Interleukin 7 (il 7)

Every 3 weeks, 45 mL peripheral blood was drawn into EDTA tubes and peripheral blood mononuclear cells (PBMC) were isolated using Ficoll gradient centrifugation for each subject. PBMCs were thawed in RPMI1640 with 10% FBS and incubated for 7 days at 37°C, 5% CO₂. On day 1 and 4, the medium was refreshed and supplemented with 5 ng/mL IL7 or 5 ng/mL IL7 and 4 ng/mL IL2 (R&D Systems), respectively. After 7 days, PBMCs were harvested and rested for 16 hours, then seeded in 2 or 3 replicates (depending on sample availability) on IFNγ/Granzyme-B/TNFα FluoroSpot plates (Mabtech) at 100,000 to 150,000 cells/well in RPMI-10% FBS. Cells were incubated either with the RPMI-10% FBS medium, or the peptides (5 μg/mL), or peptide pools (5 μg/mL per peptide) overnight at 37°C, 5% CO₂. Development was performed according to the manufacturer's instructions. Results were acquired with a Mabtech IRIS reader and analyzed using the Mabtech Apex software. In vitro stimulated (IVS) ELISpot results were considered positive when after subtracting the corresponding nonstimulated control (ΔSFU), the result was >2.5-fold higher than the DMSO negative control. A response was considered “boosted” compared with pre-vaccination when at least two-fold increase in ΔSFU was achieved.

The differentiation experiments were performed as described previously,^{30 (link)} plating 1 x 10⁴ Lin⁻cKit⁺ hematopoietic progenitor cells per 24-well plates of OP9-DL1 feeders in OP9 culture medium supplemented with 5 ng/mL Flt3 ligand (R&D Systems) and 5 ng/mL IL7 (or 1-0.2 ng/mL IL7 where indicated) (R&D Systems or Peprotech).
OP9-DL1 bone marrow stromal cells^{30 (link)} were maintained in MEMalpha medium (Gibco), supplemented with 20% fetal bovine serum (FCS; Hyclone), penicillin (100 U/mL)-streptomycin (100 μg/mL), 2 mM L-glutamine (Gibco) and incubated at 37°C with 7% CO₂ and 95% humidity. E13.5 fetal liver cells were stained with biotin-conjugated lineage cocktail antibodies [Gr-1 (Ly-6G & 6C), Ter119 (Ly-76), CD3ε, and B220 (CD45R); eBioscience], PE-conjugated streptavidin (BD Biosciences, 1:200) and CD117-APC (cKit, Immunosource, 1:200). Lin⁻cKit⁺ cells were sorted using a FACSAria II machine (BD Biosciences) and FACSDiva software. Dead cells were discarded via DAPI stain.