Raltegravir

The details of HTLV-1-infected T-lymphocyte clones used in this study are shown in Table 1. All clones were derived as previously described [11 (link)] from peripheral blood mononuclear cells (PBMCs) of donors attending the National Centre for Human Retrovirology (NCHR) at Imperial College Healthcare NHS Trust, St Mary’s Hospital, London. The identification of genomic insertion sites by LMPCR was described elsewhere [31 (link)]. The cells were maintained in RPMI-1640 (Sigma, R0883) supplemented with L-glutamine, penicillin+streptomycin and 20% fetal bovine serum (Gibco, 10500–064) in 5% CO₂ at 37°C. IL-2 (Miltenyi Biotec, 130-097-745) was supplemented (100 unit/ml) into the culture twice a week. The integrase inhibitor raltegravir (Selleck Chemicals, MK-0518) was used at 10 μM throughout the culture to prevent secondary infection.

The HTLV-1-infected T-lymphocyte clones were derived by limiting dilution from peripheral blood mononuclear cells (PBMCs) of donors attending the National Centre for Human Retrovirology (NCHR) at Imperial College Healthcare NHS Trust, St Mary's Hospital, London. All donors gave written informed consent in accordance with the Declaration of Helsinki to donate blood samples to the Communicable Diseases Research Tissue Bank, approved by the UK National Research Ethics Service (15/SC/0089). The derivation of these clones and the genomic insertion site of the single-copy HTLV-1 provirus in each clone were previously reported (Cook et al., 2012 (link)). The cells were cultured in RPMI-1640 medium (Sigma-Aldrich) with added L-glutamine (Invitrogen), penicillin and streptomycin (Invitrogen), 10% AB human serum (Biowest) at 37˚C, 5% CO₂. IL-2 (Promokine) was added to the culture every 3 days, and the retroviral integrase inhibitor raltegravir (Selleck) was maintained at 10 μM throughout cell culture, to prevent secondary infection. In addition, the cells were activated every 14 days by the addition of beads coated with antibodies against CD2, CD3 and CD28 (Miltenyi-Biotech). All experiments were carried out on cells harvested on Day 9 of this cycle, after addition of fresh media on Day 8.

Clones 11.50, 11.63 and TBX4B [34 (link)] (Table 7) were cultured in RPMI-1640 supplemented with Penicillin/Streptomycin (100 U/ml and 100 μg/ml), L-Glutamine and 20% FCS (v/v), 100 U/ml IL-2 (Miltenyi Biotec, Cat. No. 130-097-745) and 10 μM raltegravir (Selleck Chemicals, MK-0518) at 5% CO₂, 37°C. Transduced lines were maintained in 1 μg/ml puromycin (Thermo-Fisher Scientific, Cat. No. A1113803). Replacement of half the culture media with fresh media was performed 2–3 times per week.