Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

Summary

Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

Abstract

CD4⁺ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from naïve CD4⁺ T cells in vitro by activating them in the presence of the polarizing cytokines IL-1β, IL-6, IL-23, and TGFβ. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, RORγt, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.

Introduction

CD4⁺ T cells are crucial orchestrators of the adaptive immune response. Naïve CD4⁺ T cells are capable of developing into several different effector T cells (e.g. Th1, Th2, Th17, etc.), each with their own set of characteristic cytokines and transcription factors, depending upon the local microenvironment¹. The lineage decisions that T cells make are critical for both protective immunity and tolerance to self. Th17 cells are one subset of T cells known to combat extracellular bacteria and fungi, but their improper responses are also implicated in the pathogenesis of multiple autoimmune and inflammato....

Protocol

This protocol adheres to UCSF's guidelines for human research ethics.

1. Preparation of T Cell Culture, Isolation of CD4⁺ T Cells, and Th17 Polarization

1. On Day 0, coat 6-well tissue culture plates with 1.5 mL per well of anti-human CD3 (2 µg/mL) and anti-human CD28 (4 µg/mL) in PBS with calcium and magnesium for at least 2 h at 37 °C.
   1. Alternatively, coat the plates overnight at 4 °C. Wrap plates in parafilm.
2. Prepare the cord blood mononuclear cells (CBMCs) by density gradient centrifugation per manufacturer's instructions.
   CAUTION....

Results

The first step to developing a reliable system of successfully electroporating human Th17 cells was to generate robust in vitro differentiated human Th17 cell cultures. T cells cultured under Th17-polarizing conditions expressed the chemokine receptor CCR6 and the transcription factor RORγt (Figure 1A, left). These markers were not expressed when T cells were cultured under non-polarizing (ThN) conditions (Figure 1A, right). T cells cultured unde.......

Discussion

This protocol provides an improved method for the delivery of small RNAs into human Th17 cells. Although human Th17 cells were used here, this method of electroporation with small RNAs can be used with other primary human T helper subsets, such as Th1, Th2, and Tregs. It has not worked well for naïve CD4⁺ T cells so the cells must be activated in culture prior to transfection. For this protocol, we first optimized the in vitro culture system for better IL-17A production. The biggest factor was ba.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
anti-human IL-17A PE	ebioscience	12-7179-42	Clone: eBio64DEC17
anti-human IFNg FITC	ebioscience	11-7319-82	Clone: 4S.B3
anti-human CD4 eVolve605	ebioscience	83-0047-42	Clone: SK3
mouse anti-human CD196 (CCR6) BV421	BD biosciences	562515	Clone: 11A9
anti-human RORgt AF647	BD biosciences	563620	Clone: Q21-559
anti-human CD45 eFluor450	ebioscience	48-9459-42	Clone: 2D1
Foxp3/Transcription Factor Staining Buffer Set	ebioscience	00-5523-00	For intracellular transcription factor flow cytometry staining
Hu FcR Binding Inhibitor Purified	ebioscience	14-9161-71
ImmunoCult-XF T Cell Expansion Medium	Stemcell Technologies	10981	"Serum-Free Base Media"
MACS CD28 pure functional grade, human	Miltenyi Biotec	130-093-375	Clone: 15E8
anti-human CD3 Purified	UCSF monoclonal antibody core	N/A	Clone: OKT-3
LEAF purified anti-human IL-4	Biolegend	500815	Clone: MP4-25D2
anti-human IFNg, functional grade purified	ebioscience	16-7318-85	Clone: NIB42
Recombinant human IL-23	Peprotech	200-23
Recombinant human IL-1β	Peprotech	200-01B
Recombinant human TGF-β1	Peprotech	100-21C
Recombinant human IL-6	Peprotech	200-06
siGENOME Control Pool, Non-targeting #2	Dharmacon	D-001206-14-05
siGENOME SMARTpool Human RORC	Dharmacon	M-003442-00
siGENOME SMARTpool Human PTPRC	Dharmacon	M-008067-01
Dynabeads Untouched Human CD4 T Cells Kit	ThermoFisher Scientific	11346D	Human CD4+ T cell Isolation Kit
Neon Tranfection System	ThermoFisher Scientific	MPK5000	Next generation electroporation instrument
Neon Tranfection System 10 μL Kit	ThermoFisher Scientific	MPK1096
Resuspension Buffer T	ThermoFisher Scientific	Provided in kit (MPK1096)	"Transfection Resuspension Buffer"
Lymphoprep	Stemcell Technologies	#07801	Density Gradient Medium
costar 6-well tissue culture treated plates	Corning	3516	flat bottom plates
costar 48-well tissue culture treated plates	Corning	3548	flat bottom plates
BD Pharm Lyse lysing buffer, 10x	BD biosciences	555899	Must make 1x solution with distilled water prior to use