Il 21

PBMCs collected at T0 and T1 from HIV and HC were thawed and policlonally activated in vitro in complete RPMI medium (Invitrogen) supplemented with 2.5 μg/mL CpG type B (Hycult biotech), 20 ng/mL IL-4 (Peprotech) and 20 ng/mL IL-21 (ProSpec). Cells were harvested after 5 days of culture at 37°C. ELISpot 96-well filtration plates (Millipore) were coated with the addition of purified H1N1, H3N2, and B influenza inactivated virus particles and subsequently loaded with 2 × 10⁵ cells/well. Membranes were punched out with an Eli.Punch device and developed spots were scanned with an Eli.Scan and counted with the ELISpot Analysis Software V5.1 (all from A.EL.VIS).

Whole blood was first diluted at a ratio of 1:1.5 with either RPMI 1640 medium containing l-glutamine (2 mM) with antibiotics (100 IU/ml penicillin and 100 µg/ml streptomycin) (Life Technologies, Carlsbad, USA) alone or supplemented with the following cytokine cocktail: IL-2 (1000 IU/ml), IL-15 (10 ng/ml) and IL-21 (10 ng/ml) (Prospec, Ness-Ziona, Israel). Diluted blood was added in duplicates for each condition to 96-well plates pre-coated with the proteins EBNA-1 or CMV-pp65 (Prospec, Ness-Ziona, Israel) at a final concentration of 1 μg/ml and incubated for 7 days at 37 °C with 5% CO2 as previously described [26 (link), 27 (link)]. Antigen-free medium was used as negative control while phytohemagglutinin protein (PHA 5 μg/ml, Sigma Aldrich) was used positive control. Cell culture supernatants were harvested after the incubation period to quantify IFNγ production by sandwich ELISA (Mabtech, Stockholm, Sweden) according to the manufacturer’s instructions. Assay plates were analyzed using a Vmax kinetic microplate reader (Molecular Devices, USA) at 450 nm. After basal IFNγ production (medium only) subtraction, data were reported as absolute cytokine concentration values (pg/ml).

Plasma was removed following centrifugation of whole blood samples, and stored at -80°C. Glioma tumor tissue was harvested in the course of tumor surgery at the Department of Neuro-Oncology at Karolinska University Hospital, Stockholm. Tumor tissue was immediately transferred to Cellgro GMP Serum-free DC medium medium supplemented with 5% pooled human AB serum. Tumor tissue was dissected into fragments of approximately 1-2mm3 using a sterile scalpel, or processed into a tissue homogenate using the BD Medimachine. Tissue fragments of the cell suspension were washed twice with PBS and cultured in 24 well plates in Cellgro medium plus 5% pooled human AB serum supplemented with recombinant IL-2 (1000IU/ml), IL-15(10ng/ml) and IL-21 (10ng/ml) (Prospec, Ness-Ziona, Israel). Medium was changed as necessary. Irradiated (55Gy) feeder cells (allogeneic PBMCs) at the ratio of 1 (feeder cells):10 (TILs) was added on day 7. OKT3 (anti-CD3 monoclonal antibody, BioLegend, San Diego, CA) was used at 10ng/ml as TILs became visible under the microscope. TILs were transferred to 6-well plates; upon achieving > 70% confluence in the 24-well surface. They were further expanded in G-Rex flasks (Wilson Wolf, New Brighton, MN) using 30ng OKT3/mL and irradiated (55Gry) allogeneic feeder cells at the ratio of 1 (feeder cells):10 (TILs).