Fitc conjugated anti cd19

C57B/6 mice (4 to 6 weeks) were i.n. immunized with SrtA/CTB three times. 10 days after the last immunization, lymph nodes and the spleens were harvested and pooled. Single cell suspensions were made and CD4⁺ T cells were purified ( >93%) using anti-CD4 microbeads (Miltenyi Biotec) according to the manufacturer's protocol [15] (link). For purifying CD19⁺ B cells, single cell suspensions were sorted using a FACSAria sorter with FACSDiva software (BD Bioscience) after FITC-conjugated anti-CD19 (Biolegend) staining. A small volume from each sample was stained and analyzed by flow cytometry for the purity and phenotype of the cells. The purities of sorted CD4⁺ T cells and CD19⁺ B cells were 93% and 95%, respectively. Naïve female C57B/6 mice aged 4 to 6 weeks were used as recipients, and 1×10⁷ of either CD4⁺ T cells or CD19⁺ B cells were transferred to each mouse in a volume of 200 µl by tail vein injection. 24 hr later, recipient mice were challenged i.n. with 2×10⁸ CFUs of GAS M28 per mouse. All mice were killed 24 hr after the challenge; NALTs were harvested, and cell lysates were diluted and plated on sheep blood agar plates for CFU counts.

Specific T-cell phenotype and frequencies was assessed after treatment with 10 µM SnMP in an antigen-independent setting as well as before and after expansion of WT1-specific T cells. To distinguish between naïve T cells (T_N, CD62L⁺ CD45RA⁺), central memory T cells (T_CM, CD62L⁺ CD45RA⁻), effector memory T cells (T_EM, CD62L⁻ CD45RA⁻) or terminal differentiated effector memory T cells (T_EMRA, CD62L⁻ CD45RA⁺), cells were stained with peridinin chlorophyll (PerCP)-conjugated anti-CD3, allophycocyanin (APC)-conjugated anti-CD8, fluorescein isothiocyanate (FITC)-conjugated anti-CD19, APC/Cyanin 7 (Cy7)-conjugated anti-CD62L and anti-CD45RA-phycoerythrin (PE)/Cy7 (all BioLegend) and analyzed by multicolor flow cytometry (FACSCanto II, FACSDiva V8.1.2 software, BD Biosciences). Gates were set on the properties regarding light scatter of lymphocytes. At least 50,000 events were acquired in the CD3⁺ gate.

Fc receptors were blocked with Fcγ blocker (BioLegend, San Diego, CA, USA), and the surface markers were stained with BV450-conjugated anti-CD3 (BioLegend, clone:UCHT1), FITC-conjugated anti-CD19 (BioLegend, clone: HIB19), APC/Cy7-conjugated anti-CD20 (BioLegend, clone: 2H7), PE-conjugated anti-CD14 (BioLegend, clone: ΦM P9), PE-conjugated anti-CD11c (BioLegend, clone: B-ly6), PerCP/Cy5.5-conjugated anti-CD56 (BioLegend, clone: B159), FITC-conjugated anti-CD16 (BioLegend, clone:3G8), PE-conjugated anti-CD123 (BioLegend, clone: 6H6), and APC/Cy7-conjugated anti-CD4 (BioLegend, clone: RPA-T4). After fixing and permeabilization, IGBP1 was stained with Alexa 647-conjugated anti-IGBP1 (Novus Biologicals, Centennial, CO, USA).

BALF was collected according to a method described previously [14 (link)]. Briefly, the trachea was cannulated through a 22-inch intravenous catheter and the lungs were lavaged with a total volume of 1 mL PBS for three successive aspirations to obtain BALF. The BALF was centrifuged at 500×g for 8 min at 4 ℃. The cell-free supernatant was collected for cytokine analyses using ELISA kits. Cell pellets were resuspended in PBS and the total cell number was counted using CellDrop® (DeNovix, Wilmington, DE, USA). Cells were analyzed by flow cytometry using PE-conjugated anti-SiglecF (eBioscience, San Diego, CA, USA), FITC-conjugated anti-CD3 (BD Biosciences, San Jose, CA, USA), APC-conjugated anti-CD11c (Multiscience, Zhejiang, China), FITC-conjugated anti-CD19 (BioLegend, San Diego, CA, USA), Percp-cy5.5-conjugated anti-Ly6G (BioLegend) and PE-cy7-conjugated anti-MHC II (BioLegend). The flow cytometry gating strategy was according to a method described previously [15 (link)].