Megacd40l protein

Antigen-loaded MDC1 from the 4 different groups described above (Empty, Gag, Network, and Epigraph) were counted and plated separately at a concentration of 7.5 × 10⁴ MDC1 per well in a 24-well plate. Bulk T cells were negatively selected using the EasySep™ Human T Cell Enrichment Kit (STEMCELL Technologies, Cambridge, MA), and 7.5 × 10⁵ T cells were added per well to the MDC1-containing wells (MDC1 to T cell ratio = 1:10). After an incubation of 45 min at 37 °C, soluble rhCD40L was added at a concentration of 0.25 μg/ml (MEGACD40L® protein, ENZO Life Sciences, Farmingdale, NY). After a 4 to 5 d culture period, rhIL-2 (250 IU/ml) and rhIL-7 (10 ng/ml) were added to the cultures and every 3 d thereafter. At day 21 in culture, each MDC1-stimulated T cell culture was tested for responses against their respective afferent (14–21mer) or efferent (9–13mer) Gag, Network, or Epigraph stimulator peptides as determined by IFNγ ELISpot and by CD107a flow cytometry staining where specifically mentioned. The non-peptide treated (Empty) T cell cultures were also tested for responses to each of the stimulator peptide groups and served as additional non-specific activated T cell controls.

Total CD8⁺ T cells were isolated from cryopreserved PBL by negative magnetic bead separation using an EasySep™ Human CD8⁺ T Cell Enrichment Kit (STEMCELL Technologies Cat# 19053). To induce CTL responses as previously described [7 (link)], CD8⁺ T cells were cocultured with autologous differentially matured DC loaded with either HLA-A2-restricted Gag p24 Gag_151–159 9-mer peptide epitopes when using HIV-1-negative blood donors, or Gag p17/p24 overlapping 15-mer peptides (1 μg/mL, Sigma-Aldrich) when using HIV-1-infected MACS participants. The cocultures (75,000 DC: 750,000 CD8⁺ T cells) were treated with or without the addition of either 25,000 gamma-irradiated (5000 rad) CD40L-transfected J588 cells [30 (link)] or MEGACD40L® Protein (0.25 μg/mL; Enzo Life Sciences Cat# ALX-522-110-C010) where stated. On day 5, rhIL-2 (250 U/mL) and rhIL-7 (10 ng/mL) were added to the cultures and every three days thereafter. On day 12, T cell cultures were restimulated with either gamma-irradiated HLA-A2⁺ T2 cells (for induction of primary CTL responses in HLA-A2⁺ HIV-1-negative donors) or differentially matured autologous DC loaded with autologous 9-mer peptides (1 μg/mL) corresponding to the viral antigens and DC type used in the initial stimulation. Antigen-specific readout assays were performed between days 20–24 to assess CTL activity.

For naïve B cell activation and infection experiments, we obtained buffy coats from anonymous donors through the Catalan Blood and Tissue Bank (CBTB). The CBTB follows the principles of the World Medical Association (WMA) Declaration of Helsinki. Before providing the first blood sample, all donors received detailed oral and written information and signed a consent form at the CBTB. PBMCs were isolated using Ficoll-Paque gradient centrifugation. Total B cells were isolated from PBMCs using positive selection with MACS CD19 microbeads (Miltenyi Biotec). Next, cells were stained with CD27-APC (Miltenyi Biotec, clone M-T271) and IgD – PE (SouthernBiotech, Cat. No. 2032–09) and naïve B cells were sorted as CD27^–IgD⁺. For EBV infection, pure naïve B cells were incubated with B95–8 cell supernatant for 3 h at 37ºC in order to infect them with EBV, they were harvested after 30 days. For BCR activation, pure naïve B cells were cultured in RPMI medium 1640 GlutaMAXTM-1 (Gibco, Life Technologies) containing 20% fetal bovine serum (Gibco, Life Technologies), 1% sodium pyruvate and 1% Penicillin Streptomycin (Gibco Thermo Fisher Scientific, MA, USA). Cells were activated with 10 μg/ml anti-IgM (SouthernBiotech), 0.1 μg/ml MEGACD40L® Protein (Enzo) and 50 ng/ml IL21 (Tebu-bio) for 24 h (ChIP) or 7 days (bisulfite pyrosequencing).