Journal: Immunity

Article Title: Transcription Factor IRF4 Promotes CD8 + T Cell Exhaustion and Limits the Development of Memory-like T Cells during Chronic Infection.

doi: 10.1016/j.immuni.2017.11.021

Figure Lengend Snippet: Figure 4. IRF4 and NFAT Form a Positive Feedback Circuit during T Cell Exhaustion (A) Expression of Nfatc1 isoforms as identified by RNA sequencing. Graph shows expression relative to naive T cells of the short isoform (NFATc1aA, represented by exon 1–3 junctions, skipping exon 2) and the long isoform (represented by exon 2–3 junctions) in P14 T cells isolated from acutely (WE) and chronically (Docile) LCMV-infected mice at day 30 post infection (p.i.). (B) Western blot showing NFATc1, NFATc2, IRF4, BATF, and Actin (loading control) expression in CD8+ T cells flow cytometry sorted as CD62L from acutely and as CD62LPD-1+ from chronically LCMV-infected mice at day 30 p.i. The arrow marks the short isoform of NFATc1. (C) Binding of NFATc1 and NFATc2 to the Irf4 and Pdcd1 (encoding PD-1) promoters demonstrated by chromatin immunoprecipitation using specific antibodies or control IgG and RT-qPCR in total CD44+CD8+ T cells isolated from spleens of acutely and chronically LCMV-infected mice at day 8 p.i. Enrichment is expressed as percentage of total chromatin input and compares isotype control and NFATc1- and NFATc2-specific antibodies. (D) Western blot showing NFATc1, NFATc2, IRF4, BATF, and p50 NF-kB (loading control) expression in polyclonal CD8+ T cells deficient (KO) in NFATc1 (Nfatc1fl/flCd4Cre), NFATc2 (Nfatc2/), or both (DKO) after in vitro activation with anti-CD3 and anti-CD28 for 48 hr. (E) Western blot showing NFATc1, IRF4, BATF, and Actin (loading control) in polyclonal CD8+ T cells activated with anti-CD3, anti-CD28, and IL-2 with or without cyclosporine A (CsA) for 24 hr. Data in (A)–(C) are representative of 2 independent experiments and data in (D) and (E) are representative of 3 independent experiments. Error bars denote mean ± SEM. Statistical analysis was performed using unpaired two-tailed Student’s t test (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: Fluorochrome-conjugated antibodies directed against the following antigens were used for analysis by flow cytometry: CD8a (53-6.7), CD62L (MEL-14), CD71 (R17217), CD98 (RL388), Ly5.1 (A20), 2B4 (eBio244F4), PD-1 (J43), Lag3 (T47-530), TIGIT (GIGD7), CTLA4 (14D3), TIM-3 (RMT3-23), CD127 (A7R34), CXCR5 (SPRCL5), IL-2 (JES6-5H4), IFNg (XMG1.2) (from Thermo Fisher Scientific), CD44 (IM7), Ly5.2 (104), TNF (MP5-XT22) (from BD Biosciences), IRF4 (REA201) (from Miltenyi), TCF1 (C63D9), BATF (D7C5) (from Cell Signaling Technology), IRF4 (M17) (from Santa Cruz Biotechnology), and anti-goat IgG coupled to fluorescein isothiocyanate (Jackson ImmunoResearch Laboratories) and anti-rabbit IgG coupled to Alexa Fluor 647 (Thermo Fisher Scientific). e3 Immunity 47, 1–13.e1–e5, December 19, 2017 Propidium iodide, SytoxBlue or fixable viability dye eFluor506 (all Thermo Fisher Scientific) were used to exclude dead cells.

Techniques: Expressing, RNA Sequencing, Isolation, Infection, Western Blot, Control, Cytometry, Binding Assay, Chromatin Immunoprecipitation, Quantitative RT-PCR, In Vitro, Activation Assay, Two Tailed Test

Journal: Immunity

Article Title: Transcription Factor IRF4 Promotes CD8 + T Cell Exhaustion and Limits the Development of Memory-like T Cells during Chronic Infection.

doi: 10.1016/j.immuni.2017.11.021

Figure Lengend Snippet: Figure 5. IRF4 and BATF Cooperate with NFAT to Establish T Cell Exhaustion (A) Venn diagram showing overlap between genes bound by IRF4, BATF, and NFAT as identified by chromatin immunoprecipitation (ChIP) sequencing of effector CD8+ T cells (Kurachi et al., 2014; Man et al., 2013; Martinez et al., 2015). (legend continued on next page) Immunity 47, 1–13, December 19, 2017 7

Article Snippet: Fluorochrome-conjugated antibodies directed against the following antigens were used for analysis by flow cytometry: CD8a (53-6.7), CD62L (MEL-14), CD71 (R17217), CD98 (RL388), Ly5.1 (A20), 2B4 (eBio244F4), PD-1 (J43), Lag3 (T47-530), TIGIT (GIGD7), CTLA4 (14D3), TIM-3 (RMT3-23), CD127 (A7R34), CXCR5 (SPRCL5), IL-2 (JES6-5H4), IFNg (XMG1.2) (from Thermo Fisher Scientific), CD44 (IM7), Ly5.2 (104), TNF (MP5-XT22) (from BD Biosciences), IRF4 (REA201) (from Miltenyi), TCF1 (C63D9), BATF (D7C5) (from Cell Signaling Technology), IRF4 (M17) (from Santa Cruz Biotechnology), and anti-goat IgG coupled to fluorescein isothiocyanate (Jackson ImmunoResearch Laboratories) and anti-rabbit IgG coupled to Alexa Fluor 647 (Thermo Fisher Scientific). e3 Immunity 47, 1–13.e1–e5, December 19, 2017 Propidium iodide, SytoxBlue or fixable viability dye eFluor506 (all Thermo Fisher Scientific) were used to exclude dead cells.

Techniques: Chromatin Immunoprecipitation, ChIP-sequencing

Journal: International Journal of Molecular Sciences

Article Title: Transcriptional Landscape of CUT-Class Homeobox Genes in Blastic Plasmacytoid Dendritic Cell Neoplasm

doi: 10.3390/ijms25052764

Figure Lengend Snippet: Genomic activation of ONECUT2 in BPDCN. ( A ) Genomic profiling analysis of CAL-1 indicates duplication of 18q21-q23 targeting the ONECUT2 locus at 18q21.31. ( B ) Analysis of TF binding sites at ONECUT2 using the UCSC genome browser revealed potential impacts of IRF factors. Potential IRF-binding sites are indicated by red arrow heads. ( C ) RQ-PCR analysis of IRF8 and IRF4 in selected cell lines showed elevated activities exclusive to CAL-1. SiRNA-mediated knockdown experiments revealed an activatory role of IRF4, while IRF8 failed to regulate ONECUT2 expression. p-values are indicated by asterisks (*** p < 0.001, n.s. not significant). Western blot analysis confirmed IRF4 expression at the protein level in CAL-1 cells and its knockdown after siRNA treatment (below). ( D ) Genomic profiling analysis of CAL-1 indicated copy number gains for IRF4 at 6p25 and IRF8 at 16q24, probably underlying their overexpression.

Article Snippet: The following antibodies were used: alpha-Tubulin (Sigma, #T6199), CUX2 (Abnova, #H00023316-M03), IRF4 (Origene, #TA351302) and EPAS (MyBioSource, #MBS9127904).

Techniques: Activation Assay, Binding Assay, Knockdown, Expressing, Western Blot, Over Expression