Pokeweed mitogen

Fluorescence in situ hybridization (FISH) was performed using the LSI PML-RARA dual color dual fusion rearrangement probe (Vysis/Abbott Molecular Inc., Des Plains, IL) to determine the involvement of PML gene in the t(9;15) translocation. Five-μm-thick tissue sections were cut for FISH analysis. SYK break apart was determined using FISH probe (BAC clones RP11-51G20 and RP11-555F9) and a reference probe, located at 9q22.2. At least 100 nuclei were evaluated per sample using a fluorescence microscope (Olympus BX51; Olympus Optical). PML FISH break-apart assay (locus 15q24.1; 74287013-74340155) was performed in the Cytogenetics Laboratory at WCM/NYP following standard operating procedures (Empire Genomics, Williamsville, NY). Cells were isolated by the Ficoll-Paque method from peripheral blood and grown for 3 days in culture media supplemented with 50 µg/mL Pokeweed mitogen (L8777, Sigma-Aldrich). Patient cells were cultured in Colcemide (10295892001, Roche) at 0.1 µg/mL for 1 h.

Peripheral blood mononuclear cells (PBMCs) were isolated from blood 5 days after the priming (W9) and the boost (W21 for group 1 and 2 or W29 for group 3), 4 weeks after the last immunization (W24, groups 1 and 2 or W32, group 3), and 3 months after the last immunization (W32, groups 1 and 2 or W40, group 3) and assessed for antibody-secreting cells (ASCs) by B cell ELISPOT assay. Macaques were sacrificed 3 months after the last immunization (W33, groups 1 and 2 and W41, group 3) and rectal and vaginal mononuclear cells (MNCs) were prepared by enzymatic digestion of tissues with collagenase II (0.3 mg/mL) in the presence of trypsin inhibitor (0.1 mg/mL) (Sigma-Aldrich) and DNase I (0.1 mg/mL) (Roche) in RPMI medium supplemented with 3% FBS, 25 mM Hepes, antibiotics and Fungizon^® according to supplier’s recommendations (Invitrogen, Saint Aubin, France). Bone marrow (BM) MNC from iliac crest or from humerus were isolated using Ficoll density-gradient centrifugation (MSL2000, Eurobio, Les Ulis, France). PBMC and tissue-derived MNC collected 1 or 3 months after immunization were stimulated in vitro for 6 days with Pokeweed Mitogen (PWM, 1 µg/mL) and Staphylococcus aureus Cowan strain (SAC, 0.1 µg/mL) (Sigma-Aldrich) before ASC enumeration.

Peripheral blood mononuclear cells (PBMCs) were isolated from blood by density-gradient centrifugation over lymphoprep (Asis-Shield Diagnostics). Cultured and ex-vivo ELISpot assays were conducted from the PBMCs to detect antigen-specific memory and PCs respectively, using a previously described protocol (Clutterbuck et al., 2012 (link)). Briefly, MultiScreen-IP 96-well plates (Millipore) were coated with 2.5 μg/ml of purified HBsAg (GSK). For enumeration of HBsAg-specific PCs, 200,000 PBMCs were placed in each well, and incubated overnight. Cells were then washed from the plate, and IgG antibody bound to the plate detected using the AP conjugate substrate kit (Bio-Rad). For enumeration of HBsAg-specific memory cells, PBMCs were first cultured in RPMI (Sigma-Aldrich) containing 1/10 NBBS, 1/5000 Staphylococcus aureus cowans strain, 1/6000 pokeweed mitogen (Sigma-Aldrich) and 1/40 CpG class B oligonucleotide (5′-tcgtcgttttgtcgttttgtcgtt-3′; InvivoGen) for 6 days to activate antibody secretion. 200,000 cultured cells were added to each well of the plate, and detected in the same way as the ex-vivo cells. Spot counting was performed automatically using the AID ELISpot Reader System (AID Autoimmun Diagnostika). For each sample, 6 wells were seeded in parallel, and the mean spot count taken.

Sorted blood DC subsets and monocytes were adjusted to 2.5 × 10⁵ cells/ml in cRPMI and a three-fold dilution series of each population was prepared. Allogeneic PBMCs were added (5 × 10⁵ cells/well) at responder to stimulator cell ratios ranging from 2:1 to 162:1. Pokeweed mitogen (Sigma, Poole, UK) and cRPMI were added to wells containing only PBMC as positive and negative controls, respectively. After 72 h incubation at 37 °C in a humidified 5% CO₂ atmosphere, cells were pulsed with 1 μCi/well ³H-thymidine (GE Healthcare, Little Chalfont, UK) and incubated for a further 24 h. Cells were harvested onto filter mats using a Harvester 96 Mach III (TomTec Inc, Hamden, USA) and ³H-thymidine incorporation measured by addition of 25 μl/well Microscint O and counting on a MicroBeta2 (link) Plate Counter (both Perkin Elmer, High Wycombe, UK).

Freshly isolated PBMCs were washed twice and resuspended at 2 × 10⁶/mL in a serum-free medium X-VIVO 10. Cultures were plated in triplicate at 2 × 10⁵/well in 7-day assays, in 96 round-bottomed microplates (TPP, Trasadingen, Switzerland). Cells were cultured in the absence or presence of Pokeweed mitogen 10 μg/mL (Sigma) and 5 μg/mL of HIV-1 recombinant proteins gp160 and p24 (Protein Sciences, Meriden, CT). Incorporation of tritium-labeled thymidine was assessed for the last 18 hours of culture (Betaplate LKB, Wallac, Sweden). Results were expressed as mean counts per minute (cpm). The stimulation index (SI) was calculated for each sample using the formula: SI = mean cpm for cells with stimulus/mean cpm for cells without stimulus. Positive antigen-specific responses were defined as >3,000 cpm and SI >3. For analytical purposes, results were expressed as ‘positive’ or ‘negative’ CD4 proliferative responses to HIV-1 P24 protein.

Murine IFN-γ ELISPOT assays were performed ex vivo as previously described [11 (link)]. The Granzyme B ELISPOT assay was performed similarly to the IFN-γ ELISPOT assay. For this assay, the anti-mouse Granzyme capture antibody (100 ng/well, AF1865; R&D Systems, Minneapolis, USA) and the biotinylated anti-mouse Granzyme detection antibody (50 ng/well, BAF1865; R&D Systems, Minneapolis, USA) were used. Splenocytes were seeded in triplicate in 2-fold serial dilutions from 200 000 to 25 000 cells per well. One of the triplicates was left untreated (negative control), the second received 200 ng of pokeweed mitogen/well (Sigma, Deisenhofen, Germany) in 2 μl of PBS (positive control), whereas the third received 0.2 μmol of H2Db-restricted HPV-16 E749-57 peptide in 2 μl of PBS/well (test sample). Spots of the negative control (untreated) were subtracted from the spot number in the corresponding test sample.