Cutting Edge: Redox Signaling Hypersensitivity Distinguishes Human Germinal Center B Cells

Abstract

Differences in the quality of B-cell antigen receptor (BCR) signaling control key steps of B cell maturation and differentiation. Endogenously produced H2O2 is thought to fine tune the level of BCR signaling by reversibly inhibiting phosphatases. However, relatively little is known about how B cells at different stages sense and respond to such redox cues. Here, we used phospho-specific flow cytometry and high-dimensional mass cytometry (CyTOF) to compare BCR signaling responses in mature human tonsillar B cells undergoing germinal center (GC) reactions. GC B cells, in contrast to mature naïve B cells, memory B cells, and plasmablasts, were hypersensitive to a range of H2O2 concentrations and responded by phosphorylating SYK and other membrane proximal BCR effectors in the absence of BCR engagement. These findings reveal that stage specific redox responses distinguish human GC B cells.

Single Cell Analysis of Human Tissues and Solid Tumors with Mass Cytometry

“…"Fine mincing" indicates additional mechanical dissociation using scalpels. Conventional mechanical dissociation of tonsils included fine mincing and immediate filtration of tissue through a 70μm cell strainer without additional enzymatic dissociation, as previously established (8)(9)(10). Dissociation enzymes were obtained from Sigma Aldrich (Darmstadt, Germany) (collagenase II, IV, V, and XI), ThermoFisher (Waltham, MA) (TrypLE-Express), and GE Healthcare (PA) (HyQTase).…”

Preparing Viable Single Cells from Human Tissue and Tumors for Cytomic Analysis

“…Furthermore, if additional assays, such as a signaling response assay using phospho-specific flow cytometry (Krutzik and Nolan, 2003; Schulz et al, 2012), are to be performed, it is important to test different types of medium for those specific assays. For example, to preserve lymphocyte signaling capability for subsequent detection by phospho-specific flow cytometry, medium containing FBS is superior to serum-free medium (Irish et al, 2006a; Irish et al, 2010; Polikowsky et al, 2015). Conversely, multiple growth factor supplements are added to the neurosphere culture medium to ensure growth of human glioma cells (Azari et al, 2011; Lee et al, 2006).…”

“…Our group and others have implemented this technology in studies of donor and patient cells that are obtained as a suspension, such as blood and bone marrow (Bendall et al, 2011; Hardy et al, 1983; Kordasti et al, 2016; Leelatian et al, 2015; Nicholas et al, 2016; Parks et al, 1984; Qiu et al, 2011; Tung et al, 2004) or that can be disaggregated from lymphoid structures by mechanical force alone (Irish et al, 2006a; Irish et al, 2010; Myklebust et al, 2016; Polikowsky et al, 2015; Wogsland et al, 2016). Clinical diagnoses of blood malignancies use fluorescence flow cytometry characterization of cell surface marker expression, as well as cell subset quantification (Arber et al, 2016; Craig and Foon, 2008; van Dongen et al, 2012; Wood et al, 2007).…”

“…Mass cytometry-based characterization of human bone marrow (Bendall et al, 2011), blood (Newell et al, 2012), and tonsil (Polikowsky et al, 2015) cell subsets has been accomplished in prior studies and described in protocols (Leelatian et al, 2015). However, mass cytometry has just recently been developed and applied in solid tissues and organs (Diggins et al, in press ; Leelatian et al, 2016).…”

Identification of Vaccine-Altered Circulating B Cell Phenotypes Using Mass Cytometry and a Two-Step Clustering Analysis

“…+ follicular Th cells (33), B cells (27,(34)(35)(36), and vaccines (18,37). The benefits of larger Ab panels include the discovery of new subsets (31,35), the identification of coexpressed markers (27), and of signatures for differentiation, activation, and homing status of cells.…”