Cd49d

The production of T_H1 (IFNγ, TNFα, IL-2, MIP-1α, and MIP-1β) and T_H2 (IL-4 and IL-5) cytokines and IL-10 was evaluated after 20 h stimulation of T cells with S protein or human actin peptides. Briefly, PBMCs were stimulated for 20 h with SARS-CoV-2-specific peptide pools from S and a peptide pool of human actin (1 µg/mL) in the presence of co-stimulator molecules CD28 and CD49d (BD Bioscience). Cells were seeded in 96-wells round-bottom plates at a density of 0.5–1 × 10⁶ cells/200 µL culture medium per well. The concentrations of cytokines and chemokines were measured in duplicate in cell culture supernatants using BioPlex Pro Human Cytokine Screening Panel (27-Plex #M500KCAF0Y, Bio-Rad, Hercules, CA, USA) as previously described [3 (link)]. All the results were analysed with BIO-PLEX Manager software version 6.2 (Bio-Rad).

For cytometry, we used fluorochrome-labeled monoclonal antibodies with specificities for CD3, CD4, CD8 (Serotec, Kidlington, UK) and CD49d (Pharmingen/Becton Dickinson, San Diego, CA, USA). Isotype/ fluorochrome-matched unrelated antibodies were obtained from Pharmingen/Becton Dickinson. Peripheral blood mononuclear cells (PBMCs) from GRMD and healthy controls were isolated through ficoll-histopaque (Sigma-Aldrich, St Louis, MO, USA) sedimentation, using freshly obtained samples. PBMCs were first incubated in 96-well plates – with 5% fetal calf serum for 20 minutes at 4°C – and then subjected to fluorochrome-labeled primary monoclonal antibodies for 30 minutes. After washing, cells were fixed and acquisition for flow cytometry was carried out using a LSR II^® flow cytometer (Becton Dickinson, San Jose, CA, USA) equipped with FacsDiva software. A cell gate excluding cell debris and nonviable cells was determined using forward versus side scatter parameters. Analyses were done after recording 20,000 events for each sample, using the FACS Diva software.

SIV-specific CD4+ and CD8+ T cell responses in blood, lymph nodes, and spleens were tested by flow cytometric ICS assay, as described in detail [96 (link)]. Briefly, isolated CD4+ and CD8+ T cells were incubated with antigen peptides (SIVmac239 Gag ORF 15-mer peptide pool and SIVmac239 Vif ORF 15-mer peptide pool) and the co-stimulatory molecules CD28 and CD49d (BD Biosciences) for 1 hour, followed by addition of brefeldin A (Sigma-Aldrich) for an additional 8 hours. Cells incubated with Concanavalin A (ConA, Sigma-Aldrich) were used as positive controls, while cells without antigen stimulation were used as negative controls. Stimulated cells were fixed, permeabilized, and stained. The flow cytometric analysis was performed on an LSR-II instrument (BD Biosciences). Analysis was done using FlowJo software (Tree Star). In all analyses, gating on the light scatter signature of small lymphocytes was followed by progressive gating on the CD3+ population and then the CD4+/CD8- versus CD4-/CD8+ T cell subsets. Antigen-specific response frequencies for CD4+ or CD8+ T cell populations were determined from intracellular expression of TNF-α and IFN-γ.

MultiVSTs were harvested, resuspended in VST medium (2x10⁶/ml) and 200μl added per well of a round-bottom 96-well plate. Cells were incubated for 5-6 hrs with 200 ng of antigenic pepmixes pooled per virus along with CD107a, monensin (1 μg/ml), CD28 and CD49d (1 μg/ml) (BD). Next, multiVSTs were washed with PBS, pelleted, and surface-stained with CD3, CD4 and CD8 (5 μl/antibody/tube) for 15mins at 4°C. Subsequently, cells were washed, resuspended in 300 μl of PBS and at least 50,000 live cells acquired on a Gallios™ Flow Cytometer and analyzed with Kaluza^® Flow Analysis Software.

Fibroblasts were cultured and maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Paisley, UK) containing 10 % fetal calf serum (FCS) and penicillin/streptomycin, in a humidified atmosphere containing 5 % CO2 at 37 °C.
Multipotent adult stem cells were obtained from fibroblasts at early passages (SKIN-MASCs) from 4 healthy donors and 4 patients affected by NPC disease, as previously described [28 (link)]. The surface immunophenotype was determined using the following primary conjugated antibodies: CD13, CD49a, CD49b, CD49d, CD90, CD73, CD44, CD45, human leukocyte antigen-D related (HLA-DR), CD34, and CD271 (BD Biosciences, Franklin Lakes, NJ, USA); CD105 and kinase insert domain receptor (KDR; Serotec, Oxford, United Kingdom); and CD133 (Miltenyi Biotec, Bergisch Gladbach, Germany). The percentage of cells expressing all the antigens was determined by fluorescence-activated cell sorting (FACS) analysis (CyAn; Beckman Coulter, Brea, CA, USA). Properly conjugated isotype-matched antibodies were used as negative controls.