Rhil 7

Beginning with culture Step 2 (d2 of overall culture), rhIL-7 (Miltenyi Biotec premium grade, Auburn CA, #130-095-364) was added at an optimized final concentration of 50 ng/ml to fresh medium added at culture splits. Other agents tested in Step 2 but absent from the optimized formulation included rhIL-2 (aldesleukin, Prometheus Labs, San Diego, CA, final concentration 24 IU/ml); rhIL-15 (Peprotech #200-15, final concentration 5 ng/ml) and rhIL-21 (Peprotech #200-21, final concentration 3 ng/ml).

Human peripheral blood mononuclear cells (PBMCs) derived from blood discard kits of healthy donors (BloodworksNW) were isolated by Ficoll-Paque (Pharmacia Biotech). CD4⁺ and CD8⁺ cells were purified from the PBMC using RoboSep (Stemcell). In some instance, the CD4^-, CD8^- PBMC fraction was retained. CD4⁺ T cells were cultured in RPMI with 10% FBS, 5 ng/ml rhIL-7 (Miltenyi) and 0.5 ng/ml rhIL-15 (Miltenyi). CD8⁺ cells were cultured in RPMI with 10% FBS, 50 U/ml rhIL-2, (Chiron Corporation) and rhIL-15 (0.5 ng/ml). In some instances, CD4⁺ or CD8⁺ cells were cultured in 50 U/mL rhIL-2, 20 ng/mL IL-4 (Milteyni), 10 ng/mL IL-7 (Milteyni) and 20 ng/mL Il-21 (Milteyni). Medium was replaced twice a week.

DCs were generated in vitro as described [66 (link)]. The L-selectin CD62^low fraction of T-cells (effector) was isolated from lymph nodes and spleens of immunized mice. The isolated cells (2 x 10⁶/ml) were activated with antigen-pulsed DCs at T-cell to DC ratio of 8:1. Two days later, the T-cells were split (1:2) with CM, 1% MS, rhIL-2, 24 IU (4 Cetus units)/ml (Chiron); rhIL-7, 50 ng/ml (Miltenyi Biotech); and rhIL-15, 5 ng/ml (Peprotech). The cells were further stimulated with rhIL-2, 24 IU (4 Cetus units)/ml on days 5, 8 and 11. The (IFN-γ) ICC assay was performed on days 7 or 14.

Retrovirally transduced T cells were produced and expanded as previously described (11 (link)). Briefly, after isolation, PBMCs were stimulated with 600 IU/mL of recombinant human interleukin-2 (rhIL-2; Miltenyi Biotec, 130-097-745) and 50 ng/mL of anti-CD3 (eBioscience, 16-0037-81) in AIM-V (ThermoFisher Scientific, 12055091), 2% (v/v) human AB serum (Sigma-Aldrich, H6914) for 48 h, before transduction with a MP-71 vector containing the Vα34 and Vβ3 chains of the HBs183-91-specific (Ts) or the HBcore18-27-specific (Tc) TCR sequences. Bulk transduced T cells were expanded and restimulated using 0.5–1 × 10⁶ TCR-transduced T cells, 2 × 10⁵ irradiated (2,500 rads) T2 cells pulsed with 1 µg/mL of either HBs183-91 peptide (FLLTRILTI) or HBcore18-27 (FLPSDFFPSV) (Genscript, custom synthesized peptides) and 1.8 × 10⁶ irradiated PBMCs as feeders. Cells were cultured for about 2 weeks in AIM-V, 2% human AB serum supplemented with 100 IU/mL of rhIL-2, 10 ng/mL of rhIL-7 (Miltenyi Biotec, 130-095-362), and 10 ng/mL of rhIL-15 (Miltenyi Biotec, 130-095-764). In some experiments, CD8⁺ T cells were enriched through negative selection of transduced T cells using CD4 microbeads (Miltenyi Biotec, 130-045-101) following the manufacturer’s instructions.

Splenocytes of naïve C57BL/6-Thy1.1+ mice were pre-activated by 2 mM/mL Concanavalin A (ConA) (Sigma) in T cell media, RPMI 1640-GlutaMAX supplemented with 10% FBS, 1x NEAA, 1 mM sodium pyruvate, 10 mM HEPES, 50 μM β-Mercaptoethanol, 50 IU/mL Penicillin and 50 μg/mL Streptomycin (all Gibco), in the presence of 450 IU/mL rh IL-7 and 50 IU/mL rh IL-15 (both Miltenyi). 24 h after activation, cells were gently spun down (1h, 37°C, 300 x g) and incubated on MLV-E-pseudotyped gamma-retroviral vector pre-coated-RetroNectin-plates (Takara). After additional overnight cultivation, spin-down transduction was repeated on freshly coated plates with viral particles. 72 h after initial pre-activation, ConA was removed from culture and lymphocyte layer was isolated by Ficoll-Hypaque (Amersham Biosciences) density gradient centrifugation. Non-transduced T cells used as control for some experiments underwent the same ConA-activation procedure. Transgene expression on transduced murine T cells were measured via flow cytometry.