Ficoll gradient

Peripheral blood mononuclear cells (PBMC) from SCCA patients and healthy donors were isolated by density centrifugation on Ficoll gradient (Eurobio, Courtaboeuf, France). PBMC of SCCA patients were cryopreserved at a cell density of 8–15 × 10⁶ cells per vial in CryoStor (CS10 and CS5) cell preservation media (Sigma-Aldrich, St. Louis, MO, USA) and were conserved at −196 °C for flow cytometry and ELISpot analysis.
Frozen PBMC (viability > 70%) were seeded at 4 × 10⁶ cells per well in a 24-well plate in complete medium (RPMI supplemented with 10% human serum, 10,000 UI/mL penicillin and 10,000 μg/mL streptomycin, Gibco, Illkirch, France) and were exposed to PepTivator HPV16-E6 and E7 (1 μg of each peptide/mL) and the pool of peptides derived from Telomerase (5 μg/mL). A pool of the 23 peptides containing epitopes from CEF (Cytomegalovirus, Epstein-Barr and Influenza virus) (2 μg of each peptide/mL) (Cellular Technology Limited, Shaker heights, OH, USA) was used to evaluate antiviral responses. Recombinant interleukin (IL)-7 (5 ng/mL) and IL-2 (20 UI/mL, Peprotech, Neuilly-sur-seine, France) were added at days 1 and 4, respectively. At day 7, after a short-term in vitro stimulation, antigen-specific T-cell responses were monitored by IFNᵧ Enzyme-Linked ImmunoSpot (ELISpot) assay (Diaclone, Besançon, France).

EDTA-anticoagulated blood samples (5 mL) were collected (on day of initiation of empirical antibiotic treatment for infected patients) and immediately sent to the laboratory. Peripheral blood mononuclear cells (PBMCs) from infected patients and controls were isolated from whole blood using a Ficoll gradient (MSL, Eurobio) and suspended in RPMI 1640 containing 25 mM HEPES and 2 mM L-glutamine (Invitrogen) (Alingrin et al., 2016 (link)). After centrifugation at 500 × g, PBMCs were washed in sterile phosphate-buffered saline (PBS, Life Technologies) and suspended (about 5 × 10⁶ cells/ml) in RPMI 1640 supplemented with 20% fetal calf serum (FCS, Invitrogen) and 10% dimethylsulfoxide, and preserved at −80°C.

Convalescent donors were eligible for plasma donation after resolution of COVID-19. Peripheral blood samples from donors were collected at least 15 days after the end of symptoms at the Etablissement Français du Sang (EFS Besançon, France) from April to June 2020. This study is a cross-sectional research. All CPD were enrolled in COVIPLASM study (NCT04345991) after the signature of informed consent and following the EFS guidelines. Peripheral blood mononuclear cells (PBMC) from convalescent donors were isolated from the apheresis ring by density centrifugation on Ficoll gradient (Eurobio). Moreover, PBMC from convalescent donors were cryopreserved in CryoStor (CS10 and CS5) cell preservation media (Sigma-Aldrich) and were conserved in nitrogen for flow cytometry and Enzyme-linked-Immunospot (ELISpot) analysis.

CTL03.1 and N5.14 CD8+ T-cell clones specific for Melan-A and MUC1, respectively, were obtained and cultured as previously described (18 (link), 19 (link)). Polyclonal CD8+ T cells were obtained from fresh blood (Etablissement Français du Sang, ethics agreement CPDL-PLER-2018 021). Peripheral blood mononuclear cells were separated using Ficoll gradient (Eurobio, Les Ulis, France; Cat#CMSMSL01-01). Polyclonal CD8+ T cells were then sorted using EasySep Human CD8+ T Cell Isolation Kit (STEMCELL Technologies, Vancouver, Canada; Cat#17953). Purity was assessed after sorting by flow cytometry following a 20-min staining at 4°C with CD3 and CD8 antibodies directly conjugated to fluorescein isothiocyanate (FITC, BD Biosciences, Pont de Claix, France; Cat#555339, RRID : AB_395745) and phycoerythrin (PE, BD Biosciences, Pont de Claix, France; Cat#555367, RRID : AB_395770), respectively. Polyclonal T cells were considered acceptable for further experiments when CD3+CD8+ population among viable cells represented over 90%.

We constructed 1928zT2 CARs with a CAR linking FMC63-scFv, CD28 transmembrane, endodomain, CD3ζ signaling domain, and Toll/interleukin-1 receptor domain (amino acid 639–784) of TLR2 (1 (link)). Five samples from each source were selected for in vitro and in vivo experiments. Mononuclear cells were isolated from samples by centrifugation using a Ficoll gradient (Eurobio, Les Ulis, France, CMSMSL01-01). T cells were isolated and stimulated by MACS GMP TransAct CD3/CD28 Kit RUO and IL-2 (200 IU/ml) for 48 h and then transfected into third-generation CAR-T cells by lentivirus carrying 1928zT2 CARs. Transduced cells were then cultured in media containing Roswell Park Memorial Institute (RPMI) 1640 (Gibco-BRL, Gaithersburg, MD, USA), 10% fetal bovine serum (Hyclone Laboratories Inc., Logan, UT, USA), 1% l-glutamine (Invitrogen, Carlsbad, CA, USA), and IL-2 (200 IU/ml) for 9–11 days before subsequent analysis. The efficiency of transfection was evaluated by green fluorescent protein (GFP).

Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy volunteers from the Etablissement Français du Sang (Besançon, France) by Ficoll gradient centrifugation (Eurobio, Courtaboeuf, France). NK cells were purified from PBMCs by negative selection with magnetic enrichment kit (Stemcell, Grenoble, France) and FcγIII+ CD14- non-classical monocytes were isolated from PBMCs using Slan-(M-DC8)+ Monocyte Isolation Kit, (Miltenyi Biotec, Paris, France)⁸² . All sorted cell populations exhibited high purity (>90%), as revealed by flow cytometry.

Whole blood from healthy adult donors was obtained from the Etablissement Francais du Sang. Peripheral blood mononuclear cells (PBMCs) were recovered from the interface of a Ficoll gradient (Eurobio, Evry, France). T cells were isolated with Pan T cell isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and then activated for 2 days with T Cell TransAct^TM (Miltenyi Biotec, Bergisch Gladbach, Germany), a polymeric nanomatrix conjugated to humanized CD3 and CD28 agonists. Cells were subsequently cultivated in Roswell Park Memorial Institute Medium (RPMI; Gibco by Thermo Fischer Scientific, Waltham, MA, United States) 1640 supplemented with 25 mM GlutaMax (Gibco by Thermo Fischer Scientific, Waltham, MA, United States), 10% fetal calf serum (FCS; Gibco by Thermo Fischer Scientific, Waltham, MA, United States) at 37°C, and 5% CO₂ in the presence of IL-2 (50 ng/mL; Miltenyi Biotec, Bergisch Gladbach, Germany) and used 7 days after activation. At the time of use, the cells were >99% positive for pan-T lymphocyte marker CD3 and assessed for activation and proliferation with CD25, CD45RO, CD45RA, and CD69 makers as judged by flow cytometry. ROCK inhibitor Y-27632 dihydrochloride was obtained from Sigma–Aldrich (St. Louis, MO, United States) and Myosin II inhibitor Blebbistatin from Fisher Bioblock Scientific (Illkirch, France).