Cd40l

PBMC were suspended in culture medium (complete medium) for viability test with trypan blue (Gibco) and rested in an incubator overnight at 37°C, with 5% CO2. After counting, cells were suspended in culture medium and stimulated with 4μg/ml of GMZ2 vaccine antigen (Henogen S.A. Belgium) for 18h for the T cell panel using 1μg/ml of Staphylococcus Enterotoxin B (Sigma-Aldrich) as positive control, and 6h for the B cell panel using 10μg/ml CPG (Invivogen) + 50ng PMA (Thermo Fisher) + 1μg IONO (Thermo Fisher) as positive control. Unstimulated cells as negative control were incubated in culture medium alone. For the T and B cell panels, cells were cultured in the presence of anti-CD28/CD49d antibodies (BD Biosciences) and CD40L (BD Biosciences) respectively at a concentration of 1μg/ml. In order to assess Forkhead box P3 (Foxp3) expression and IL-10 production, 1μg/ml of Golgi Plug (BD Biosciences) was added two hours after PBMC stimulation. Each plate used for cell stimulation included PBMC isolated both on D0 and on D84.

T-cell lines were analyzed with monoclonal antibodies to: CD3, CD4, CD8, CD56/16, CD45RA, CD62L (Becton Dickinson). To detect CMV-pp65- and CMV-IE-1-specific T-cells in the T-cell lines, we used the soluble CMV-pp65 pentamers HLA-A*02-NLV, HLA-A*02-LQT, HLA-A*02-MLN, HLA-A*24-QYD, HLA-B*7-TPR, HLA-B*7-RPH, and HLA-A*01-YSE (prepared by Proimmune Inc). Tetramers for HLA-A*02-LQT, HLA-A*02-MLN, and HLA-B*35-DAN were first created by the Protein Core Facility of Baylor College of Medicine and the Dan L. Duncan Cancer Center. Functionality of the T-cells was assessed by the flow cytometric detection of intracellular accumulation of IFN-γ, granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 2 (IL-2), tumor necrosis factor-α (TNF-α), and CD40 ligand (CD40L) (all from BD Biosciences) as previously described. (48 (link), 49 (link)) Briefly, T-cells were stimulated with 1 μg/mL final concentration of each Pepmix; medium alone was used as the negative control. After incubation for 1 hour at 37°C, 10 μg/mL of Brefeldin A (Sigma) was added for an addition 5 hours. Cells were then washed, fixed, and analyzed using a FACSAria or LSRII flow cytometer (BD Biosciences). When possible at least 200,000 live events were acquired per tube. Data analysis was performed using FlowJo.

Fluorochrome conjugated anti-human mAb specific to HLA-A2, CD3, CD8, CD28, CD38, CD40L, CD69, ICOS, TCRαβ, CCR7, CD45RO, CD80, CD86, ICOS ligand, IFN-γ, CTLA-4, PD-1, PD-L1, PD-L2, T-bet, granzyme B or Akt were purchased from Becton Dickinson (San Diego, CA). Fluorochrome conjugated anti-human Eomes mAb was purchased from eBioscience (San Diego, CA). Recombinant human IL-2, IL-4, IFN-α and TNF-α were purchased from R&D Systems, and human GM-CSF was obtained from Immunex (Seattle, WA). Lenalidomide (Celgene, Summit, NJ) was reconstituted in 1% DMSO and was used at a final concentration of 5 μM to treat either XBP1-CTL or various tumor cell lines.