Journal: PLoS ONE

Article Title: The Human CD8β M-4 Isoform Dominant in Effector Memory T Cells Has Distinct Cytoplasmic Motifs That Confer Unique Properties

doi: 10.1371/journal.pone.0059374

Figure Lengend Snippet: ( A ) HEK-293T cells co-transfected with plasmids expressing HA-tagged ubiquitin, CD8α and each CD8β isoform or mutant proteins. After 48 hours cells were lysed with 1% BRIJ 97 and immunoprecipitated with anti-CD8β mAb, run on a polyacrylamide gel, transferred to a membrane and probed with the anti-HA antibody. Gels were reprobed with an anti-CD8β antibody (5F2). Small differences in size reflect different lengths of the isoforms. ( B ) Co-transfection of HEK-293T cells with either wild type HA-Ub or I 44 A mutant of HA-Ub. The immunoprecipitation and Western blotting procedures were performed as in (A). ( C ) Determination of direct ubiquitination of CD8β isoforms. Cells expressing individual isoforms were immunoprecipitation with anti-HA mAb followed by blotting with anti-CD8β antibody. An aliquot of total cell lysate was similarly analyzed. ( D ) Effect of lysine mutants on M-4 ubiquitination. Cells expressing M-4 wild-type, single lysine (K 234 R, K 242 G) or double lysine (K 234 R/K 242 G labeled KRKG) mutants were lysed and protein analyzed as described in (C). ( E ) The JM T cells expressing CD8α and either CD8β M-4 or the double lysine mutant KRKG were incubated with the anti-CD8α (OKT8) and anti-CD3 (OKT3) mAbs for 0, 2.5 or 7 minutes at 37°C. Cells were lysed, immunoprecipitated with the anti-Ub mAb followed by Western blotting with the anti-CD8β antibody. A representative of three independent experiments is shown in each panel.

Article Snippet: Antibodies used include: anti-CD8β 5F2 (Santa Cruz, Santa Cruz, CA) or SIDIBEE (eBioscience, San Diego, CA), anti-CD8αβ 2ST8.5H7 (Beckman Coulter Inc., Brea, CA), anti-CD8α OKT8 (ATCC hybridoma, FITC labeled), Alexa-Fluor647 conjugated anti-CD3, anti-GAPDH (Sigma-Aldrich, St. Louis, MO), anti-HA (6E2) (Cell Signaling, Danvers, MA), and anti-Ub (P4D1, Biolegend, San Diego, CA).

Techniques: Transfection, Expressing, Ubiquitin Proteomics, Mutagenesis, Immunoprecipitation, Membrane, Cotransfection, Western Blot, Labeling, Incubation

Journal: Cancer Immunology Research

Article Title: Immuno-PET Monitoring of Lymphocytes Using the CD8-Specific Antibody REGN5054

doi: 10.1158/2326-6066.cir-21-0405

Figure Lengend Snippet: Figure 1. Characterization of REGN5054 and DFO-REGN5054 binding to primary cells and in vitro functionality. A, Binding to primary human (top) and cynomolgus monkey (bottom) CD8þ T cells shown by flow cytometry. Mean fluorescence intensities (MFI) are plotted. REGN5054 (blue), DFO-REGN5054 (red), and their isotype controls (IgG4P-PVA control, gray, open triangles, and DFO-IgG4P-PVA control, gray, closed triangles). Values indicated are the concentration of antibodies at half-maximum binding. B, Immobilized or soluble DFO-REGN5054 does not induce the proliferation of human PBMCs, as determined by a viability luciferase assay. DFO-REGN5054 (red), DFO-isotype control (DFO-IgG4P-PVA control, gray), TeGenero (TGN) superagonist anti-CD28 IgG4P (orange; contains S228P mutation), and IgG4P control (black). RLUs are plotted. Data represent average RLU values SD from 4 donors. Statistical significance was determined on Log10 transformed data using a 2-way ANOVA with Tukey post hoc test. Asterisks indicate statistically significant (P < 0.05) differences as compared with the respective isotype-matched control groups. C, A T-cell/antigen-presenting cell–based reporter assay was used to assess concentration-dependent inhibition of human CD8-dependent TCR signaling. These experiments were performed using JRT3.T3.5/AP1-Luc/1G4AB/hCD8AB T cells stimulated by parental 3T3 or 3T3/hb2M/HLA-A2/NYESO1p cells. Black closed squares and green open squares represent the commercial anti-CD8 antibody RPA-T8 and its isotype control (mIgG1). Other reagents used are as in A. RLUs are plotted. D, Mitomycin-treated human PBMCs were treated with purified allogeneic CD8þ T cells in an MLR. Various antibodies were titrated at a range of concentrations (0.8–100 nmol/L). Supernatant was collected after 120 hours and was tested in a human IFNg alphaLISA assay.

Article Snippet: For assessment of anti-CD8A with different functional properties on primary infection virus clearance, mice were treated as indicated with either a depleting anti-human CD8A (OKT8; BE0004-2, Bio X Cell) or with Regeneron CD8A antibodies with different blocking activities as defined previously by in vitro functional assay.

Techniques: Binding Assay, In Vitro, Cytometry, Control, Concentration Assay, Luciferase, Mutagenesis, Transformation Assay, Reporter Assay, Inhibition

Journal: Cancer Immunology Research

Article Title: Immuno-PET Monitoring of Lymphocytes Using the CD8-Specific Antibody REGN5054

doi: 10.1158/2326-6066.cir-21-0405

Figure Lengend Snippet: Figure 4. 89Zr-DFO-REGN5054 specifically localizes to lymphoid tissues in immunocompetent mice humanized for CD8. VelociT mice received a single dose of 89Zr-DFO- REGN5054, and PET/CT was acquired on days 0, 1, 4, and 6 after injection. Quantitative ex vivo biodistribution of the mice (n ¼ 4 per dose group) was conducted on day 6 after injection. A, Representative PET/CT images demonstrate the localization of 89Zr-DFO-REGN5054 over time when dosed at 0.1, 0.5, or 1.5 mg/kg. Blue, yellow, and green arrows highlight the spleen, axillary, and inguinal lymph nodes, respectively. B, Tissue biodistribution values presented as percent injected dose per gram (%ID/g). C, Tissue biodistribution values presented as tissue-to-blood ratios (T:B). n ¼ 3–4 mice per group, and data are representative of 4 experiments.

Article Snippet: For assessment of anti-CD8A with different functional properties on primary infection virus clearance, mice were treated as indicated with either a depleting anti-human CD8A (OKT8; BE0004-2, Bio X Cell) or with Regeneron CD8A antibodies with different blocking activities as defined previously by in vitro functional assay.

Techniques: Positron Emission Tomography-Computed Tomography, Injection, Ex Vivo

Journal: European journal of immunology

Article Title: A human immune system (HIS) mouse model that dissociates roles for mouse and human FcR + cells during antibody-mediated immune responses.

doi: 10.1002/eji.202350454

Figure Lengend Snippet: Figure 2. Human immune cell reconstitution in BRGSA2DR2 Fcer1g−/−mice. Quantification of human immune cell populations in human immune system (HIS) Fcer1g wt mice (black) versus HIS Fcer1g−/−mice (red). (A–C) In blood at 12 weeks postgrafting (wpg), (A) percentage of hCD45 calculated as %hCD45 cells = 100*hCD45/(hCD45 + mCD45), (B) frequency of CD19+ B cells in hCD45 cells, and (C) frequency of CD3+ T cells in hCD45 cells. (D–I) For the spleen, BM and liver at 16 wpg. (D) Absolute numbers of hCD45 per organ (for BM for one femur and one tibia). Frequency of (E) CD19+ B cells, (F) CD3+ T cells, (G) NKp46+ and CD94+ NK cells in hCD45 and (H) CD4+ and (I) CD8+ in total CD3+ T cells. Each dot represents one mouse, and data from the blood are representative for 21 mice (>6 independent experiments) and from the organs for 2 independent experiments. Pairwise comparison in the blood was done by Wilcoxon test, and Kruskal–Wallis test was used for multiple comparisons of means in the organs.

Article Snippet: Antigen Fluorochrome Clone Reactivity Supplier Catalog number CD3 BV510 SK7 Human BioLegend 344828 CD3 APC-Vio770 REA613 Human Miltenyi Biotec 130-113-136 CD4 APC RPA-T4 Human BD 555349 CD4 BUV496 SK3 Human BD 612936 CD11b AF700 M1/70 Mouse/human eBioscience 56-0112-82 CD16 BUV737 3G8 Human BD 564434 CD16 BUV805 3G8 Human BD 748850 CD16/CD32 PE 2.4G2 Mouse BD 561727 CD19 PE HIB19 Human BioLegend 302208 CD19 PE-CF594 HIB19 Human BD 562294 CD20 BV786 2H7 Human BioLegend 302356 CD45 APC-Cy7 30-F11 Mouse BD 557659 CD45 PerCP-Cy5.5 HI30 Human BD 564105 CD45 FITC 30-F11 Mouse BioLegend 103108 CD45 BUV395 HI30 Human BD 563792 CD45 BV650 30-F11 Mouse BD 563410 CD45 BUV805 HI30 Human BD 612891 CD64 PE X54-5/7.1 Mouse BD 558455 CD8a PE-CF594 RPA-T8 Human BD 562282 CD8a BV786 RPA-T8 Human BioLegend 301046 CD8b PE-Cy7 SIDI8BEE Human eBioscience 25-5273-42 CD94 FITC DX22 Human BioLegend 305504 CD94 PerCP-Vio700 REA113 Human Miltenyi Biotec 130-119-763 FCER1A PE MAR-1 Mouse eBioscience 12-5898-82 FCER1G FITC Human/mouse Merck- Millipore FCABS400F Gr-1 FITC RB6-8C5 Mouse BD 553127 NKp46 BV421 9E2/NKp46 Human BD 564065 Antibody-mediated depletion of human cell subsets in vivo For CD8a+ cell depletion, HIS mice were prebled 3 days before i.p. (intraperitoneal) injection with 100 μg/mouse of antihuman CD8a antibody OKT8 (BioXcell) in PBS, and control mice were injected with only PBS and bled at day 4 (d4) postinjection.

Techniques: Comparison