Anti cd8 fitc

Bronchoalveolar lavage (BAL) was performed as described [21 (link)]. In short, cytokines were determined by FlowCytomix assay and cells were stained for FACS analysis with the following markers: anti-CD19-PE/Cy7, anti-CD45-PerCP/Cy5.5, anti-CD4-APC/Cy7, anti-Gr1-APC (all BioLegend, London, UK), anti-CD8-FITC (eBioscience, Schwechat, Austria), and anti-CD25-PE (BD Biosciences, Schwechat, Austria). Red blood cells were lysed and cells were analyzed on a FACSCanto II flow cytometer (BD Biosciences, San Jose, CA). The eosinophil population was distinguished by CD45^medGr1^lowside-scatter^high phenotype. Neutrophils were identified as a CD45^high, Gr1^high cell population.

Cells (1 × 10⁶) were washed in PBS containing 0.5% BSA. To prevent nonspecific binding cells were incubated with 5% normal rat serum for 10 min at RT. Cells were then stained (45 min, on ice) with the appropriate fluorochrome-conjugated Ab (anti-CD11c-APC, anti-CD11b FITC, anti-CD86 PE, anti-PDCA-1 FITC, anti-Gr-1 PB, anti-MHC-II PE, anti-CD4 PB, anti-CD8 FITC, anti-CD25 PE, and anti-CD3 FITC Ab (eBioscience)). Cells were washed and analyzed using a LSRII cytometer (BD Biosciences). Data analysis was performed with the FlowJo software (version 5.7.2).

Splenocytes or lymphocytes were stained using anti-PD-L1 PE, anti-PD-1 PE, anti-CD4 FITC, anti-CD8-FITC and anti-CD107a-PE (eBioscience, San Diego, CA) as indicated by the manufacturer. Specific mAb isotype-matched control was also used. Sera from naïve and mice cured by combined anti-PD-1 and anti-CD4 mAb therapy were used to assess reactivity against Neuro2apc or NXS2pc cells at dilutions ranging from 1:10 to 1:10,000. A FITC-conjugated goat anti-mouse antiserum (Jackson Labs,West Grove, PA) was used as a second-step reagent. Anti-GD2 mAb supernatant (M36.1-S2a) (ATCC) was used as positive control.
Tumors from Neuro2a- and NXS2 bearing mice were dissociated mechanically and immune cells infiltrates were stained with anti-CD4 FITC, anti-CD25 APC, anti-LAG-3 PE, anti-Gr1 FITC and anti-CD11b PE (eBioscience, San Diego, CA) as indicated by the manufacturer. Samples were analyzed by a FACScan or FACSCalibur analyzer (Becton Dickinson).

IMQ was purchased from InvivoGen (SanDiego, CA, USA). 3-MA was purchased from Sigma-Aldrich (St. Louis, MO, USA). For western blot analysis, anti-β-actin, anti-LC3B, anti-Beclin-1 and anti-Atg5-12 antibodies were purchased from Cell Signaling Technologies (Danvers, MA, USA). Anti-phospho-Erk1/2, anti-Erk1/2, anti-phospho-p38, anti-p38 and anti-phospho-IκBα were purchased from Cell Signaling Technologies (Danvers, MA, USA). Anti-phospho-NF-κB p65, anti-NF-κB p65 and β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The other reagent sources were as follows: diphenyleneiodonium chloride (DPI) (Calbiochem, USA), apocynin (Sigma-Aldrich, USA), and DCFH-DA (Calbiochem, USA). Anti-CD4-FITC, anti-CD8-FITC, anti-CD11b-FITC, anti-FOXP3-Alexa 488 (126406), anti-Ly6C-APC, anti-CD45 (103124), anti-Ly6G-PE/Cy7 (15-5931-82), anti-TNFα-APC (17-7321-82), anti-IFN-γ-APC (17-7311-82), anti-Gr-1-Alexa 488 (108417) and anti-CD25-Alexa 647 (102020) antibodies were purchased from eBioscience (San Diego, CA, USA). For immunostaining, an anti-LC3 antibody was purchased from MBL International Corporation (Nagoya, Japan), and a FITC-conjugated anti-rabbit secondary antibody was purchased from Jackson Immuno Research (West Grove, PA, USA).

Resting PBMCs or purified NK cells obtained from healthy donors were co-incubated with or without 5 × 10⁸ exosomes for 24 hours, then cells were stained with anti NKG2D APC and anti-CD8 FITC or anti-CD16 FITC/anti-CD56 PE (all from eBiosciences) and tested by flow cytometry. In a separate set of experiments, SW480- and SW620-derived exosomes were pre-incubated for 15 min at 37 °C with 5 µg/ml of anti-MICA/B neutralizing antibody (R&D system) before adding to cells.

The anti‐CD69‐PE, anti‐CD8‐FITC, and anti‐CD3‐FITC were provided by eBioscience, anti‐human B7H3‐PE mAb, and mouse IgG1‐PE isotype antibody were provided by R&D System. The cells were analyzed by the flow cytometer (CytoFLEX, Beckman Coulter), and data were processed using the accompanying software (CytExpert, Beckman Coulter, BeiJing, China).