Interleukin 2 (il 2)

PBMCs were isolated from leucocyte cones of healthy adult donors by density centrifugation using a Ficoll gradient. The PBMC layer was carefully removed and washed to remove platelets. CD4+ and CD8 + T cells were subsequently isolated by negative selection using either a CD4 + T cell isolation kit (130-096-533; Miltenyi Biotec) or a CD8 + T cell isolation kit (130-096-495; Miltenyi Biotec). Cells were maintained in RPMI-1640 Medium supplemented as per Jurkat cells. Primary cells were maintained in 37 °C, 5% CO₂ tissue culture incubators. To stimulate T cells to induce receptor expression, 48-well plates were coated with poly-L-lysine (0.01%; P8920; Sigma-Aldrich) for 10 mins, washed 3 times with ddH₂O, and then coated with 5 ug/mL OKT3 (produced in house; RRID:AB_467057) and 1 ug/mL CD28.2 (16-0289-81; RRID:AB_468926; Thermo Fisher Scientific) in DPBS (D8537; Sigma-Aldrich) for 1 h, washed 3 times in DPBS before cells were plated at 1 × 10⁶/mL and supplemented with 200 U/mL interleukin-2 (Roche). Cells were used on day 3.

Avidity levels reported in the literature for TCR interactions with the relevant peptide/HLA-A2 complexes were confirmed as previously described by serial dilution of the peptides in cell-mediated cytotoxicity assays
[21 (link)]. Briefly, cytotoxic T cells (CTL) specific for the peptide were generated by incubating 5 × 10⁶ PBMC from a reactive subject in a small volume of medium for 1 hr with 100 μM peptide and then culturing the cells at 2.5 × 10⁶/mL in medium supplemented with 25 ng/mL recombinant interleukin-7 (R&D Systems). After 3 days, interleukin-2 (Roche) was added to 10 U/mL and the cells expanded for a further 7 days. The effector cells were first tested against autologous or HLA-A2 matched ⁵¹Cr-labelled BLCL pulsed for 1 hr with 10 μM peptide as in reference
[20 (link)] to determine an appropriate effector-to-target (E:T) ratio and then were tested against target cells pulsed with a serial dilutions of the test peptide beginning at 1 μM and progressing through 5 fold dilutions to < 0.02 nM. The avidity of the TCR/peptide-Class I HLA complex interaction was determined by graphing percent specific cytotoxicity against peptide concentration and estimating the concentration of peptide at which specific cytotoxicity fell to 50% of maximum cytotoxicity observed.

Virus was produced by transiently transfecting 293T cells with replication-competent HIV-1 proviral clones. Virus-containing supernatants were passed through 0.45-μm filters, normalized for p24 antigen with an HIV-1 p24 enzyme-linked immunosorbent assay (ELISA) kit (PerkinElmer or XpressBio), and used to infect target cells. T lymphoid cells (2 × 10⁵) were infected in T25 flasks in 5 ml medium. PBMC were isolated from the blood of healthy donors by Ficoll-Hypaque density gradient centrifugation, seeded into 6-well plates at a density of 10 × 10⁶ cells/well in 4 ml medium, and immediately infected at a p24 concentration of 0.25 ng/ml. On day 4 after infection, the cells were stimulated with 1 μg/ml PHA (Sigma). The next day, the PHA-containing culture medium was replaced with medium containing 10 U/ml interleukin 2 (Roche Applied Science). Alternatively, PBMC were prestimulated with 1 μg/ml PHA immediately after isolation and infected 24 h later in the presence of 10 U/ml interleukin 2. Virus replication was examined by comparing Gag protein expression levels in infected cells by Western blotting using anti-CA antibody 183-H12-5C and by measuring p24 antigen in the culture supernatants by a p24 ELISA.

Peripheral blood leukocyte cones from healthy human donors were acquired from the National Health Service blood service under ethics license REC 05/Q0401/108 (University of Manchester). Peripheral blood mononuclear cells (PBMCs) were isolated using density gradient centrifugation with Ficoll Paque (GE Healthcare). NK cells were isolated by negative selection (NK Cell Isolation Kit; Miltenyi Biotec) and cultured in DMEM with 30% Ham’s F-12, 10% human serum (Sigma), 1% nonessential amino acids (Sigma), 1 mM sodium pyruvate (Sigma), 2 mM L-glutamine, 50 U/mL penicillin–streptomycin, and 50 μM 2-mercaptoethanol (Gibco). Media were supplemented with 200 U/mL interleukin-2 (Roche) on day 0, and NK cells were used in assays on day 6.

The CD4⁺ T-cell lines Jurkat (from acute lymphoblastic leukemia) [43 (link)], Molt-4 [44 (link)], MT-2 (HTLV-1 in vitro transformed) [45 (link)], Tesi (Tax-1-transformed) [46 (link)], the Jurkat-derived cell line SVT35 (carrying an NF-κB-driven CD14 reporter gene) [47 (link)], and the HTLV-2-transformed T-cell lines MoT [48 (link)] and C3-44-Mo [49 (link)] were cultured in RPMI 1640M with L-glutamine (0.35 g/L), penicillin/streptomycin, and 20% (Tesi) or 10% (all other cell lines) fetal calf serum. For cultivation of Jurkat, SVT35, Molt-4, and Tesi, media were supplemented with 45% Panserin 401 (PAN-Biotech, Aidenbach, Germany), and Tesi cells were additionally cultured in media supplemented with 40 U/mL interleukin-2 (Roche Diagnostics GmbH, Mannheim, Germany).