Tetrameric Fluorescent Antigen Arrays for Single-Step Identification of Antigen-Specific B Cells

Summary

This protocol details the customizable production and use of fluorescent probes for labeling antigen-specific B cells.

Abstract

Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and the use of four-arm, fluorescent PEG-antigen conjugates to selectively identify antigen-specific B cells through avid engagement with cognate B cell receptors. Using modular click chemistry and commercially available fluorophore kit chemistries, we demonstrated the versatility of preparing customized fluorescent PEG-conjugates by creating distinct arrays for proteolipid protein (PLP_139-151) and insulin, which are important autoantigens in murine models of multiple sclerosis and type 1 diabetes, respectively. Assays were developed for each fluorescent conjugate in its respective disease model using flow cytometry. Antigen arrays were compared to monovalent autoantigen to quantify the benefit of multimerization onto PEG backbones. Finally, we illustrated the utility of this platform by isolating and assessing anti-insulin B cell responses after antigen stimulation ex vivo. Labeling insulin-specific B cells enabled the amplified detection of changes to co-stimulation (CD86) that were otherwise dampened in aggregate B cell analysis. Together, this report enables the production and use of fluorescent antigen arrays as a robust tool for probing B cell populations.

Introduction

The adaptive immune system plays a critical role in the progression or regression of many disease states, including autoimmunity, cancer, and infectious diseases¹. For broad applications including the study of immunopathology or the development of new precision treatments, it is often critical to assess antigen-specific B and T cell responses underlying disease progression^2,3,4. Major histocompatibility complex (MHC) tetramers are widely and commercially available for identifying antigen-specific T cell clones⁵. These fluorophor....

Protocol

All animal procedures represented in this work were approved by the Institutional Animal Care and Use Committee at the University of Kansas.

1. Antigen array synthesis (4–6 days)

1. Functionalize unmodified antigen with an alkyne handle (1 h). Add 1 equivalent insulin (100 mg, 17.4 μmol) to a 20 mL glass vial with a stir bar and dissolve in anhydrous dimethylsulfoxide (DMSO) (2 mL) with gentle heating to 40–50 °C using a heat-gun or water bath.
   1. Add 1,.......

Discussion

We developed a protocol (Figure 1) to construct customized FAAs for identifying antigen-specific B cells, simplifying the generation of B cell probes for difficult antigen targets. We selected 4-arm PEG polymers with terminal azide groups as a facile substrate for building FAAs, as PEG confers water solubility while the functional azide handles enable simple click conjugation reactions³³. The defined number of functional handles (4 arms) is conducive to simplified che.......

Materials

Name	Company	Catalog Number	Comments
1,1,3,3-tetramethylguanidine	Alfa Aesar	AAA12314AC
12 M hydrochloric acid	Fisher Chemical	A508-4
12% Mini-PROTEAN TGX Precast Protein Gels, 10-well, 30 ul	Bio-Rad	4561043
20 kDa 4-arm PEG azide	Jenkem USA	A7185-1
3.5 kDa MWCO dialysis tubing (regenerated cellulose)	Fisher Scientific	25-152-10
Acetonitrile, HPLC grade	Fisher Chemical	A998-4
Alexa Fluor 647 anti-mouse CD19 Antibody	BioLegend	115522
anhydrous dimethylsulfoxide	ACROS Organics	AC610420010
barium chloride	ACROS Organics	203135000
Brilliant Blue G-250 Dye	Fisher BioReagents	BP100-50
Cell Prime r-insulin	EMD Millipore	4512-01	Recombinant human insulin for insulin FAA synthesis
Copper (II) sulfate pentahydrate	ACROS Organics	AC197722500
dimethylsulfoxide	Fisher Chemical	S67496
fluorescein isothiocyanate (FITC) isomer 1	Sigma-Aldrich	F7250-1G
Glacial acetic acid	Fisher Chemical	A38-212
Glycerol	ACROS Organics	15892-0010
Glycine	Fisher Chemical	G46-500
homopropargyl-PLP	Biomatik	NA	Custom synthesis (sequence: homopropargyl-HSLGKWLGHPDKF; purity: 97.29%)
iodine	Sigma-Aldrich	207772-100G
Methanol, HPLC grade	Fisher Chemical	A452-4
NHS-Rhodamine (5/6-carboxy-tetramethyl-rhodamine succimidyl ester), mixed isomer	Thermo Scientific	46406	A commercially available analog of the Rhodamine-B NHS ester used in the paper
PE/Cyanine7 anti-mouse CD3 Antibody	BioLegend	100220
potassium iodide	Sigma-Aldrich	30315
propargyl N-hydroxysuccinimide ester	Sigma-Aldrich	764221
sodium ascorbate	Sigma-Aldrich	A7631
sodium biocarbonate	Sigma-Aldrich	S5761-1KG
sodium dodecyl sulfate	Fisher BioReagents	BP166-100
Sodium phosphate monobasic monohydrate	Fisher Chemical	S468-500
trifluoroacetic acid	Sigma-Aldrich	302031-10x1mL
Tris base	Fisher BioReagents	BP152-500
Tris(3-hydroxypropyltriazolylmethyl)amine	ClickChemTools	1010-1000
xBridge BEH C18 3.5 um, 4.6 x 150 mm column	Waters	186003034
xBridge BEH C18 5um OBD Prep Column, 19 x 250 mm	Waters	186004021