Cd62l pe

CD3⁺ T, CD4⁺ T, CD8⁺ T, CD19⁺ B, and CD16⁺ CD56⁺ NK cells were stained using BD Multitest 6-color TBNK reagent in Trucount tubes. All anti-human antibodies were purchased from BD Biosciences: CD45-APC-Cy™7 (#557833), CD3-APC (#555342), CD4-FITC (#566802), CD8-Percp-cy5.5 (#565310), CD14-AF488 (#562689), CD16-PE (#561313), CD107a-APC (#560664), CD45RA-BV605 (#562886), CD62L-PE (#555544), CD197-BV421 (#566743). Total cells were Fc-blocked and stained with indicated combinations of antibodies for 30 min on ice, then washed three times and resuspended in 1% FBS/PBS. The flow cytometric data were collected on a BD Calibur flow cytometer and analyzed using FlowJo software or Summit software.

Eugenol, RPMI 1640 medium, M-199 medium, penicillin G sodium salt, streptomycin sulphate, 3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide (MTT), carboxyfluoresceinsuccinimidyl ester (CFSE), AmB, anti-mouse IgG and isotype antibodies, o-phenylenediaminedihydrochloride (OPD) were procured from Sigma-Aldrich (St Louis, MO, USA). Fetal bovine serum (FBS) was obtained from Gibco-BRL, DMSO from SRL, methanol from Merck, limulus amebocyte lysate (LAL) kit from Pierce, Thermo Scientific. Fluorochrome conjugated anti-mouse antibodies such as CD4-phycoerythin (PE), CD8-fluorescein isothiocyanate (FITC), CD80-allophycocyanin (APC), CD86-phycoerythin cyanine dye 7, APC-CD4 and PE-CD8, FITC-IFN-γ, CD8-APC, CD62L-PE and CD44-FITC, isotype controls and Brefeldin A and cytokine bead array kit (CBA) were procured from BD Pharmingen, USA. Aspartate aminotransaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), creatinine and urea kits were purchased from Span Diagnostics Ltd (Surat, Gujarat, India). Besides these, the analytical grade reagents were used.

Evaluation of T cell proliferation in immunized mice was conducted using a FACSCalibur flow cytometer (BD Biosciences, Milpitas, CA, USA). Briefly, a total of 1,000,000 mouse splenocytes were stimulated with the SARS-CoV-2 RBD peptides pool (4 μg/mL of each peptide) for 4 h at 37 °C with 5% CO₂. Brefeldin A (1 mg/mL, BD Sciences, Milpitas, CA, USA) was, then, added to the splenocytes and incubated for an additional 4 h. The splenocytes were washed twice with the PBS, and then stained with fluorescently conjugated antibodies to CD3-FITC (BD Pharmingen, San Diego, CA, USA), CD4 (PerCP-Cyanine5.5, BD Pharmingen, San Diego, CA, USA), CD8-PE-Cyanine7 (BD Pharmingen, San Diego, CA, USA), CD44-APC (BD Pharmingen, San Diego, CA, USA), and CD62L-PE (BD Pharmingen, San Diego, CA, USA). Zombie NIR™ Fixable Viability Kit (BioLegend, San Diego, CA, USA), whose stain has similar emission to APC/Cy7, was used to evaluate live or dead status of mammalian cells. Data were analyzed using FlowJo software (Version 10.8.1).

Panel 1 included antibodies allowing the gating of PMN-MDSCs in the PBMCs and the analysis of expression of surface markers. It comprised of CD3-BV510, CD56-BV510, CD19-BV510, CD11b-APCH7, CD15-BV711, CD14-BV650, HLA-DR-BV605, CD10-PE-CF594 (Becton Dickinson), CD16-AF700 (BioLegend, Ozyme, Saint-Cyr-l’Ecole, France), CD33-PC7 (eBiosciences, Thermo Fisher), LIVE/DEAD fixable aqua (Life Technologies, Villebon-sur-Yvette, France). LOX-1-PE, CD11b-activated-PE (clone CBRM1/5) (Bio-Legend, Ozyme), CD62L-PE, CD66b-PE, PDL-1-BV421, CXCR1-PE, CD172ab-AF647 (BD Biosciences) were alternately included in Panel 1.
Panel 2 was used for functional analysis. It contained CD15-FITC (eBiosciences, Thermo Fisher), CD3-PC5 (BD Biosciences) and LIVE/DEAD near-IR (Live Technologies,). CD66b-PE, CD10-PE-CF594 and CD16-AF700 were occasionally added. PBMCs were stained 20 min at room temperature with fluorescent reagents pre-mixed in PBS, washed, then fixed with 4% paraformaldehyde and analyzed by FACS within 4 days on LSRII-SORP cytometer (BD Biosciences) equipped with four lasers (405 nm/100 mW, 488 nm/100 mW, 560 nm/50 mW and 630 nm/40 mW). PMT were set using unstained and fully stained samples. Compensations were performed with beads stained with corresponding reagents. Data were exported and analyzed with FlowJo (version 9-2, MacOS X).

For the eight elderly donors, PBMCs were isolated from fresh blood samples using density gradient centrifugation with Ficoll (GE Healthcare). CD3⁺ T cells were enriched from PBMCs by immunomagnetic selection using CD3 MicroBeads (Miltenyi Biotec, Auburn, CA). Cells were stained in the dark for 15 min with the following anti-human Abs: CD45RO PE-Cy7 (BD Biosciences, San Jose, CA), CD3 Alexa Fluor 700 (BD Biosciences), CD62L-PE (BD Biosciences), CD45RA-allophycocyanin (BD Biosciences), CD8-Pacific Blue (BD Biosciences), CD4 allophycocyanin-Cy7 (BD Biosciences), and LIVE/DEAD Aqua fluorescent reactive dye (Invitrogen, Grand Island, NY).
CD8⁺ T cell subsets were isolated using the BD FACSAria cell-sorting system (BD Biosciences), including CD8⁺CD45RA⁻CD45RO⁺ (for CD8⁺ memory), CD8⁺CD45RA⁺CD45RO⁻CD62L^hi (CD8⁺ naive), and CD8⁺CD45RA⁺CD62L^lo/− (CD8⁺ T_EMRA). FlowJo (TreeStar, Ashland, OR) analysis was used to determine the proportions of the different subsets as a fraction of total CD8⁺ T cells.

Tumor cells and T cells were phenotyping with CD3‐APC (OKT3, UCH1), CD19‐APC CY7 (SJ26C1), CD4‐PE Cy7 (RPA‐T4, A161A1), CD8‐pacific blue (RPA‐T8, SK1), CD10‐APC (HI10a), CD22‐PE (HIB22), CD38‐PECy7 (HTT2), CD56‐PE CY7 (MEM‐188), CD62L‐BV510 (DREG‐56), PDL1‐PE (29E.2A3), PD1‐FITC (EH12.2H7), streptavidin‐APC‐Cy7, and 7AAD, which were purchased from BioLegend. CD20‐FITC (2H7), CD45RA‐FITC (HI100), and CD62L‐PE (DREG‐56) were purchased from BD. CD137‐PE, CD34‐APC, and antibiotin‐PE were purchased from MACS. The percentage of CAR‐CD19 was determined by biotinylated Erbitux. All cells were analyzed MACSquant with a filter set for eight fluorescence signals and analyzed with FlowJo software (Tree Star).