Flow Cytometric Methods for the Detection of Intracellular Signaling Proteins and Transcription Factors Reveal Heterogeneity in Differentiating Human B Cell Subsets.

Casper Marsman,S Marieke Van Ham,Anja Ten Brinke,Tineke Jorritsma

doi:10.3390/cells9122633

Casper Marsman, S Marieke Van Ham + Show 2 more

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https://doi.org/10.3390/cells9122633

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Abstract

The flow cytometric detection of intracellular (IC) signaling proteins and transcription factors (TFs) will help to elucidate the regulation of B cell survival, proliferation and differentiation. However, the simultaneous detection of signaling proteins or TFs with membrane markers (MMs) can be challenging, as the required fixation and permeabilization procedures can affect the functionality of conjugated antibodies. Here, a phosphoflow method is presented for the detection of activated NF-κB p65 and phosphorylated STAT1, STAT3, STAT5 and STAT6, together with the B cell differentiation MMs CD19, CD27 and CD38. Additionally, a TF-flow method is presented that allows the detection of the B cell TFs PAX5, c-MYC, BCL6 and AID and antibody-secreting cell (ASC) TFs BLIMP1 and XBP-1s, together with MMs. Applying these methods on in vitro-induced human B cell differentiation cultures showed significantly different steady-state levels, and responses to stimulation, of phosphorylated signaling proteins in CD27-expressing B cell and ASC populations. The TF-flow protocol and Uniform Manifold Approximation and Projection (UMAP) analysis revealed heterogeneity in TF expression within stimulated CD27- or CD38-expressing B cell subsets. The methods presented here allow for the sensitive analysis of STAT, NF-κB p65 signaling and TFs, together with B cell differentiation MMs, at single-cell resolution. This will aid the further investigation of B cell responses in both health and disease.

Highlights

One of the cornerstones of the adaptive immune system is the ability of B cells to respond to pathogens and differentiate into antibody-secreting cells
The antigen is internalized and presented on MHC-II in order to form a cognate interaction with the TFH cell [6]. This interaction provides multiple stimuli indispensable for GC B cell proliferation and differentiation, such as membrane-bound CD40L, which activates CD40 on the B cell [7,8], and the TFH hallmark cytokine IL-21, which binds to the IL-21 receptor [9,10,11]
We present two methods, one that allows the monitoring of signaling via STAT molecules and NF-κB and one that allows for the detection of transcription factors (TFs) dynamically expressed throughout in vitro

Summary

Introduction

One of the cornerstones of the adaptive immune system is the ability of B cells to respond to pathogens and differentiate into antibody-secreting cells. B cells are activated upon antigen recognition by the B cell receptor (BCR). The antigen is internalized and presented on MHC-II in order to form a cognate interaction with the TFH cell [6]. This interaction provides multiple stimuli indispensable for GC B cell proliferation and differentiation, such as membrane-bound CD40L, which activates CD40 on the B cell [7,8], and the TFH hallmark cytokine IL-21, which binds to the IL-21 receptor [9,10,11].

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