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

Podsumowanie

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

Streszczenie

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.

Wprowadzenie

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 inflammatory diseases such as multiple sclerosis and psoriasis^2,3. Human Th17 cells can be generated from naïve CD4⁺ T cells in vitro by providing them with an appropriate polarizing environment⁴. Various combinations of the cytokines IL-1β, IL-23, TGFβ, and IL-6 have been used for the development of human Th17 cells. Human Th17 cells express CCR6, a chemokine receptor that is commonly used to identify this cell population and are defined by the expression of their principal transcription factor, RORγt (encoded by RORC)^5,6. Th17 cells have the ability to express multiple cytokines, but IL-17A is the lineage-defining effector cytokine produced by these cells. We examined the expression of all three Th17-associated markers (CCR6, RORγt, IL-17A) to assess the robustness of our human Th17 in vitro differentiation assay. Additionally, we cultured human CD4⁺ T cells under non-polarizing conditions, where no cytokines or blocking antibodies were added to the culture media to use as a negative control since expression of these Th17 markers should be very low or absent.

One way to study normal human T cell development and biology is to manipulate gene expression during their development. Short-interfering RNA (siRNA) are synthetic small RNA molecules that target protein-coding mRNAs and can be utilized to reduce specific gene expression. MicroRNAs (miRNAs) are endogenous non-coding small RNAs known to modulate gene expression post-transcriptionally. miRNAs have been shown to play an important role in both murine and human T cell biology, including in Th17 cells^7,8,9. It is crucial to have reliable methods of manipulating small RNA activity in human T cells to study their effects on gene expression and ultimately on human T cell biology. Here, we describe an easy-to-use, consistent and reliable protocol that we developed for introducing small synthetic RNAs and locked nucleic acids (LNAs, chemically modified nucleic acids with increased stability) into immune cells, and specifically into human Th17 cells.

There are several alternative methods of introducing small RNAs into mammalian cells, which generally fall into chemical, biological, or physical categories¹⁰. Commonly used chemical methods, including lipid-based transfections and calcium-phosphate transfections, rely on creating chemical-DNA complexes that are more efficiently taken up by cells. In general, chemical methods are not as efficient for the transfection of primary T cells. The most common biological method is to use a viral vector (e.g. retrovirus or lentivirus), which directly inserts foreign RNA into a host as a part of its natural replication cycle. Viral transduction typically takes longer to complete, especially when one factors in time for molecular cloning of proviral plasmids. Additionally, viral transduction vectors can be potentially harmful to human researchers. Electroporation is a physical method of inducing membrane permeabilization by subjecting cells to high voltage pulses, allowing nucleic acids to transiently enter into the cell where they can act on their target. Traditional electroporation instruments were not effective for transfecting primary lymphocytes. However, optimized next generation electroporation has proven to be capable of transfecting T cells at very high efficiency, especially when the material to be transfected is small RNA. The term next generation is loosely used to differentiate the two newer platforms (e.g., Neon, Amaxa) from traditional electroporation machines. Additionally, this method is easily scalable for moderate throughput screens with up to approximately 120 small RNAs in a single experiment, often using validated synthetic reagents. Importantly, successful transfections can be achieved in as little as 16 h after T cell activation. The disadvantage of this method, however, is that it does not result in stable genomic incorporation, and is therefore transient. Hence, it is worth the extra effort to create a stable expression construct that can be packaged into a viral vector and successfully expressed in T cells in cases where long-term expression of a small RNA is required.

We have used a next generation transfection (e.g., Neon) to deliver diverse synthetic single or double-stranded RNA or LNA oligonucleotide tools for different purposes^11,12,13. Efficient RNA interference can be induced in primary mouse and human T cells using double-stranded short-interfering RNA (siRNA). This protocol describes optimized conditions for using this technique in human Th17 cells. In addition to siRNAs, commercially available synthetic miRNA mimics and inhibitors can be used to study miRNA gain and loss of function. miRNA mimics are double-stranded RNA molecules very similar to siRNAs, but designed with the sequence of endogenous mature miRNAs. miRNA inhibitors are chemically-modified RNA and/or LNA based single stranded oligonucleotides that bind to native miRNAs and antagonize their function. We have found that all of these tools can be used effectively in cultured primary T lymphocytes, including but not limited to human Th17 cells.

Protokół

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: Work carefully and ensure proper personal protective equipment (PPE) is worn when handling human blood to avoid any risk of exposure to blood-borne pathogens.
3. Once the mononuclear cells have been isolated and washed, perform human CD4⁺ T cell isolation by negative selection using a commercial Human CD4⁺ T cell kit.
   NOTE: If there is red blood cell contamination after mononuclear cell isolation, an optional red blood cell lysis may be performed prior to the CD4⁺ T cell isolation steps. Resuspend the mononuclear cells in 1 mL of isolation buffer (2% FBS in PBS). Add 5 mL of 1x lysing solution. Incubate for 15 min at room temperature. Then add 5 mL of isolation buffer and centrifuge at 300 x g for 5 min at 4 °C to pellet the cells.
   1. Resuspend the mononuclear cells at a density of 50 x 10⁶ per 500 µL in the isolation buffer in a new 5 mL tube.
   2. Add 100 µL of FBS and 100 µL of the Antibody Mix per tube then incubate each tube at 4 °C for 20 min on an orbital shaker to mix well.
   3. After the incubation, add 3-4 mL of isolation buffer to wash the cells. Centrifuge the cells at 300 x g for 8 min at 4 °C to pellet the cells. Carefully aspirate the supernatant.
   4. During this spin, pre-wash the magnetic beads. Transfer the desired amount of magnetic beads into a new tube (used at 1:1 with the cells) and add an equal volume of isolation buffer to wash the beads. Mix well using a 1 mL micropipette and then place the tube in the magnet for at least 1 min. Carefully aspirate the supernatant. Resuspend the magnetic beads in the same volume initially transferred prior to the wash.
   5. Resuspend the cells in each tube with 500 µL of the isolation buffer and add 500 µL of pre-washed magnetic beads.
   6. Incubate the cells with the magnetic beads for 15 min on an orbital shaker at room temperature (18 °C to 25 °C) to mix well.
   7. After the incubation, thoroughly pipet the cells using a 1 mL micropipette at least 10 times. Then add 3-4 mL of isolation buffer and place each tube in the magnet for 2 min.
   8. Carefully transfer the negatively selected CD4⁺ T cells that are in the supernatant to a new tube.
4. Once CD4⁺ T cell isolation is completed, count the cells with a hemocytometer and keep the cells on ice.
5. Wash the antibody-coated plates two times with PBS. Then add 1.5 mL of 2x mix of Th17-polarizing media to each well: anti-human IFNγ (20 µg/mL), anti-human IL-4 (20 µg/mL), human TGFβ (10 ng/mL), human IL-1β (40 ng/mL), human IL-23 (40 ng/mL), human IL-6 (50 ng/mL) all diluted in a serum-free base media (supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 10 mM HEPES, 1 mM sodium pyruvate and 100 µM 2-mercaptoethanol).
   NOTE: Transfection and RNA interference does work in serum-containing media. The purpose of using serum-free media with this protocol is to achieve better IL-17A production.
6. Centrifuge the human CD4⁺ T cells at 500 x g for 5 min at 4 °C to pellet cells. Carefully aspirate the supernatant. Then resuspend the cells in serum-free base media and plate the cells at a density of 3 x 10⁶ in 1.5 mL per well so that the final volume is 3 mL per well and polarizing cytokines are now at a 1x final concentration.
   1. Place the plates in 5% CO₂, 37 °C incubator for two days.

2. Electroporation of In Vitro Polarized Human Th17 Cells

1. On Day 2, coat 48-well tissue culture plates with 250 µL 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 small RNAs for transfection. For each transfection, aliquot 1 µL of a 5 µM stock solution of siRNA into a 1.5 mL microcentrifuge tube. Include appropriate chemistry-matched small RNA control. Keep all tubes on ice.
3. Wash the antibody-coated plates two times with PBS. Then add 500 µL of 1X Th17-polarizing media to each well: anti-human IFNγ (10 µg/mL), anti-human IL-4 (10 µg/mL), human TGFβ (5 ng/mL), human IL-1β (20 ng/mL), human IL-23 (20 ng/mL), human IL-6 (25 ng/mL) all diluted in a serum-free base media (supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 10 mM HEPES, 1 mM sodium pyruvate and 100 µM 2-mercaptoethanol).
4. After the culture plates and transfection reagents are prepared, resuspend the cells with a 1 mL micropipette, pipetting gently but ensuring that all the cells are detached from the bottom of the wells. Pool the cells into a conical tube and centrifuge at 500 x g for 5 min at 4 °C.
   NOTE: For culture periods longer than the four-day protocol presented herein, the cells should be transfected approximately every 3 days using this same protocol. Step 2.4 should be modified however since the cells treated with different small RNAs should not be pooled. All of the conditions being tested must be collected, counted, and transfected separately.
5. Carefully aspirate the supernatant, and then resuspend the cells in at least 1 mL of PBS to wash. Count the live Th17 cells (e.g. with a hemocytometer using Trypan Blue exclusion to assess viability) and then transfer the cells to a microcentrifuge tube.
6. Centrifuge at 500 x g for 5 min at room temperature to pellet the cells.
7. Carefully aspirate the supernatant, and then resuspend the cells using the provided resuspension buffer from the transfection system 10 µL kit at a density of 2.5-4 x 10⁷ cells per mL. Keep cells at room temperature.
   NOTE: As a guideline, try to prepare only as many cells as can be transfected within 30 min. Multiple batches can be compared.
8. Add 9 µL of cells to 1 µL of small RNA in each microcentrifuge tube. Pipet once to mix the cells and small RNAs for transfection then load into the provided pipette electrode tip.
   NOTE: Typically, add 9.5 µL of cell suspension to ensure there is enough of the mixture to prevent creating bubbles in the pipet electrode tip prior to transfection.
9. Fill the provided cuvette with 3 mL of room temperature Electrolytic Buffer E. Place the cuvette inside the pipette station and then place the pipette into position inside the cuvette.
10. Immediately electroporate each 10 µL mix of cells and small RNA using the following parameters: pulse voltage 1,500-1,550 V, pulse width 10 ms, and 3 pulses total on the transfection device.
11. After the electroporation is complete, directly add the cell mixture to 500 µL of 1X Th17-polarizing media in prepared wells of a culture plate. Place plates in a 5% CO₂, 37 °C incubator for two more days.
    NOTE: Wash the pipette electrode tip by pipetting up and down in PBS in between each transfection.

3. Harvesting Human Th17 Cells

1. Resuspend the cells in culture media with a micropipette, pipetting gently but ensuring that all the cells are detached from the bottom of the wells. Prepare the cells for functional and/or gene expression assays.
   NOTE: Routinely, enough cells are yielded from a single well of transfected Th17 cells to be used for flow cytometric analysis of surface marker and intracellular cytokine and transcription factor staining or for preparation of RNA for gene expression analysis.

Wyniki

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...

Dyskusje

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...

Materiały

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