Anti cd107a

The ex vivo frequency of NK and NKT cell populations and expression of CD16 and CD107a (a degranulation marker) were determined using PBMCs from HC, HAM/TSP patients, and SN. Cells were stained with monoclonal antibodies (anti-CD3, anti-CD56, anti-CD16, and anti-CD107a) (eBioscience or R&D Systems) for 20 minutes at 4°C. PBMCs were washed with PBS and then fixed with 2% paraformaldehyde. Cells were then analyzed on the FacsCanto II flow cytometer (BD Biosciences, San Jose, CA). Analyses were performed using FlowJo software version 10 (Tree Star, Ashland, OR). Lymphoid cells were selected by size (SSC) and granularity (FSC) and then characterized as CD56⁺CD3⁻, CD56⁺CD16⁺, and CD56⁺ cells were further divided as CD56^dim or CD56^bright. These cells are herein called NK-like cells for having strong similarities with and core functions known to be associated with classical NK cells. To look specifically at NKT cells, we analyzed a subset of cells gated from lymphoid cells and then gated for CD3⁺ cells, classified as CD56⁺CD3⁺(CD16⁺/⁻). The gating strategy is shown in Figures 1 and 2. In this study, fluorescence minus one (FMO) was used as an internal experiment control for flow cytometry in the evaluation of CD107a expression, with the limit of fluorescence as a negative control.

For ILC function assays, spleen cells were stimulated for 4 h with plate bound anti-NK1.1 in the presence of 1 × protein transport inhibitor cocktail (PTIC) containing Brefeldin A/Monensin (eBioscience, Thermo Fisher Scientific) and anti-CD107a (eBio1D4B, eBioscience, Thermo Fisher Scientific) in complete Iscove's DMEM medium (Corning). Cells were incubated during stimulation at 37°C in 5% CO₂ for 4 h. Cells were then first surface stained then intracellularly stained to measure function. ILC phenotypes were measured directly ex vivo. Spleen cells were stained following procedures indicated below after fixable Live/Dead staining (Invitrogen).

Splenocytes and liver mononuclear cells were isolated as described previously [16 (link)]. Flow cytometric analysis was performed using BD FACSCalibur and FACSAria III instruments. Antibodies used in this study included fluorescein isothiocyanate (FITC)-labeled anti-mouse CD49b (DX5), anti-NK1.1, anti-CD4, and anti-CD11c; Phycoerythrin (PE)-labeled anti-mouse-CD69, anti-CD107a, anti-CD44, anti-CD86, and anti-IFN-γ; PE-cyanine 5.5-labeled anti-mouse CD3e and anti-CD8; and allophycocyanin (APC)-labeled anti-mouse CD314 (NKG2D), anti-CD69, anti-CD25, anti-CD80, anti-CD62L, and anti-TNF-α, these antibodies were obtained from eBioscience (San Diego, CA, USA). FITC-B540-labeled anti-CD3, APC-cy7-labeled anti-NK1.1, PE-YG582-labeled anti-CTLA4, PE-cy7-YG780-labeled anti-TIGIT, APC-R660-labeled anti-PD-1, V450-labeled anti-LAG-3, Percpcy5.5-B695-labeled anti-Tim-3, YG780-labeled anti-Granzyme B, and B540-labeled anti-perforin were obtained from Biolegend (California,USA) and BD (New York, USA). For the analysis of intracellular molecules, cells stained with anti-CD4 and anti-CD8 antibodies were fixed using Fix/Perm Buffer (eBioscience, San Diego, CA, USA) for 30 min, incubated with anti-mouse-IFN-γ and -TNF-α for 30 min at 4 °C, and then analyzed using flow cytometry.