Cd80 fitc

We used the following antibodies: CD4-PErCP (100538), CD8-APC/Cy7 (100713), CD69-FITC (104506), CD25-APC (101910), IFNγ-APC (505810), IL17-PE/Cy7 (506922), CD11b-APC/Cy7 (101226), CD11c-APC (117310), CD80-FITC (104706), MHCII-PE (107607), CD3-PacificBlue (100214), CD4-PE (100512), CD4-APC (100516) and CD4-FITC (100510) from Biolegend, USA.
SIRT2 (ab211033), β-actin (ab8227), acH3K18 (ab40888), α-tubulin (ab184613), acetyl-α-tubulin (ab179484), acetyl-NFκB p65 (ab19870), Alexa Fluor 647 (ab150075) and IgG-APC isotype control (ab232814) from Abcam.
ERK1/2 (9102), Phospho-ERK1/2 (9101), p-38 (9212) and phospho-p38 (4511) from Cell Signaling Technology.

Spleens were removed from sacrificed mice according to Kalli et al.^{45 (link)}. Frequency of memory T lymphocytes was assessed ex vivo on splenocytes stained by the following conjugated mAbs: CD3-BV510 (BD PharMingen, Haryana, India), CD4-APC-eFluor 780, CD8-PE-Cy7, CD44- PE (Thermo Scientific,Waltham, MA, USA). To assess ex vivo the percentages of non-lymphoid APCs (monocytes and DCs), splenocytes were labelled with the following conjugated mAbs: CD80- FITC (Biolegend, San Diego, CA, USA), HLA-DR-PE, CD86-APC (Thermo Scientific,Waltham, MA, USA). The samples were analyzed by a FACS Canto II flow-cytometer (BD Bioscience, Frankin Lake, NJ, USA) using FACS DIVA software. In both analyses, splenocytes were stained with Live/dead fixable violet dead cell stain (Thermo Scientific,Waltham, MA, USA) to exclude dead cells. Statistical analyses were performed by the Mann-Whitney unpaired T test for nonparametric measures using the GraphPad Prism 4.0 Software (GraphPad Software, Inc, La Jolla, CA).

Surface antigen phenotyping of WJ-MSCs was assessed using flow cytometry (FACSCalibur™, BD Biosciences, USA) and analyzed with CellQuest Pro software. The cells were stained with anti-human antibodies labeled with phycoerythrin (PE), allophycocyanin (APC), fluorescein isothiocyanate (FITC), or PerCP. The specific antibodies used for staining included CD73-PE, CD44-FITC, CD29-PE, CD166-PE, CD45-FITC, CD34-PE, CD14-FITC, CD79a-APC, HLA-DR-PerCP (BD Pharmingen™, BD Biosciences, USA), CD105-APC (eBioscience™, ThermoFisher Scientific, USA), CD90-FITC, CD31-PE, HLA-ABC-APC, CD80-FITC, CD40-PE, and CD86-APC (Biolegend®, BioLegend Inc., USA). Before and following staining, samples were washed with 1× PBS. Isotype antibodies from the same manufacturers for mice or rats were used as controls.

To detect the in vivo immune response, 4T1 xenotransplant tumors were divided into eight groups and received treatments identical to in vivo therapeutic evaluation. On the 9th day, both primary tumors and distant metastases were harvested for single-cell suspensions fabrication. The prepared cells were further stained with CD11c-FITC (eBioscience, Catalog: N418), CD86-PE (eBioscience, Catalog: GL1), CD80-APC (eBioscience, Catalog: 16-10A1), F4/80-APC (Biolegend, Catalog: BM8), CD11b-PE (Biolegend, Catalog: M1/70), CD80-FITC (Biolegend, Catalog: 16-10A1), CD206-FITC (Biolegend, Catalog: C068C2), CD3-FITC (Biolegend, Catalog:17A2), CD8a-APC (Biolegend, Catalog: QA17A07) and CD4-PC5.5 (Biolegend, Catalog: RM4-5) antibody and then analyzed by flow cytometry. Serum was collected from different groups of mice, and cytokines including IL-6, IL-12, TNF-α and IL-10 were analyzed using ELISA kits according to the manufacturer's protocols. In addition, immunofluorescence staining was further conducted to investigate the infiltrating immune cells in tumor tissues.

Dendritic cells were stimulated with inactivated A. fumigatus conidia and germ tubes (MOI = 1), 100 µg/ml zymosan depleted [a yeast cell wall preparation, which was treated with hot alkali to remove all TLR-stimulating properties to selectively activate Dectin-1 (dZym, InvivoGen)] or 1 mg/ml lipopolysaccharid (LPS, Sigma) for 24 h. Subsequently, cells were harvested, washed, and resuspended in cold Hank’s balanced salt solution (HBSS, Sigma) containing 2 mM EDTA (Sigma). The following antibodies were used for extracellular staining: HLA-ABC-PE (BD Biosciences), HLA-DR-PE (BD Biosciences), CD1a-APC (BD Biosciences), CD14-FITC (BD Biosciences), CD80-APC (Miltenyi Biotec), CD86-PE (BD Biosciences), Dectin-1-PE (R&D) (human cells) and HLA-2K^b-FITC (BD Biosciences), HLA-Ia/I-E-PE (BioLegend), CD11c-APC (BioLegend), CD80-FITC (BioLegend), CD86-PE (BD Biosciences), and Dectin-1-PE (R&D) [murine cells]. 5 × 10⁵ cells were stained for 15 min at 4°C. Subsequently, cells were washed twice and 10⁴ viable cells according to FSC-SSC properties were acquired using a FACS Calibur (BD Biosciences) flow cytometer and CellQuest Pro software (version 5.2). Data were analyzed with FlowJo software (Tree Star Inc., Ashland, OR, USA).

Co-cultures were seeded as described previously, but onto non-tissue culture-treated plastics to enable trypsinization of macrophages. Co-cultures were washed in ice-cold PBS, trypsinized at room temperature (RT) and rapidly rinsed in cold media before centrifugation at 4 °C. Cells were resuspended in cold PBS and vortexed, then run through the BD Accuri C6 cytometer (BD Biosciences). Debris was filtered by SSC/FSC gating with 50,000 cellular events collected. Macrophages and fibroblasts without any fluorescent tags were used to establish an exclusion region from final co-culture sort, while eGFP⁺, DsRed⁺ and dual-positive gates were drawn with each cell type seeded alone. For macrophage polarization markers, BMDMs were polarized as described, then scraped and resuspended in ice cold FACS buffer (1% FBS, 0.1% sodium azide in PBS). Cells were treated with Fc block (BD Biosciences) for 10 mins before incubation for 1 hr on ice in the dark with appropriate antibodies. Cells were washed three times in FACS buffer before immediately reading on the cytometer. Antibodies are as follows: F4/80 PE (Biolegend 565410), CD80 FITC (Biolegend 104706), CD126/IL6Rα APC (115811), CD206 PE (Biolegend 141795).

The primary antibodies used were CD14 VioBlue (BD Biosciences, Heidelberg, Germany; catalog number 558121), CD16 FITC (Miltenyi Biotec, Bergisch Gladback, Germany; catalog number 130‐113‐392), CD80 FITC (BioLegend, San Diego, CA, USA; catalog number 305205), CD86 PE (BioLegend, San Diego, CA, USA; catalog number 305405), CD163 PE (Miltenyi Biotec, Bergisch Gladback, Germany; catalog number 130‐097628), CD169 APC (Miltenyi Biotec, Bergisch Gladback, Germany; catalog number 130‐098‐643), CD206 APC (BD Biosciences, Germany; catalog number 550889) and CD209 VioBlue (BioLegend, San Diego, CA, USA; catalog number 330102). Antibodies were used as per manufacturer's instructions and incubated for 30 min at 4°C. Data were acquired using a MACSQuant flow cytometer (Miltenyi Biotec, Bergisch Gladback, Germany) and analyzed using FlowJo ™ version 10.7 (BD Biosciences, San Jose, CA, USA).