Lipofectamine 2000 transduction reagent

To prevent mispairing of the exogenous and endogenous TCR, α9 and β5 chains contain 9 amino acid-mutated C regions amplify by the above overlapping PCR methods have been connected by 2A and inserted into the retrovirus empty vector PMX-IRES-GFP. Construction of the recombinant retroviral vector was performed as previously described [25 (link)]. Retrovirus was packaged by co-transfection of the recombinant vector pMX-β5-2A-α9 and the VSV-G envelope protein vector into the retroviral packaging cell line GP2-293 with Lipofectamine 2000 Transduction Reagent (Invitrogen, Carlsbad, CA, USA) following manufacturer’s instructions and concentrated and the virus titer was determined as described before [25 (link)].

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.

To determine the function of the TCR gene‐modified T cells upon antigen stimulation, experiment groups were set up as indicated in figure legends. Peptide (10 μg/ml, or as described in figure legends)‐loaded or peptide‐unloaded autologous DCs were seeded at 5 × 10³ cells/well in a 96‐well plate (Nunc) and cocultured with CD8⁺ T cells according to the different effector: target (E:T) ratio, 7 in IFN‐γ assays and 20 in TNF‐α and granzyme B (GrB) assays, respectively. 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), the pCAGGS‐Env plasmid (gifted by Dr. James M. Binley in Torrey Pines Institute for Molecular Studies, San Diego, CA, USA) or the pCI‐OVA plasmid (kindly provided by Dr. Yukio Koide, Hamamatsu University School of Medicine, Hamamatsu, Japan), respectively, using Lipofectamine 2000 Transduction Reagent (Invitrogen). The culture supernatants were harvested 18 hrs later for determining IFN‐γ levels and 24 hrs later for TNF‐α and GrB. Cytokines were measured using IFN‐γ, TNF‐α ELISA kits (Bender MedSystems, Vienna, Austria) and GrB ELISA Kit (R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacture's protocols. Both E:T ratios and incubation time were determined according to our previous study 20.