The following antibodies were used: FITC anti–human CD4 (BD Biosciences, catalog 550628), APC mouse anti–human CD69 (BD Biosciences, catalog 555533), PE mouse anti–human CD154 (BD Biosciences, catalog 555700), PE anti–human IL-17A (BD Biosciences, catalog 560487), Alexa Fluor 647 anti–human IFN-γ (BioLegend, catalog 502516), PE anti–human IL-4 (BioLegend, catalog 500704), APC-Cy7 anti–human Bcl-6 (BD Biosciences, catalog 563581), PE-CY7 anti–human CD185 (BioLegend, catalog 356923), Alexa Fluor 647 anti–human FOXP3 (BD Biosciences, catalog 561184), PE anti–human CD278 (Thermo Fisher Scientific, catalog 12-9185), APC anti–human CD279 (Thermo Fisher Scientific, catalog 85-17-2799-42), human E4BP4 (NFIL3) monoclonal antibody (Thermo Fisher Scientific, catalog 12-9812-42), FITC anti–mouse CD4 (BioLegend, catalog 100406), PerCP-Cy5.5 rat anti–mouse Foxp3 (BD Biosciences, catalog 563902), APC-Cy7 mouse anti–Bcl-6 (BD Biosciences, catalog 563581), Biotin rat anti–mouse CXCR5, APC-Cy7 rat anti–mouse CD62L (BD Biosciences, catalog 560514), PE-Cy7 rat anti–mouse CD44 (BD Biosciences, catalog 560569), APC hamster anti–mouse CD279 (PD-1) (BD Biosciences, catalog 562671), PE rat anti–mouse CD278 (ICOS) (BD Biosciences, catalog 552146), PE hamster anti–mouse CD95 (BD Biosciences, catalog 554258), Alexa Fluor 647 anti–mouse T and B cell activation antigen (BD Biosciences, catalog 561529), FITC anti–mouse CD45R/B220 (BD Biosciences, catalog 553087), and mouse E4BP4 (NFIL3) monoclonal antibody (BD Biosciences, catalog 551960; Thermo Fisher Scientific, catalog 12-5927-82).

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: E4BP4-mediated inhibition of T follicular helper cell differentiation is compromised in autoimmune diseases

doi: 10.1172/JCI129018

Figure Lengend Snippet: (A) Flow cytometric analysis of CXCR5+PD-1+ in naive CD4+ T cells from the E4bp4-cKO and WT mice. Statistical analysis is indicated in B. (C–F) Flow cytometric analysis of in vitro–polarized Tfh-like cells from the E4bp4-cKO and WT mice and from the E4bp4-cKI and WT mice. Representative histograms of BCL6 and IL-21 expression in CD4+CXCR5+PD-1+ Tfh-like cells are shown in D and F (n = 6). (G) Gene set enrichment analysis of gene signatures (either upregulation or downregulation) in Tfh cells relative to their expression in non–Tfh cells from published data (GEO accession code GSE16697), and differentially expressed genes between the in vitro–polarized E4bp4-cKO Tfh-like cells and WT Tfh-like cells. Red/blue rectangles indicate enriched genes in the E4bp4-cKO Tfh-like cells. (H) RNA-seq analysis of gene expression of in vitro–polarized Tfh-like cells; colors indicate upregulated (red) or downregulated (blue) genes. (I) Clustered heatmap of 39 Tfh signature genes regulated by E4BP4. (J) Clustered heatmap of 10 Tfr signature genes regulated by E4BP4. The data were normalized from 2 replicates (n = 2). Genes with the most transcriptional changes are listed. (A–F) Data are representative of 3 independent experiments. Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The following antibodies were used: FITC anti–human CD4 (BD Biosciences, catalog 550628), APC mouse anti–human CD69 (BD Biosciences, catalog 555533), PE mouse anti–human CD154 (BD Biosciences, catalog 555700), PE anti–human IL-17A (BD Biosciences, catalog 560487), Alexa Fluor 647 anti–human IFN-γ (BioLegend, catalog 502516), PE anti–human IL-4 (BioLegend, catalog 500704), APC-Cy7 anti–human Bcl-6 (BD Biosciences, catalog 563581), PE-CY7 anti–human CD185 (BioLegend, catalog 356923), Alexa Fluor 647 anti–human FOXP3 (BD Biosciences, catalog 561184), PE anti–human CD278 (Thermo Fisher Scientific, catalog 12-9185), APC anti–human CD279 (Thermo Fisher Scientific, catalog 85-17-2799-42), human E4BP4 (NFIL3) monoclonal antibody (Thermo Fisher Scientific, catalog 12-9812-42), FITC anti–mouse CD4 (BioLegend, catalog 100406), PerCP-Cy5.5 rat anti–mouse Foxp3 (BD Biosciences, catalog 563902), APC-Cy7 mouse anti–Bcl-6 (BD Biosciences, catalog 563581), Biotin rat anti–mouse CXCR5, APC-Cy7 rat anti–mouse CD62L (BD Biosciences, catalog 560514), PE-Cy7 rat anti–mouse CD44 (BD Biosciences, catalog 560569), APC hamster anti–mouse CD279 (PD-1) (BD Biosciences, catalog 562671), PE rat anti–mouse CD278 (ICOS) (BD Biosciences, catalog 552146), PE hamster anti–mouse CD95 (BD Biosciences, catalog 554258), Alexa Fluor 647 anti–mouse T and B cell activation antigen (BD Biosciences, catalog 561529), FITC anti–mouse CD45R/B220 (BD Biosciences, catalog 553087), and mouse E4BP4 (NFIL3) monoclonal antibody (BD Biosciences, catalog 551960; Thermo Fisher Scientific, catalog 12-5927-82).

Techniques: In Vitro, Expressing, RNA Sequencing Assay

Journal: The Journal of Clinical Investigation

Article Title: E4BP4-mediated inhibition of T follicular helper cell differentiation is compromised in autoimmune diseases

doi: 10.1172/JCI129018

Figure Lengend Snippet: Age-matched WT or E4bp4-cKO mice were immunized with KLH for 14 days, then lymph node and spleen cells were harvested. (A and B) Flow cytometric analysis of FoxP3+ Treg cells, CD4+CXCR5+PD-1+ Tfh cells, and FoxP3+ Tfr cells in follicular T cells. (C) Analysis of B220+Fas+GL-7+ GC B cells. (D) Summary of the percentage of FoxP3+ Treg cells, Tfh cells, and FoxP3+ Tfr cells in follicular T cells, as described in A and B, and GC B cells, as described in C. (E) Immunofluorescence of GCs from WT and E4bp4-cKO mice, representative images of CD3 and PNA staining of LNs. Scale bars: 200 μm. Quantification of PNA+ GC areas and number of PNA+ follicles per lymph node. (F) Detection of serum anti-KLH–specific IgG1, IgG2a, IgG2b, IgG3, and IgM by ELISA. (G) Draining lymph node cells were restimulated with KLH for 3 days, and supernatant IL-21 expression was detected by ELISA. (H) Il21 and Bcl6 mRNA expression levels were analyzed by qPCR (n = 6). Data are representative of 4 independent experiments. Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The following antibodies were used: FITC anti–human CD4 (BD Biosciences, catalog 550628), APC mouse anti–human CD69 (BD Biosciences, catalog 555533), PE mouse anti–human CD154 (BD Biosciences, catalog 555700), PE anti–human IL-17A (BD Biosciences, catalog 560487), Alexa Fluor 647 anti–human IFN-γ (BioLegend, catalog 502516), PE anti–human IL-4 (BioLegend, catalog 500704), APC-Cy7 anti–human Bcl-6 (BD Biosciences, catalog 563581), PE-CY7 anti–human CD185 (BioLegend, catalog 356923), Alexa Fluor 647 anti–human FOXP3 (BD Biosciences, catalog 561184), PE anti–human CD278 (Thermo Fisher Scientific, catalog 12-9185), APC anti–human CD279 (Thermo Fisher Scientific, catalog 85-17-2799-42), human E4BP4 (NFIL3) monoclonal antibody (Thermo Fisher Scientific, catalog 12-9812-42), FITC anti–mouse CD4 (BioLegend, catalog 100406), PerCP-Cy5.5 rat anti–mouse Foxp3 (BD Biosciences, catalog 563902), APC-Cy7 mouse anti–Bcl-6 (BD Biosciences, catalog 563581), Biotin rat anti–mouse CXCR5, APC-Cy7 rat anti–mouse CD62L (BD Biosciences, catalog 560514), PE-Cy7 rat anti–mouse CD44 (BD Biosciences, catalog 560569), APC hamster anti–mouse CD279 (PD-1) (BD Biosciences, catalog 562671), PE rat anti–mouse CD278 (ICOS) (BD Biosciences, catalog 552146), PE hamster anti–mouse CD95 (BD Biosciences, catalog 554258), Alexa Fluor 647 anti–mouse T and B cell activation antigen (BD Biosciences, catalog 561529), FITC anti–mouse CD45R/B220 (BD Biosciences, catalog 553087), and mouse E4BP4 (NFIL3) monoclonal antibody (BD Biosciences, catalog 551960; Thermo Fisher Scientific, catalog 12-5927-82).

Techniques: Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Expressing

Journal: The Journal of Clinical Investigation

Article Title: E4BP4-mediated inhibition of T follicular helper cell differentiation is compromised in autoimmune diseases

doi: 10.1172/JCI129018

Figure Lengend Snippet: Age-matched WT and E4bp4-cKO mice were administered pristane. After 32 weeks, the mice were sacrificed for the development of autoimmune traits. (A) The spleens and lymph nodes were weighed. (B) The number of CD4+ T cells from splenocytes and lymphocytes was estimated. (C) The level of proteinuria was measured by ELISA. (D and E) Serum anti-snRNP and anti-dsDNA antibodies were measured by ELISA. Flow cytometric analysis of (F) Tfh cells (CD4+ CXCR5+PD-1+) and (G) GC B cells (B220+GL-7+FAS+). (H) Statistical analysis of F and G. (I) Representative images of H&E- or PAS-stained kidney sections were collected at the end of the observation period. Scale bars: 50 μm. (J) C3 and IgG deposition in the kidney sections were assessed by immunofluorescence staining. Scale bars: 25 μm (n = 8). Data are representative of 2 to 3 independent experiments. Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The following antibodies were used: FITC anti–human CD4 (BD Biosciences, catalog 550628), APC mouse anti–human CD69 (BD Biosciences, catalog 555533), PE mouse anti–human CD154 (BD Biosciences, catalog 555700), PE anti–human IL-17A (BD Biosciences, catalog 560487), Alexa Fluor 647 anti–human IFN-γ (BioLegend, catalog 502516), PE anti–human IL-4 (BioLegend, catalog 500704), APC-Cy7 anti–human Bcl-6 (BD Biosciences, catalog 563581), PE-CY7 anti–human CD185 (BioLegend, catalog 356923), Alexa Fluor 647 anti–human FOXP3 (BD Biosciences, catalog 561184), PE anti–human CD278 (Thermo Fisher Scientific, catalog 12-9185), APC anti–human CD279 (Thermo Fisher Scientific, catalog 85-17-2799-42), human E4BP4 (NFIL3) monoclonal antibody (Thermo Fisher Scientific, catalog 12-9812-42), FITC anti–mouse CD4 (BioLegend, catalog 100406), PerCP-Cy5.5 rat anti–mouse Foxp3 (BD Biosciences, catalog 563902), APC-Cy7 mouse anti–Bcl-6 (BD Biosciences, catalog 563581), Biotin rat anti–mouse CXCR5, APC-Cy7 rat anti–mouse CD62L (BD Biosciences, catalog 560514), PE-Cy7 rat anti–mouse CD44 (BD Biosciences, catalog 560569), APC hamster anti–mouse CD279 (PD-1) (BD Biosciences, catalog 562671), PE rat anti–mouse CD278 (ICOS) (BD Biosciences, catalog 552146), PE hamster anti–mouse CD95 (BD Biosciences, catalog 554258), Alexa Fluor 647 anti–mouse T and B cell activation antigen (BD Biosciences, catalog 561529), FITC anti–mouse CD45R/B220 (BD Biosciences, catalog 553087), and mouse E4BP4 (NFIL3) monoclonal antibody (BD Biosciences, catalog 551960; Thermo Fisher Scientific, catalog 12-5927-82).

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Immunofluorescence

Journal: The Journal of Clinical Investigation

Article Title: E4BP4-mediated inhibition of T follicular helper cell differentiation is compromised in autoimmune diseases

doi: 10.1172/JCI129018

Figure Lengend Snippet: (A) Venn diagram showing the overlap between the genes with E4BP4 enrichment in promoter (green) and differentially expressed genes revealed by expression profile analysis (yellow). (B) ChIP-Seq analysis of E4BP4-binding peaks located at gene loci Bcl6 and Il13 in the E4bp4-cKI CD4+ T cells. The Bcl6-binding site motif is shown (right). (C–F) E4bp4-cKI/E4bp4-cKO naive CD4+ T cells were cultured under Tfh-polarizing conditions for 5 days. (C) ChIP-PCR was performed with IgG and anti-FLAG antibody to analyze the binding activity of E4BP4 to Bcl6. (D) The H3 acetylation and H3K27 trimethylation levels in the Bcl6 promoter region in WT versus E4bp4-cKI mice are shown. (E) The H3 acetylation and H3K27 trimethylation levels in the Bcl6 promoter region in WT versus E4bp4-cKO mice are shown. (F) The enrichments of HDAC1 and EZH2 in the Bcl6 promoter region in WT versus E4bp4-cKO mice are shown (n = 3). (G–I) Anti-FLAG antibody, anti-HDAC1, and anti-EZH2 antibodies were used for coimmunoprecipitation experiments to analyze the interaction between E4BP4 and HDAC1 or EZH2 in E4bp4-cKI Tfh-like cells. Each result corresponds to data pooled from 3 independent experiments. Student’s t test. ***P < 0.001; **P < 0.01; *P < 0.05.

Article Snippet: The following antibodies were used: FITC anti–human CD4 (BD Biosciences, catalog 550628), APC mouse anti–human CD69 (BD Biosciences, catalog 555533), PE mouse anti–human CD154 (BD Biosciences, catalog 555700), PE anti–human IL-17A (BD Biosciences, catalog 560487), Alexa Fluor 647 anti–human IFN-γ (BioLegend, catalog 502516), PE anti–human IL-4 (BioLegend, catalog 500704), APC-Cy7 anti–human Bcl-6 (BD Biosciences, catalog 563581), PE-CY7 anti–human CD185 (BioLegend, catalog 356923), Alexa Fluor 647 anti–human FOXP3 (BD Biosciences, catalog 561184), PE anti–human CD278 (Thermo Fisher Scientific, catalog 12-9185), APC anti–human CD279 (Thermo Fisher Scientific, catalog 85-17-2799-42), human E4BP4 (NFIL3) monoclonal antibody (Thermo Fisher Scientific, catalog 12-9812-42), FITC anti–mouse CD4 (BioLegend, catalog 100406), PerCP-Cy5.5 rat anti–mouse Foxp3 (BD Biosciences, catalog 563902), APC-Cy7 mouse anti–Bcl-6 (BD Biosciences, catalog 563581), Biotin rat anti–mouse CXCR5, APC-Cy7 rat anti–mouse CD62L (BD Biosciences, catalog 560514), PE-Cy7 rat anti–mouse CD44 (BD Biosciences, catalog 560569), APC hamster anti–mouse CD279 (PD-1) (BD Biosciences, catalog 562671), PE rat anti–mouse CD278 (ICOS) (BD Biosciences, catalog 552146), PE hamster anti–mouse CD95 (BD Biosciences, catalog 554258), Alexa Fluor 647 anti–mouse T and B cell activation antigen (BD Biosciences, catalog 561529), FITC anti–mouse CD45R/B220 (BD Biosciences, catalog 553087), and mouse E4BP4 (NFIL3) monoclonal antibody (BD Biosciences, catalog 551960; Thermo Fisher Scientific, catalog 12-5927-82).

Techniques: Expressing, ChIP-sequencing, Binding Assay, Cell Culture, Activity Assay

Journal: The Journal of Clinical Investigation

Article Title: E4BP4-mediated inhibition of T follicular helper cell differentiation is compromised in autoimmune diseases

doi: 10.1172/JCI129018

Figure Lengend Snippet: (A–D) CD4+ T cells isolated from healthy donors were transfected with E4BP4 overexpression (E4BP4-over) plasmid or empty vector (EV), as well as with E4BP4 siRNA and negative control (NC). The cells were then cultured under Tfh polarization conditions for 5 days. (A) Flow cytometric analysis of CD4+CXCR5+PD-1+ Tfh cells in EV and E4BP4-over group. Representative histograms of BCL6 and IL-21 expressions in CD4+CXCR5+PD-1+ Tfh cells are shown on the right. (B) Statistical analysis of A. (C) Analysis of CD4+CXCR5+PD-1+ Tfh cells in NC and siRNA groups. Representative histograms of BCL6 and IL-21 expressions in CD4+CXCR5+PD-1+ Tfh cells are shown. (D) Statistical analysis of C (n = 6). (E) The luciferase reporter assay in HEK293T cells by electroporation with the reporter plasmids pEGFP-C1 empty vector plus pGL3-basic BCL6 WT or pEGFP-C1 E4BP4 plus pGL3-basic BCL6 WT or pEGFP-C1 E4BP4 plasmid plus pGL3-basic BCL6 mutant or pEGFP-C1 empty vector plasmid plus pGL3-basic BCL6 mutant (n = 3). (F) Phosphomimetic mutation of E4BP4 putative serine sites at ser301 or ser353 mutants or double mutants (S to A). (G) Mutant and WT versions of E4BP4 were expressed in human CD4+ T cells, separately. Cells were then cultured under Tfh-polarizing conditions for 5 days and the percentage of CD4+CXCR5+PD-1+ Tfh cells was analyzed by flow cytometry. (H) BCL6 MFI expression in CD4+CXCR5+PD-1+ Tfh cells indicated in G was analyzed by flow cytometry (n = 6). (I) Mutant and WT versions of E4BP4 were expressed in Jurkat cells. ChIP-PCR was used to detect levels of E4BP4 enrichment, H3 acetylation, and H3K27 trimethylation in the BCL6 gene promoter (n = 3). Data are representative of 3 independent experiments. For B, D, and E, Student’s t tests were used. For G–I, 1-way ANOVA analysis with Dunnett’s post hoc test was used. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The following antibodies were used: FITC anti–human CD4 (BD Biosciences, catalog 550628), APC mouse anti–human CD69 (BD Biosciences, catalog 555533), PE mouse anti–human CD154 (BD Biosciences, catalog 555700), PE anti–human IL-17A (BD Biosciences, catalog 560487), Alexa Fluor 647 anti–human IFN-γ (BioLegend, catalog 502516), PE anti–human IL-4 (BioLegend, catalog 500704), APC-Cy7 anti–human Bcl-6 (BD Biosciences, catalog 563581), PE-CY7 anti–human CD185 (BioLegend, catalog 356923), Alexa Fluor 647 anti–human FOXP3 (BD Biosciences, catalog 561184), PE anti–human CD278 (Thermo Fisher Scientific, catalog 12-9185), APC anti–human CD279 (Thermo Fisher Scientific, catalog 85-17-2799-42), human E4BP4 (NFIL3) monoclonal antibody (Thermo Fisher Scientific, catalog 12-9812-42), FITC anti–mouse CD4 (BioLegend, catalog 100406), PerCP-Cy5.5 rat anti–mouse Foxp3 (BD Biosciences, catalog 563902), APC-Cy7 mouse anti–Bcl-6 (BD Biosciences, catalog 563581), Biotin rat anti–mouse CXCR5, APC-Cy7 rat anti–mouse CD62L (BD Biosciences, catalog 560514), PE-Cy7 rat anti–mouse CD44 (BD Biosciences, catalog 560569), APC hamster anti–mouse CD279 (PD-1) (BD Biosciences, catalog 562671), PE rat anti–mouse CD278 (ICOS) (BD Biosciences, catalog 552146), PE hamster anti–mouse CD95 (BD Biosciences, catalog 554258), Alexa Fluor 647 anti–mouse T and B cell activation antigen (BD Biosciences, catalog 561529), FITC anti–mouse CD45R/B220 (BD Biosciences, catalog 553087), and mouse E4BP4 (NFIL3) monoclonal antibody (BD Biosciences, catalog 551960; Thermo Fisher Scientific, catalog 12-5927-82).

Techniques: Isolation, Transfection, Over Expression, Plasmid Preparation, Negative Control, Cell Culture, Luciferase, Reporter Assay, Electroporation, Mutagenesis, Flow Cytometry, Expressing

Journal: The Journal of Clinical Investigation

Article Title: E4BP4-mediated inhibition of T follicular helper cell differentiation is compromised in autoimmune diseases

doi: 10.1172/JCI129018

Figure Lengend Snippet: (A) Analysis of E4BP4 MFI expression in CD4+ T cells in healthy subjects (n = 11), inactive SLE patients (n = 20), and active SLE patients (n = 20) by flow cytometry. (B) Gating strategy of SLE PBMC CD45RA+, CD45RA–CXCR5lo, and CD45RA–CXCR5hi subsets in CD4+ T cells. Analysis of E4BP4 MFI expression in indicated subsets was shown (right) by flow cytometry. Dots indicate individual donors (n = 10). (C) Immunoprecipitating with antiphosphoserine antibody before Western blot analysis of anti-E4BP4 in SLE CD4+ T and NC CD4+ T cells (n = 8). Representative results of Western blot analysis (IP/Input) are shown (right). Data are representative of 3 independent experiments. For A and B, 1-way ANOVA with Tukey’s test was used. For C, Student’s t test was used. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The following antibodies were used: FITC anti–human CD4 (BD Biosciences, catalog 550628), APC mouse anti–human CD69 (BD Biosciences, catalog 555533), PE mouse anti–human CD154 (BD Biosciences, catalog 555700), PE anti–human IL-17A (BD Biosciences, catalog 560487), Alexa Fluor 647 anti–human IFN-γ (BioLegend, catalog 502516), PE anti–human IL-4 (BioLegend, catalog 500704), APC-Cy7 anti–human Bcl-6 (BD Biosciences, catalog 563581), PE-CY7 anti–human CD185 (BioLegend, catalog 356923), Alexa Fluor 647 anti–human FOXP3 (BD Biosciences, catalog 561184), PE anti–human CD278 (Thermo Fisher Scientific, catalog 12-9185), APC anti–human CD279 (Thermo Fisher Scientific, catalog 85-17-2799-42), human E4BP4 (NFIL3) monoclonal antibody (Thermo Fisher Scientific, catalog 12-9812-42), FITC anti–mouse CD4 (BioLegend, catalog 100406), PerCP-Cy5.5 rat anti–mouse Foxp3 (BD Biosciences, catalog 563902), APC-Cy7 mouse anti–Bcl-6 (BD Biosciences, catalog 563581), Biotin rat anti–mouse CXCR5, APC-Cy7 rat anti–mouse CD62L (BD Biosciences, catalog 560514), PE-Cy7 rat anti–mouse CD44 (BD Biosciences, catalog 560569), APC hamster anti–mouse CD279 (PD-1) (BD Biosciences, catalog 562671), PE rat anti–mouse CD278 (ICOS) (BD Biosciences, catalog 552146), PE hamster anti–mouse CD95 (BD Biosciences, catalog 554258), Alexa Fluor 647 anti–mouse T and B cell activation antigen (BD Biosciences, catalog 561529), FITC anti–mouse CD45R/B220 (BD Biosciences, catalog 553087), and mouse E4BP4 (NFIL3) monoclonal antibody (BD Biosciences, catalog 551960; Thermo Fisher Scientific, catalog 12-5927-82).

Techniques: Expressing, Flow Cytometry, Western Blot