Nlvpmvatv

Manufactured by ProImmune

Sourced in United Kingdom

The NLVPMVATV is a laboratory equipment product. It serves a core function, but a detailed description while maintaining an unbiased and factual approach is not available.

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Lab products found in correlation

8 protocols using nlvpmvatv

CMV-specific CD8 T cell Activation by pDCs

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pDCs from donors positive for CMV reactivity were transferred into 96-well plates (2.5 x 10⁴ cells/well) and stimulated with ICs generated by combining 0.5 μg/mL CG50 plasmid DNA with 10 μg/mL of IgG^D, IgE^D, or 5 μg/mL of each isotypes (IgG^D + IgE^D) for 4 hours in the presence of 10 μg/mL of a cytomegalovirus (CMV) peptide (NLVPMVATV, ProImmune). pDCs were then washed and incubated for 4 days with autologous CD8 T cells (2.5 x 10⁵ cells/well) in the presence of 100 IU/ml recombinant human IL-2 (Peprotech). CMV-specific CD8 T cells were identified using CMV Dextramer HLA-A*0201/NLVPMVATV (Immudex). CMV-specific CD8 T cell proliferation was assessed by flow cytometry using CellTrace^™ CFSE Cell Proliferation Kit (Life Technologies).

Henault J., Riggs J.M., Karnell J.L., Liarski V.M., Li J., Shirinian L., Xu L., Casey K.A., Smith M.A., Khatry D.B., Izhak L., Clarke L., Herbst R., Ettinger R., Petri M., Clark M.R., Mustelin T., Kolbeck R, & Sanjuan M.A. (2015). Self-reactive IgE exacerbates interferon responses associated with autoimmunity. Nature immunology, 17(2), 196-203.

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CMV-specific CD8 T cell Activation by pDCs

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T Cell Cytokine Reactivity Assay

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CD8⁺ cells were tested for reactivity in cytokine-release assays using ELISA kits [IFN-γ, TNF-α, granulocyte-macrophage colony-stimulating factor (GM-CSF); ExCell Bio, Shanghai, China; granzyme B (GrB); eBioscience, San Diego, California, USA]. T2 were pulsed with HLA-A∗0201-restricted MTB Ag85B_199–207 (KLVANNTRL), HIV-1 Env_120–128 (KLTPLCVTL) or CMV pp65_495–503 peptide (NLVPMVATV) (Proimmune, Oxford, UK) at indicated concentrations for 3 h at 37°C. Stimulator cells and responder cells were cocultured in a 96-well U-bottom plate (Nunc) for 24 h, except IFN-γ, which was detected after 18 h of incubation. In some groups, DCs were transfected with the pV1J.ns-tPA-Ag85B (gifted by Dr Kris Huygen in Pasteur Institute of Brussels, Brussels, Belgium) or the pCAGGS-Env plasmid (gifted by Dr James M. Binley in Torrey Pines Institute for Molecular Studies, San Diego, California, USA) respectively using Lipofectamine 2000 Transduction Reagent (Invitrogen, Carlsbad, California, USA).

Zhou C.Y., Wang R.N., He W.T., Luo D.R., Yuan S.R., Wen Q., Hu S.F., Zhou X.Y, & Ma L. (2022). Functional analysis of the antigen binding sites on the MTB/HIV-1 peptide bispecific T-cell receptor complementarity determining region 3α. AIDS (London, England), 37(1), 33-42.

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Cytokine Production Assay for Antigen-Specific CD8+ T Cells

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T2 cells pulsed with HLA-A*0201-restricted MTB Ag85B_199–207 peptide (KLVANNTRL), HIV-1 Env_120–128 peptide (KLTPLCVTL), or CMV pp65_495–503 peptide (NLVPMVATV) (10 µg/ml, or as described in figure legends; Proimmune, Oxford, UK) for 3 h at 37°C were incubated with CD8⁺ T cells. If IL-2 was the cytokine measured, the cells were washed with media without IL-2 prior to coculture. For these assays, 1 × 10⁵ effector cells (CD8⁺ T cells) and 1 × 10⁵ stimulator cells (T2 cells) were incubated in individual wells of 96-well U-bottom plate (Nunc) with a total volume of 0.2 ml for 24 h, except interferon-γ (IFN-γ), which was measured after 18 h of incubation. In some groups, DCs were transfected with the pV1J.ns-tPA-Ag85B plasmid (gifted by Dr. Kris Huygen in Pasteur Institute of Brussels, Brussels, Belgium) or the pCAGGS-Env plasmid (gifted by Dr. James M. Binley in Torrey Pines Institute for Molecular Studies, San Diego, CA, USA), respectively, using Lipofectamine 2000 Transduction Reagent (Invitrogen, Carlsbad, CA, USA).Cytokines were measured using IFN-γ, tumor necrosis factor-α (TNF-α), IL-2, granulocyte-macrophage colony-stimulating factor (GM-CSF) ELISA kits (ExCell Bio, ShangHai, China), and granzyme B (GrB) ELISA Kit (eBioscience, San Diego, CA, USA) according to the manufacture’s protocols.

Zhou C.Y., Wang R.N., Wen Q., He W.T., Zhang S.M., Du X.L., Yang J.H, & Ma L. (2017). Alanine Mutagenesis in the Complementarity Determining Region 3 of the MTB and HIV-1 Peptide-Bispecific T Cell Receptor Beta Chain Affects Ligand Recognition. Frontiers in Immunology, 8, 983.

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Detecting CMV and WT1-Specific T Cells

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Responder T cells from HLA-A*0201 expanded in the MLR culture were stained with APC-labeled HLA-A*0201/CMV-pp65 pentamer (NLVPMVATV; ProImmune) or Wilms’ tumor 1 (WT1) pentamer (HLA-A*0201/WT1 pentamer RMFPNAPYL; ProImmune) according to manufacturer's instructions. Cells were washed twice in MLR culture medium and were subsequently stained with anti-CD8-FITC and analyzed in a Becton Dickinson FACScalibur flow cytometer. For assessment of EBV-specific responses HLA-A*0201 positive naïve, cord blood T cells were used as responders and were cultured with autologous APC and a pool of EBV peptides corresponding to EBV immunogenic epitope BMLF1, in the presence or in the absence of roscovitine. Detection of EBV-specific T cells was performed using HLA-A*0201/GLCTLVAML-APC conjugated Dextramer (#RX05APC; Immudex, Copenhagen, Denmark), specific for BMLF1.

Nellore A., Liu B., Patsoukis N., Boussiotis V.A, & Li L. (2014). The cyclin dependent kinase inhibitor (R)-roscovitine mediates selective suppression of alloreactive human T cells but preserves pathogen-specific and leukemia-specific effectors. Clinical immunology (Orlando, Fla.), 152(0), 48-57.

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HBV- and CMV-specific T-cell Identification

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Patients were tested for their HLA-A2 status and PBMC from HLA-A2+ patients were stimulated with peptides representing HLA-A2-restricted HBV epitopes (HBVenv: FLLTRILTI, WLSLLVPFV, LLVPFVQWFV, GLSPTVWLSV; HBVcore: FLPSDFFPSV; HBVpol: GLSRYVARL, KLHLYSHPI) or the CMV pp65-encoded NLVPMVATV epitope (Proimmune). Virus-specific cells were identified by multicolour flow-cytometry (BD LSR II): surface staining with CD3/Pe-Cy7, CD8-AF700, CD4-APC Cy7 (eBioscience, Hatfield, UK), CD38-PE Texas Red, CD14-V500, CD19-V500 (Biolegend, London, UK) in the presence of fixable live/dead stain (Invitrogen).

Gill U.S., Peppa D., Micco L., Singh H.D., Carey I., Foster G.R., Maini M.K, & Kennedy P.T. (2016). Interferon Alpha Induces Sustained Changes in NK Cell Responsiveness to Hepatitis B Viral Load Suppression In Vivo. PLoS Pathogens, 12(8), e1005788.

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CMV and EBV Peptide Stimulation

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Antigenic HLA-A*0201 restricted CMV-specific pp65_495–504 (NLVPMVATV) and EBV-specific BMLF1_259–267 (GLCTLVAML) peptides were purchased from ProImmune Ltd. (Oxford, UK). Peptides were dissolved in endotoxin-free DMSO (Sigma-Aldrich, Munich, Germany) and had a purity of >98%. Corresponding PE-labeled MHC class I CMV tetramer as well as EBV Dextramer were obtained from Beckman Coulter Inc. (Fullerton, CA, USA) and Immudex (Copenhagen, Denmark), respectively. For in vitro stimulations, PEG–IFN-alfa-2a (Pegasys; Roche) was used. Additionally, lyophilized recombinant human ICAM-1 and VCAM-1 were obtained from PeproTech Inc. (Rocky Hill, NJ, USA) and reconstituted in 0.1% bovine serum albumin solution according to the manufacturer’s instructions.

Owusu Sekyere S., Suneetha P.V., Hardtke S., Falk C.S., Hengst J., Manns M.P., Cornberg M., Wedemeyer H, & Schlaphoff V. (2015). Type I Interferon Elevates Co-Regulatory Receptor Expression on CMV- and EBV-Specific CD8 T Cells in Chronic Hepatitis C. Frontiers in Immunology, 6, 270.

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Analyzing B-CLL Cell Activation by EVs

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500,000 B-CLL cells were cultivated with 500 ng of EVs in a final volume of 2 ml in a 6-well plate for 48 h. Induction of surface accessory molecules was measured by flow cytometry using a FACSCanto flow cytometer (Becton Dickinson, Franklin Lanes, NJ,) and analysed with FlowJo (Tree Star Inc., Ashland, ORE). The cells were washed in PBS and incubated for 20 min at 4°C with the following antibodies diluted 1:50 in PBS/3% FCS: mouse anti-CD19–APC (HIB19; BioLegend), mouse anti-CD80-PE (2D10; BioLegend), mouse anti-CD86-PE (37301; R&D Systems, Wiesbaden, Germany), mouse anti-CD54-PE (HCD54; BioLegend), mouse anti-CD95-PE (DX2; BD Pharmingen). The gp350 antibody 72A1 was developed in our own group. To study the activation of bystander CLL cells, cells were stained with CellTracker^TM Green CFMDA dye (Thermo Fisher Scientific, Waltham, MA), according to the manufacturer’s protocol. T cells were detected by staining with mouse anti-CD4-PE (RPA-T4; BioLegend), mouse anti-CD8-APC (RPA-T8; BioLegend), unlabelled Pro5 HLA-A*02:01/NLV pentamer (1:30; pp65 495–504; NLVPMVATV; ProImmune, Oxford, UK) and PE-labelled Pro5 Fluorotag (1:10; ProImmune). Cell surface expression of the human leucocyte antigens on primary CLL cells was analysed by using the antibodies mouse anti-HLA-A2-PE (BB7.2; BioLegend) and FITC-coupled mouse anti-HLA-DR, DP, DQ (Tu39; BD Pharmingen).

Gärtner K., Luckner M., Wanner G, & Zeidler R. (2019). Engineering extracellular vesicles as novel treatment options: exploiting herpesviral immunity in CLL. Journal of Extracellular Vesicles, 8(1), 1573051.

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