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OMIP-068

High‐Dimensional Characterization of Global and Antigen‐Specific B Cells in Chronic Infection

Katherine Cascino, Mario Roederer, Thomas Liechti Cytometry PART A, Volume 97, Issue 10, 1037-1043 (2020)

PURPOSE: This 24‐color flow cytometry panel focuses on characterizing antigen‐specific B cells and precise delineation of B‐cell subsets in chronic infections and is applicable to other chronic diseases such as autoimmunity. The panel was optimized for human cryopreserved peripheral blood mononuclear cells (PBMCs). Markers were chosen to extensively distinguish B‐cell lineages (CD19, CD20, CD10, CD38, CD24, IgM, IgD, CD27, CD21, CD43, CD5). Inclusion of antigen‐specific probes was of high priority in order to assess hepatitis B virus (HBV) antigen‐specific B cells for our purposes. These probes can be readily exchanged for other pathogen‐specific probes or additional markers for the panel to be tailored to desired research questions beyond HBV. In addition, we included a comprehensive and unique set of functional markers such as chemokine receptors (CXCR3, CXCR5), co‐stimulatory molecule (CD86), Fc receptor (CD32), regulatory molecules (BTLA, CD39), and inhibitory markers associated with chronic infections (PD‐1, FcRL5, CD11c, CD22) to enable in‐depth analysis of global and antigen‐specific B cells during chronic infection.

CELL TYPE: Cryopreserved PBMC

MACHINE: BD FACSymphony A5

Marker Target Clone Fluorochrome Purpose
Dead cells Human Live/Dead Fix Aqua n/a
PD-1 Human EH12.2H7 FITC n/a
CD11c Human Bu15 BB630-P2 n/a
FCRL5 Human 509f6 BB660-P2 n/a
CD272 Human J168-540 BB700 n/a
IgD Human IA6-2 BB790-P n/a
CD27 Human M-T271 PE-CF594 n/a
CD10 Human HI10a PE-Cy5 n/a
CD5 Human CD5-5D7 PE-Cy5.5 n/a
CD185 Human J252D4 PE-Cy7 n/a
IgM Human Alexa Fluor 700 n/a
CD19 Human APC-H7 n/a
CD24 Human ML5 BUV395 n/a
CD21 Human B-ly4 BUV496 n/a
CD38 Human HIT2 BUV661 n/a
CD86 Human BUV737 n/a
CD22 Human HIB22 BUV805 n/a
CD183 Human G025H7 Brilliant Violet 421 n/a
CD3 Human UCHT1 Brilliant Violet 510 n/a
CD14 Human MfP9 Brilliant Violet 510 n/a
CD43 Human 1G10 Brilliant Violet 605 n/a
CD39 Human TU66 Brilliant Violet 711 n/a
CD32 Human 3D3 Brilliant Violet 750 n/a
CD20 Human Brilliant Violet 786 n/a