Cd45r b220

Splenocyte subpopulations were analyzed by evaluating the relative proportions of Tregs, macrophages, and B cells. Single-cell suspensions of splenocytes were stained with anti-mouse CD4 (BioLegend) and CD25 (eBioscience, Waltham, MA, USA) antibodies to analyze Tregs. To analyze macrophages and their polarization, splenocytes were stained with anti-mouse F4/80 (BioLegend), CD80 (eBioscience), and CD206 (BioLegend) antibodies. Splenocytes were stained with an antibody against CD45R (B220; BioLegend) for B cell detection. To eliminate nonspecific staining, isotype- control antibodies, matched to the surface marker antibody’s host species and class, were used. FACS analysis was performed using a BD FACSCanto II and BD FACSVerse flow cytometers (BD Biosciences, San Diego, California, USA). Data collected were analyzed with FlowJo software (Tree Star, Inc., Ashland, Oregon, USA).

To analyze the immune cell components, spleens and tumors of MFC tumor-bearing mice were sampled. Spleen lymphocytes were prepared as described by Li et al. (2020) (link). Briefly, fresh spleen was gently scraped with sterile cover glasses in PBS and the cells were filtered with a 70 µm strainer after erythrocytes were lysed. Tumor tissue was dissociated into single-cell suspensions according to the protocol of the Tumor Dissociation Kit (Miltenyi Biotec, cat: 130-096-730). Single cells were stained with fluorescein-conjugated monoclonal antibodies for 30 min at 4°C, and subsequently analyzed on Flow Cytometer (Cytek Aurora 3,000). The monoclonal antibodies include CD45 (Biolegend, cat: 103138), CD3 (Biolegend, cat: 100210), CD4 (Thermo, cat: 48-0041-82), CD8 (BD Pharmingen, cat: 557959), CD25 (Biolegend, cat: 102008), PD-1 (Biolegend, cat: 109118), NK1.1 (Biolegend, cat: 108710), CD11b (Biolegend, cat: 101228), Gr-1 (Biolegend, cat: 108426), CD19 (Biolegend, cat: 115543), CD45R/B220 (Biolegend, cat: 103244), CD11c (Biolegend, cat: 117348), F4/80 (Biolegend, cat: 123118), and MHC-Ⅱ (Biolegend, cat: 107643).

Eight days after adoptive transfer and infection one third of the spleens was fixated in 4% formaldehyde for two hours, dehydrated in 30% sucrose for 24 hours and frozen with liquid nitrogen in Tissue-tek O.C.T. compound (Sakura Finetek Europe; Alphen aan den Rijn, Netherlands). On glass slides cryostat sections of 7 μm thickness were blocked with 5% BSA (Sigma-Aldrich) for 30 minutes, followed by staining with PE conjugated CD90.1 antibody diluted 1:500, FITC conjugated CD45R/B220 antibody and APC conjugated CD4 antibody both diluted 1:200 in 5% BSA, containing DAPI (all from BioLegend) diluted 1:8000. After one hour the slides were washed with PBS three times for three minutes and mounted with mounting medium (PermaFlour by Thermo Scientific, ThermoFisher). Images were taken at an Axioplan 2 fluorescence microscope at 20x magnification using an AxioCam camera and Axiovision LE software Rel. 4.9 (all Axio devices were from Zeiss, Oberkochen, Germany). Stitching as well as computational cell detection and counting were performed in QuPath-0.2.3 (University of Edinburgh). Single cells were identified by DAPI signal and B cells, CD4 T cells and SMARTA CD4 T cells by their respective fluorescence coupled antibody (FITC/B220, APC/CD4 and PE/CD90.1). B cell zones were marked manually, and cell numbers counted computationally.