Anti cd19 v500

Venous blood samples were collected in sodium heparin tubes and peripheral blood mononuclear cells isolated using Ficoll gradient centrifugation and cryopreserved in 10% Dimethyl Sulfoxide. Batched flow cytometry staining of frozen samples were performed including an even fraction of control and patient samples using the following antibodies for MAIT cell identification: Dead Cell Marker Aqua (Thermofisher, ratio 1:100), anti-CD14-V500 (BD Biosciences, clone: M5E2, ratio 1:100), anti-CD19-V500 (BD Biosciences, clone: HIB19, ratio: 1:100), anti-CD3-BV570 (Biolegend, clone:UCHT1, ratio: 1:50), anti-CD4-BUV615 (BD Biosciences, clone: SK3, ratio: 1:100), anti-CD161-BV650 (BD Biosciences, clone: DX12, ratio: 1:25), and anti-TCR Vα7.2-PE (Biolegend, clone: 3C10, ratio: 1:50). Cells were acquired using a BD FACSymphony A5. After compensation, MAIT cells were defined by first removing dead cells, B cells and monocytes to avoid background and unspecific binding of antibodies. Thereafter, total CD3-expressing cells were identified, CD4-positive cells excluded and MAIT cells defined as CD161 and TCR Vα7.2 double-expressing cells. A gating scheme to identify MAIT cells out of CD3⁺CD4^- cells and plots for CD161 and Vα7.2 in individuals with and without bacterial infection is presented in Fig 2.

Serum immunoglobulins (Ig) levels (IgG, IgA and IgM) were measured by immunoturbidimetry using the automatic analyzer Alinity c system (Abbott Laboratories, Chicago, IL, USA). For cellular evaluation, EDTA whole blood samples were collected. Lymphocyte subpopulations (CD4+ T, CD8+ T, B and NK cells) were performed using BD Trucount tubes and the BD Multitest 6 Color BTNK kit (BD Biosciences, San Diego, CA, USA), which included the following mixtures of fluorophore-conjugated monoclonal antibodies (mAb): anti-CD45-PerCP-Cy5.5, anti-CD3-FITC, anti-CD8-APC-Cy7, anti-CD4-PE-Cy7, anti-CD19-APC, and anti-CD16+CD56-PE. B cell phenotype was performed with an eight-color panel of the following mAb: anti-CD45-APC-H7, anti-CD19-V500, anti-CD10-V450, anti-CD38-PE-Cy7, anti-CD21-PE, anti-CD27-PerCP-Cy5, anti-IgD-FITC, and anti-IgM-APC (BD Biosciences, San Diego, CA, USA), following EURO-Class classification. Cells were acquired on a BD FACSCanto II Flow Cytometer (BD Biosciences, San Diego, CA, USA), and the InfinicytTM22.0 software was employed for multiparametric analysis (Cytognos SL, Salamanca, Spain).