Human ab serum

Gag‐specific CD4⁺ T cell clones (F12) are specific for HIV Gag‐p24 (gag2: aa 271–290) and restricted by HLA‐DRβ1*01 as previously described (Moris et al, 2006 (link); Coulon et al, 2016 (link)). HCMV‐specific CD4⁺ T cell clones are specific for HCMV pp65 antigen (pp65: aa 108–127) and restricted by HLA‐DRβ1*01. Pp65‐specific clones were isolated from PBMCs of healthy donors after several rounds of in vitro stimulation with synthetic peptide corresponding to immunodominant epitopes from the pp65 protein. Pp65‐specific cells were isolated using the IFNγ secreting assay from Miltenyi Biotec and cloned by limiting dilution. F12 and pp65 clones were restimulated and expanded, as previously described (Moris et al, 2006 (link)), using irradiated feeders and autologous or HLA‐matched lymphoblastoid cell lines loaded with cognate peptides in T cell cloning medium: RPMI 1640 containing 5% human AB serum (Institut Jacques Boy), recombinant human IL‐2 (100 IU/ml, Miltenyi Biotec), PHA (0,25 μg/ml, Remel), nonessential amino acids, and sodium pyruvate (both from Life Technologies). At least 1 h before co‐culture with HeLa‐CIITA cells, T cell clones were thawed and allowed to rest at 37°C in RPMI containing DNAse (5 μg/mL, New England Biolabs).

Human peripheral blood mononuclear cells (PBMC) were isolated from the blood using Ficoll Paque Plus (Sigma-Aldrich). The subtypes of MHC I molecules of Human PBMC were examined and HLA-A2⁺ human PBMC were identified by flow cytometry. Primary T cells were activated and expanded by culturing 1 × 10⁶ PBMC in TexMACS medium (Miltenyi Biotec, Germany) supplemented with 500 IU/ml human recombinant Interleukin 2 (rhIL2) (Miltenyi Biotec), with 10 µl TransAct (Miltenyi Biotec) which contains anti-CD3 and CD28 antibodies, for 3 days. The T cells were then purified with a pan-T cell isolation kit (Miltenyi Biotec) and cultured in TexMACS medium + rhIL2 for 2 weeks before CD3/28 antibody re-stimulation or DC cell-based antigen stimulation. Primary NK cells were activated and expanded by culturing 1 × 10⁶ PBMC in NK MACS medium (Miltenyi Biotec) supplemented with 5% human AB serum (Invitrogen) and 500 IU/ml rhIL2, with 5 × 10⁵ microbeads conjugated with anti-CD335 and CD2 antibodies (Miltenyi Biotec) for 7 days. The NK cells were purified with NK cell isolation kit (Miltenyi Biotec) and cultured in NK MACS medium + human AB serum + rhIL2 for 1 week before hypoxic culture. The purity of the immune cells was examined by flow cytometry analysis.

Retrovirally transduced T cells were produced and expanded as previously described (11 (link)). Briefly, after isolation, PBMCs were stimulated with 600 IU/mL of recombinant human interleukin-2 (rhIL-2; Miltenyi Biotec, 130-097-745) and 50 ng/mL of anti-CD3 (eBioscience, 16-0037-81) in AIM-V (ThermoFisher Scientific, 12055091), 2% (v/v) human AB serum (Sigma-Aldrich, H6914) for 48 h, before transduction with a MP-71 vector containing the Vα34 and Vβ3 chains of the HBs183-91-specific (Ts) or the HBcore18-27-specific (Tc) TCR sequences. Bulk transduced T cells were expanded and restimulated using 0.5–1 × 10⁶ TCR-transduced T cells, 2 × 10⁵ irradiated (2,500 rads) T2 cells pulsed with 1 µg/mL of either HBs183-91 peptide (FLLTRILTI) or HBcore18-27 (FLPSDFFPSV) (Genscript, custom synthesized peptides) and 1.8 × 10⁶ irradiated PBMCs as feeders. Cells were cultured for about 2 weeks in AIM-V, 2% human AB serum supplemented with 100 IU/mL of rhIL-2, 10 ng/mL of rhIL-7 (Miltenyi Biotec, 130-095-362), and 10 ng/mL of rhIL-15 (Miltenyi Biotec, 130-095-764). In some experiments, CD8⁺ T cells were enriched through negative selection of transduced T cells using CD4 microbeads (Miltenyi Biotec, 130-045-101) following the manufacturer’s instructions.