Fluorescent anti cd3

On day 0 (D0), peripheral blood mononuclear cells (PBMCs) from 3 healthy women donors (20, 21 and 24 years old, referred to as #1, #2, and #3, respectively) were incubated at the density of 200,000 cells/well in 96-well plates, in complete RPMI medium (Panbiotech, Aidenbach, Germany, P04-17500) supplemented with HEPES buffer (10 mM, Lonza, Basel, Switzerland, 17-737E), non-essential amino acids (1X, Lonza, 13-114E), sodium pyruvate (1 mM, Lonza, 13-115E), L-glutamine (2 mM, Biowest, Nuaillé, France X0550-100), human decomplemented serum AB (2% v/v, SIGMA, H3667-100) in the presence of HPV16_(L1) (PepMix^TM HPV16 (L1), JPT Peptide Technologies GmbH, Berlin, Germany) at the final dilution of 1/400 v/v. On D5, the cells were harvested, saturated with FcBlock solution (BD, 564220), immune-stained with fluorescent anti-CD3 (BioLegend, San Diego, CA, USA, 300325), anti-CD4 (BioLegend, 300519), and anti-CD8 antibodies (BioLegend, 344722), fixed, and analyzed by flow cytometry on a BD FACS Canto II (BD Biosciences, Franklin Lakes, NJ, USA), configuration 4/2/2.

On D0, PBMCs from 6 healthy women donors (from 25 to 35 years old) were incubated at the density of 200,000 cells/well in 96-well plates in Roswell Park Memorial Institute (RPMI) 1640 medium (Panbiotech, P04-17500), added with 2% human decomplemented serum, 1 mM non-essential amino acids, 1 mM pyruvate, 2 mM L-glutamine, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, in the presence of HPV16_(L1) peptide mix at the final dilution of 1/450 v/v. On D2, either the Veh., MIM-1; -2; -3; -4 or -5 were added to the medium and incubated for the next 72 h at the final sucrose–lactose concentration of 11 mM. Controls with either HPV16_(L1) or IL-2 (20 ng/mL) were also run in parallel. On D5, the cells were harvested, saturated with FcBlock solution (BD, 564220), immune-stained with fluorescent anti-CD3, anti-CD4, anti-CD8, anti-CD71, anti-CD95, anti-CD28 and anti-HLA-DR antibodies (all purchased from BioLegend), fixed, and analyzed by flow cytometry. The discrimination of the cell sub-populations is as follows: CD4⁺: CD3^high; CD4^high; CD8^low; SSC^low, CD8⁺: CD3^high, CD4^low, CD8^high, SSC^low, CD4⁻/CD8⁻: CD3^high; CD4^low; CD8^low; SSC^low. The supernatants (SNs) were retrieved and the cytokine levels of IFN-γ, IL-6, and interferon- γ inducible protein (IP-10) were assessed by enzyme-linked immunosorbent assay (ELISA).