Anti ifn γ pe

1*10⁵ MUTZ-DC were cultured overnight in the presence of the maturation-inducing cytokine cocktail with different concentrations of a 25-mer MART-1 synthetic long peptide (SLP) (aa16-40L), which was synthesized in-house, as described previously [22 (link)]. Next, loaded MUTZ-DC were harvested and cultured with a MART-1_aa26-35 recognizing cytotoxic T cell line (>95% pure by dextramer binding analysis) for 5 hours, in the presence of 1 μl/ml of the golgi inhibitor GolgiStop (BD Biosciences). The cells were subsequently washed and stained with MART-1_aa26-35 dextramer (Immudex, Copenhagen, Denmark) for 15 minutes, followed by 15 minutes of labeling with CD3 Horizon and CD8 (BD Biosciences). After washing, the cells were fixed and permeabilized using BD Cytofix/Cytoperm solution (BD Biosciences), following manufacturer’s protocol. The intracellular IFNγ levels were determined by staining for 30 minutes at 4°C with anti-IFNγ-PE (BD Biosciences), after which the cells were washed and IFNγ production was analyzed using flow cytometry (LSRFortessa, BD Biosciences), as a measure of activation.

IFN-γ ELISPOT and cytometric bead array (CBA) assays were carried out as previously described [37 (link)] and intracellular cytokine staining and staining of cell surface molecules was carried out as previously described by Burgers et al., 2006 [47 (link)]. The following antibodies were used; anti-CD3⁺ Alexa 700, anti-CD4⁺ PE-Cy7, anti-CD8⁺ APC-Cy7, anti-CD62L APC, and anti-CD44 FITC). The cytokine antibodies were all PE-conjugated (0.2μg anti-TNF-PE, 0.06 μg anti-IL-2-PE, 0.06 μg anti-IFN-γ-PE) and were obtained from BD biosciences. The anti-CD4⁺ PE-Cy7, anti-CD8⁺ APC-Cy7 were obtained from BD biosciences. All the other antibodies were obtained from eBioscience.

Activated T cells were re-stimulated with 20 nM phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) and 1 μM ionomycin (Sigma-Aldrich). After incubation for 1 h at 37 °C, a protein transport inhibitor Brefeldin-A (Invitrogen) was added, followed by 4-h incubation. Surface Fc receptors were blocked for 15 min at 4 °C using Human TruStain FcX™ (Biolegend). Cells were stained with anti-CD8-APC/Fire™750, fixed with BD Cytofix/Cytoperm (BD Biosciences) and stained with anti-CD4-AF488 (BD Biosciences), anti-IFN-γ-PE (BD Biosciences), anti-TNF-α-BV650 (BD Biosciences), and IL-2-APC (eBioscience). Cells were acquired on the NovoCyte flow cytometer and analyzed using FlowJO v10.

Purified murine B cells (5 × 10⁵ cells/well) were stimulated with ArtinM (0.312–5.0 µg/mL), PMA (50 ng/mL) plus ionomycin (1 µM), or culture medium alone. After 12 h of incubation at 37 °C, the protein transport inhibitor was added (BD GolgiStopTM; 1 µL every 1.5 mL of the culture), and after 12 h the cells were washed and fixed/permeabilized with BD Cytofix/CytopermTM Plus (20 min at 4 °C). The cells were washed with wash buffer (BD Perm/WashTM buffer) and resuspended in Perm/WashTM buffer with anti-IFN-γ PE (BD Pharmingen™) and anti-IL-12 FITC (BD PharmingenTM) antibodies. After 40 min of incubation at 4 °C, the cells were washed and resuspended in Perm/WashTM buffer, and the frequency of positive B cells for IL-12 and IFN-γ (IL-12+/IFN-γ+) was measured using flow cytometry (Guava EasyCyte™ Mini System).

Intracellular staining for the cytokines Interferon-γ (IFN-γ), Tumor necrosis factor α (TNF-α) and IL-2 was performed as originally mentioned by Jung et al. [59 (link)]. Briefly, cells after one week MLPC were restimulated with appropriate antigens in the presence of Brefeldin A for six hours (Biolegend, San Diego, CA, USA). Cells were harvested for live-dead NEAR (Invitrogen, Thermo Fisher Scientific, Waltham, MA USA) and surface marker staining followed by fixation using paraformaldehyde (PFA, Sigma Aldrich). For surface marker staining the following antibodies were used: CD3 Alexa fluor700 (Biolegend, Fell, Germany), CD8 Peridinin chlorophyll (PerCP, Biolegend), CD197 (CCR7) phycoerythrin Cy7 (eBioscience, San Diego, CA, USA) and CD45RA fluorescein isothiocyanate (FITC, BD Biosciences, Heidelberg, Germany). After permeabilization cells were blocked by FcR blocking reagent (Miltenyi Biotec) then stained for intracellular cytokines using anti-IFN-γ PE, anti-TNF-α Pacific Blue and anti-IL-2 AmCyan antibodies (BD Biosciences, Heidelberg, Germany). Samples were measured with the flow cytometer BD LSR II and analyzed using the BD FACSDiva analysis software (BD Biosciences).