Golgi stop and golgi plug

Mouse and human cell suspensions were incubated with LPS EB-ultrapure from E. coli O111:B4 (1 μg/mL, In vivogen) or CpG-ODN 2395 (5μM, Invivogen) at 37°C for 4 hours in presence of Golgi stop and Golgi plug (BD Biosciences) and then assessed for cytokine expression by intracellular staining.

The iNKT kinetics assay was set up with 2 million PBMCs in 1 ml R10 per condition in a 24-well plate. PBMCs from each donor (n = 4) were stimulated with 1 μg/ml α-Galactosyl Ceramide glycolipid (α-GalCer, also known as KRN7000, Cayman Chemicals, USA). PBMC activation was assessed at 2 h, 4 h, 6 h, 8 h, and 10 h post-stimulation. Further, PBMCs were stimulated for 10 h, centrifuged for 5 min (1500 rpm) and resuspended in R10 for a rest period of 2 h, 4 h and 6 h, respectively. The negative control was an unstimulated ex vivo control and positive control was a 6 h stimulation using Phorbol 12-myristate (PMA—25 ng/ml) and 13-acetate plus ionomycin (Io—500 ng/ml) (Sigma Aldrich, USA). Prior to stimulation, all conditions and controls were pre-stained with the anti-Vα24-Jα18 TCR antibody (6B11, Biolegend, USA), as short-term iNKT stimulation causes iNKT TCR internalization^{21 (link)}. Addition of Golgi stop and Golgi plug (BD Biosciences) which allowed intracellular IFN-γ accumulation, were added 2 h, 1 h, and 30 min post-stimulation in the 8 h and 10 h timepoints, 4 h and 6 h timepoints, and 2 h timepoint, respectively. iNKT cell IFN-γ production was the primary outcome. At the end of each timepoint, cells were stained for flow cytometry.

K562 cells devoid of MHC-I stably (ATCC) expressing HLA-E^*0101 (K562-E^*0101; Applied Biological Materials Inc.) cells were incubated with 50 µM of SIVmac_239/251 Env peptide (NQLLIAILL) at 26 °C for 15–20 h^{7 (link)}. Isolated NK cells were cultured for 6 h in the presence of an anti-CD107a-PE-Cy5 mAb (clone HA43, 5 μL, cat. 555802; BD Biosciences), either alone (NK), or co-cultured at a 5:1 ratio with 2 × 10⁴ K562-E^*0101 cells that were either unpulsed (NK+K562^*E) or loaded with SIVmac_239/251 Env peptide (NK+K563^*E+ENV). GolgiStop and GolgiPlug (BD Biosciences) was added 1 h following culture initiation. The frequency of NK cells expressing surface CD107a was measured by flow cytometry per each culture condition to assess the background (%CD107a⁺_NK), the maximum (%CD107a⁺_NK+K562*E), and the peptide-specific degranulation activity (%CD107a⁺_{NK+K562*E+ENV}; Supplementary Fig. 6). From these measurements the Env-specific NK cell activity was calculated as previously described^{30 (link),55 (link)}: ${\log }_{10}\left[100\times \frac{\%\mathrm{CD}107{\mathrm{a}}_{\mathrm{NK}+\mathrm{K}562*\mathrm{E}+\mathrm{ENV}}^{+}-\%\mathrm{CD}107{\mathrm{a}}_{\mathrm{NK}}^{+}}{\%\mathrm{CD}107{\mathrm{a}}_{\mathrm{NK}+\mathrm{K}562*\mathrm{E}}^{+}-\%\mathrm{CD}107{\mathrm{a}}_{\mathrm{NK}}^{+}}\right]=\mathrm{Env\; -\; specific}\mathrm{NK}\mathrm{cell}\mathrm{activity}$

Most T cell responses were evaluated by using an IFN-γ ELISPOT Set from BD-Biosciences as described (30 (link)). In brief, spleens were removed, homogenized, erythrocytes were lysed and cells (8×10⁵/well) were stimulated with different peptide concentrations (10-0.01 μM). To analyze recognition of neoantigen-presenting tumor cells, irradiated (200 Gy) MC703 cells (8×10⁴ cells/well), transduced with retroviruses encoding neoantigens or with control vector, were cocultured with splenocytes (8×10⁵/well) obtained from immunized mice. In all cases, 24 h later ELISPOT plates were developed and spot-forming cells were counted with an ImmunoSpot automated counter using Immunospot Image Acquisition 4.5 and Immunospot 3 software.
In some cases, T cell activation was determined by flow cytometry. Splenocytes were stimulated for 4 hours with peptides (10 μM) in the presence of GolgiStop and GolgiPlug (BD-Biosciences) and antiCD107a-FITC (BD-Pharmingen). Next, they were stained with anti-CD3ϵ-Percp-Cy5, CD4-FITC and CD8-BV421 (BioLegend). Then, cells were fixed and permeabilized with BD Cytofix/Cytoperm™ Fixation/Permeablization Kit and intracellularly stained with IFNγ-PE and TNFα-BV510 antibodies (BioLegend). Dead cells were excluded from the analysis using Maleimide (PromoKine). Samples were acquired with a Cytoflex cytometer (Beckman Coulter) and data analyzed using FlowJo software.