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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Epigenetic factors can interact with genetic programs to modulate gene expression and regulate B cell function. By combining in vitro B-cell stimulation, qRT-PCR, and high-throughput microRNA-sequence and mRNA-sequence approaches, we can analyze the epigenetic modulation of miRNA and gene expression in B cells.

Abstract

Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA recombination (CSR), and plasma cell differentiation. In recent years, epigenetic modifications or factors, such as histone deacetylation and microRNAs (miRNAs), have been shown to interact with B-cell genetic programs to shape antibody responses, while the dysfunction of epigenetic factors has been found to lead to autoantibody responses. Analyzing genome-wide miRNA and mRNA expression in B cells in response to epigenetic modulators is important for understanding the epigenetic regulation of B-cell function and antibody response. Here, we demonstrate a protocol for inducing B cells to undergo CSR and plasma cell differentiation, treating these B cells with histone deacetylase (HDAC) inhibitors (HDIs), and analyzing mRNA and microRNA expression. In this protocol, we directly analyze complementary DNA (cDNA) sequences using next-generation mRNA sequencing (mRNA-seq) and miRNA-seq technologies, mapping of the sequencing reads to the genome, and quantitative reverse transcription (qRT)-PCR. With these approaches, we have defined that, in B cells induced to undergo CSR and plasma cell differentiation, HDI, an epigenetic regulator, selectively modulates miRNA and mRNA expression and alters CSR and plasma cell differentiation.

Introduction

Epigenetic marks or factors, such as DNA methylation, histone posttranslational modifications, and non-coding RNAs (including microRNAs), modulate cell function by altering gene expression1. Epigenetic modifications regulate B lymphocyte function, such as immunoglobulin class-switch DNA recombination (CSR), somatic hypermutation (SHM), and differentiation to memory B cells or plasma cells, thereby modulating the antibody and autoantibody responses2,3. CSR and SHM critically require activation-induced cytidine deaminase (AID, encoded as Aicda), which is highly induced in B cells....

Protocol

The protocol follows the animal care guidelines of The Institutional Animal Care and Use Committees of the University of Texas Health Science Center at San Antonio.

1. Stimulation of Mouse B Cells for CSR, Plasma Cell Differentiation, and HDI Treatment

  1. Preparation of spleen cell suspensions
    NOTE: Perform all steps in a laminar flow hood except for mouse euthanasia and dissection.
    1. Euthanize the specific pathogen-free C57BL/6J mouse (8-12 weeks of .......

Representative Results

Using our protocol, purified B cells placed with LPS (3 µg/mL) and IL-4 (5 ng/mL) for 96 h can induce 30-40% of CSR to IgG1 and ~10% of plasma cell differentiation. After treatment with HDI (500µM VPA), the CSR to IgG1 decreased to 10-20%, while plasma cell differentiation decreased to ~2% (Figure 1). HDI-mediated inhibition of CSR was further confirmed by decreased numbers of post-recombination Iμ-Cγ1 and mature VHDJH-Cγ1 tr.......

Discussion

This protocol provides comprehensive approaches to induce B cell class switching and plasma cell differentiation; to analyze their impact by epigenetic modulators, namely HDI; and to detect the effect of HDI on mRNA and miRNA expression in these cells. Most of these approaches can also be used to analyze the impact of epigenetic factor on human B-cell function and mRNA/miRNA expression. The qRT-PCR and mRNA-seq/miRNA-seq approaches can also be used to analyze B cells isolated from mice treated with epigenetic modulators,.......

Acknowledgements

This work was supported by NIH grants AI 105813 and AI 079705 (to PC), the Alliance for Lupus Research Target Identification in Lupus Grant ALR 295955 (to PC), and the Arthritis National Research Foundation research grant (to HZ). TS was supported by the Pediatrics Medical Center, Second Xiangya Hospital, Central South University, Changsha, China, in the context of the Xiangya-UT School of Medicine San Antonio medical student visiting program.

....

Materials

NameCompanyCatalog NumberComments
C57BL/6 miceJackson Labs664
Corning cellgro RPMI 1640 Medium (Mod.) 1X with L-Glutamine (Size: 6 x 500mL; With L-Glutamine)Fisher ScientificMT 10-040-CV 
FBSHycloneSH300
HyClone Antibiotic Antimycotic Solution 100 mLFisher Scientific - HycloneSV3007901 
β-MercaptoethanolFisher Scientific44-420-3250ML
Falcon Cell StrainersFisher Scientific21008-952  
Trypan Blue Stain 0.04%GIBCO/Life Technologies/Inv15250
ACK Lysis Buffer Fisher ScientificBW10-548E 
Hausser Scientific Bright-Line Counting ChamberFisher Scientific02-671-51B
EasySep MagnetStem Cell Technologies18000
Falcon Round-Bottom Polystyrene Tubes with CapFisher Scientific14-959-1A
EasySep Mouse B cell Isolation KitStem Cell Tech19854
BD Needle Only 18 Gauge 1.5 inch SHORT BEVEL 100/box BD Biosciences 305199
PE/Cy7 anti-mouse CD138 (Syndecan-1) AntibodyBioLegend 142513 (25 ug)
PE-Cy7 B220 antibodyBioLegend103222
7-AAD (1 mg)Sigma AldrichA9400-1MG
APC anti-mouse/human CD45R/B220 antibodyBiolegend103212
Mouse APC-IgG1 200 µgBiolegend406610
FITC anti-mouse IgM AntibodyBiolegend406506
FITC anti-mouse/human CD45R/B220 AntibodyBiolegend103206
PE Anti-Human/Mouse CD45R (B220) (RA3-6B2)Biolegend103208
HBSS 1XFisher ScientificMT-21-022-CM
Bovine Serum Albumin, Fraction V, Heat Shock Treated Fisher ScientificBP1600-100
LPS 25mg (Lipopolysaccharides from Escherichia coli 055:B5)Sigma AldrichL2880-25MG
Recombinant mouse IL-4 (carrier-free)BioLegend574302 (size: 10 ug)
Valproic acid sodium saltSigma AldrichP4543
SterilGARD e3 Class II Type A2 Biosafety CabinetThe Baker CompanySG404
Large-Capacity Reach-In CO2 Incubator Thermo Scientific3950
Isotemp Digital-Control Water Baths: Model 205Fisher Scientific15-462-5Q
5mL Round Bottom Polystyrene Test TubeFisher Scientific 14-959-5
Corning CentriStar 15ml Centrifuge Tubes Fisher Scientific 05-538-59A
1.7 mL Microtube, clearGenesee22-282
Higher-Speed Easy Reader Plastic Centrifuge Tubes 50mlFisher Scientific06-443-18
ELMI SkySpin CM-6MTELMICM-6MT
Rotor 6MELMI6M
Rotor 6M.06ELMI6M.06
Drummond Portable Pipet-Aid XP Pipet ControllerDrummond Scientific4-000-101
25 mL serological pipette tipsFisher Scientific89130-900
10 mL serological pipette tipsFisher Scientific89130-898
5 mL serological pipette tipsFisher Scientific898130-896
48-well platesFisher Scientific07-200-86
Allegra 6 Benchtop Centrifuge, Non-RefrigeratedBeckman Coulter366802
GH-3.8A Rotor, Horizontal, ARIES Smart BalanceBeckman Coulter366650
Allegra 25R Benchtop Centrifuge, RefrigeratedBeckman Coulter369434
TA-15-1.5 Rotor, Fixed AngleBeckman Coulter368298
Fisher Scientific AccuSpin Micro 17Fisher Scientific13-100-675
Fisher Scientific Analog Vortex Mixer Fisher Scientific02-215-365
miRNeasy Mini Kit (50)Qiagen217004
Direct-zol RNA MiniPrep kitZymo ResearchR2050
Chloroform (Approx. 0.75% Ethanol as Preservative/Molecular Biology)Fisher ScientificBP1145-1
Rnase-Free Dnase set (50)QIAGEN79254
NanoDrop 2000 SpectrophotometersThermo ScientificND-2000
Superscript III First-strand Synthesis System RT-PCRInvitrogen175013897
iTaq Universal SYBR Green SupermixBio-rad 172-5121
Fisherbrand 96-Well Semi-Skirted PCR Plates, case of 25Fisher14-230-244
Microseal 'B' Adhesive SealsBio-RadMSB-1001
MyiQ Optics ModuleBio-Rad170-9744
iCycler ChassisBio-Rad170-8701
Optical KitBio-Rad170-9752
BD LSR II Flow Cytometry AnalyzerBD Biosciences
FACSDiva softwareBD Biosciences
FlowJo 10BD Biosciences
2100 BioanalyzerAgilent TechnologiesG2943CA
S200 Focused-ultrasonicatorCovarisS200
SPRIworks Fragment Library System I for IlluminaBeckman CoulterA288267
cBot Cluster Generation StationilluminaSY-312-2001
HiSeq 2000 Genome SequencerIlluminaSY-401-1001
TruSeq RNA Library Prep Kit v2IlluminaRS-122-2001
TruSeq Small RNA Library Prep KitIlluminaRS-200-0012
NEXTflex Illumina Small RNA Sequencing Kit v3Bioo Scientific5132-05
2200 TapeStationAgilentG2964AA

References

  1. Allis, C. D., Jenuwein, T. The molecular hallmarks of epigenetic control. Nat Rev Genet. 17, 487-500 (2016).
  2. Li, G., Zan, H., Xu, Z., Casali, P. Epigenetics of the antibody response. Trends Immunol. 34, 460-470 (2013).
  3. ....

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