Anti cd5 apc

C57BL/6 (WT) mice were purchased from The Jackson Laboratories (Bar Harbor, ME).
Cd244^-/- mice on the C57BL/6 background [12 (link)], were kindly provided by Dr. Raymond Welsh (University of Massachusetts Medical Center, Worcester, MA). Mice were maintained in the Translational Biomedical Research Center of the Medical College of Wisconsin and both genders were used for experiments in an age (6–12 week) and gender-matched manner. anti-CD45R-PE-Texas Red, anti-CD45R-PE, anti-GL7-FITC and anti-CD5-APC were purchased from BD Biosciences (San Jose, CA). CD21-eFluor 450, anti-CD23-PE-Cy7, anti-CD93-biotin, anti-CD4-APC-eFluor 780, anti-CD8-PE-Cy7, anti-TCRβ-FITC, anti-TCRβ-PE, anti-CD11b-Alexa Fluor 488, anti-CD244, anti-Foxp3-PE, anti-CD19-Alexa 700, anti-CD4-PE, anti-IgG-FITC and Streptavidin PE-Cy5.5 were purchased from eBioscience (San Diego, CA). Anti-CD11b-Brilliant Violet 605, anti-CD11c-PE, anti-NK1.1-APC, anti-IgM-APC Cy7, anti-IgD-Pacific Blue, anti-CD38-Alexa Fluor 647, anti-CD138-APC and rat anti-mouse IgG_2a-biotin were purchased from Biolegend (San Diego, CA). Anti-IgM-FITC and the SBA Clonotyping System-B6/C57J-HRP were purchased from Southern Biotech (Birmingham, AL). 4-Hydroxy-3-nitrophenylacetyl (NP)-ficoll, NP-Chicken Gamma Globulin (CGG), NP(24)-PE and NP-BSA were purchased from Biosearch Technologies (Novato, CA).

Splenocytes, thymocytes, and peritoneal cavity cells were resuspended in PBS containing 1% FBS and Rat Anti-Mouse CD16/CD32 Mouse BD Fc Block (BD Biosciences). The following antibody combinations were used: anti-CD4-PE (BD Biosciences, clone GK1.5) and anti-CD8a-PerCP (BD Biosciences, clone 53–6.7); anti-IgD-PE (BD Biosciences, clone 11-26c.2a) and anti-IgM-APC (SouthernBiotech, clone 1B4B1); anti-CD5-APC (BD Biosciences, clone 53–7.3) and anti-IgM-PE (SouthernBiotech, clone 1B4B1); and anti-CD21-APC (BD Biosciences, clone 7G6) and anti-CD23-FITC (BD Biosciences, clone B3B4). Flow cytometry was performed with a BD FACSCaliber and Flojo software (Treestar).

The flow cytometry analysis was made using thawed single-cell tumour suspension (patient no. 11, Table 1) obtained after collagenase digestion of fresh tumour material. Bregs were identified using anti-CD19 BV510, anti-CD5 APC, anti-CD38 PERCPCy5.5, anti-CD45 APCH7, anti-GZMB FITC (all from BD Biosciences, Franklin Lakes, New Jersey, USA), and anti-IL10 PE (BioLegend, San Diego, CA, USA). The cells were stained for cell surface markers after blocking nonspecific antibody binding to the Fc receptors using the FcR Blocking Reagent (Miltenyi, Bergisch Gladbach, Germany), fixed and permeabilised with Cytofix/Cytoperm buffer (BD Biosciences), and stained with the intracellular markers (IL10 and GZMB). Production of IL10 and GZMB was measured after overnight stimulation in the presence or absence of LPS (10 μg/mL), PMA (50 ng/mL), and ionomycin (1 μg/mL). GolgiStop (0.7 μL/mL) was added after one-and-a-half hours' stimulation. Dead cells were identified using the LIVE-DEAD® Fixable Violet Dead Cell Stain Kit (Thermo Fisher Scientific) according to the manufacturer's instructions and excluded from the analysis. Data were acquired by flow cytometry (Gallios™, Beckman Coulter, Brea, California, USA), and analysis was done by Kaluza® Software (Beckman Coulter).