Journal: Cell

Article Title: RAB11FIP5 Expression and Altered Natural Killer Cell Function Are Associated with Induction of HIV Broadly Neutralizing Antibody Responses

doi: 10.1016/j.cell.2018.08.064

Figure Lengend Snippet:

Article Snippet: Human KIR3DL2/CD158k APC-conjugated Antibody, Clone # 539304 , R and D Systems , Cat# FAB2878A.

Techniques: Recombinant, Staining, Cell Isolation, Enzyme-linked Immunosorbent Assay, Marker, Library Quantification, Gene Expression, Sequencing, Software

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Cancer cells suppress NK cell activity by actin-driven polarization of inhibitory ligands to the immunological synapse

doi: 10.1073/pnas.2503259122

Figure Lengend Snippet: Inhibition of NK cell polarization by cognate HLA–iKIR interactions is associated with synaptic actin polarization. Emerald-LifeAct-expressing MDA-MB-231 cells (green) were cocultured for 60 min with CTV-labeled primary NK cells, either KIR2DL1 + or KIR2DL1 − cells. After coculture, cells were immunolabeled for Granzyme B (red), γ-Tubulin (yellow), and KIR2DL1 (magenta). Data were collected from NK cells isolated from three distinct donors, with n = 100 cell-to-cell conjugates analyzed per condition. ( A ) Representative IFC images of cell-to-cell conjugates between primary NK cells (KIR2DL1 + or KIR2DL1 − ) and MDA-MB-231 cells, with or without synaptic actin cytoskeleton remodeling (AR+ and AR−, respectively). (Scale bars, 10 µm.) ( B and C ) Emerald-LifeAct relative intensity at the synapse was used to classify MDA-MB-231 cells into AR+ and AR− groups (ratio >1 and ratio <1, respectively). The percentage of conjugates of each subgroup is indicated for each condition. NK cell lytic machinery polarization was evaluated by measuring the distance between the MTOC and the synapse center ( B ) and the distance between the Granzyme B centroid and the IS center ( C ). Distances for AR+ and AR− subgroups are presented. Statistical significance was determined using the Kruskal–Wallis test.

Article Snippet: Cells were then washed and incubated for 30 min in the following antibody staining mix: FITC-conjugated anti-CD56 antibody (BioLegend #318304), PE-conjugated anti-CD3 antibody (BioLegend #300330), and PE-conjugated anti-CD158a (KIR2DL1) antibody (Miltenyi #130-120-446).

Techniques: Inhibition, Expressing, Labeling, Immunolabeling, Isolation

Journal: Cell host & microbe

Article Title: SMAC mimetics induce autophagy-dependent apoptosis of HIV-1-infected resting memory CD4+ T cells

doi: 10.1016/j.chom.2018.09.007

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: α-human HLA-DR microbeads , Miltenyi Biotec , Cat# 130-046-101.

Techniques: Virus, Recombinant, Marker, Enzyme-linked Immunosorbent Assay, Cell Isolation, shRNA, Transduction, Software

Journal: bioRxiv

Article Title: LSD1 inhibitors induce neuronal differentiation of Merkel cell carcinoma by disrupting the LSD1-CoREST complex and activating TGFβ signaling

doi: 10.1101/2020.04.14.041657

Figure Lengend Snippet: a , Bulk mouse skin labeled with IgM-PE mouse isotype control. b , Bulk mouse skin labeled with NCAM-PE. DAPI - cells were sorted. c , Merkel cells labeled with IgM-PE mouse isotype control. d , Merkel cells labeled with NCAM-PE. DAPI - /NCAM + cells were sorted. Each plot shows the subpopulation gated for in the preceding plot to the left.

Article Snippet: Merkel cells were stained with anti-PSA-NCAM-PE antibody (Miltenyi Biotec, Cat. 130-117-394) or with mouse IgM-PE isotype control (Miltenyi Biotec, Cat. 130-120-070) according to manufacturer’s instructions, for 10 min at 4°C.

Techniques: Labeling, Control

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