Isolation and Th17 Differentiation of Naïve CD4 T Lymphocytes

Summary

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether naïve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Abstract

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-β. After incubation for at least 72 hours and for up to five days at 37 °C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Introduction

CD4+ T lymphocytes (T cells) play a critical role in immune system-mediated defense against infectious microorganisms. Conversely, T cells are also intimately associated with the onset and progression of autoimmune diseases such as type 1 diabetes, systemic lupus erythematosus, and rheumatoid arthritis. CD4+ T lymphocytes become activated through a combination of T cell receptor (TCR) interactions with cognate antigen/major histocompatibility complex II (MHCII) molecules, and CD28 receptor interactions with B7.1/B7.2 ligands¹⁵. In addition to the provision of TCR stimulation and CD28 co-stimulation, antigen-presenting cells also provide a cytokine environment, which determines the differentiation state of the T lymphocyte, thereby directing the T lymphocyte's response to the given antigen. Distinct pathogen/antigen-presenting cell interactions create distinct cytokine environments, which skew T lymphocytes down distinct pathways focused on the elimination of the initiating pathogen. Unfortunately, T lymphocyte effector pathways, originally designed to eradicate invading pathogens, can be erroneously directed against self-tissues¹⁵. Therefore, better understanding of each distinct CD4+ T cell subset's differentiation state is critical for our understanding of how to modulate the balance between elimination of pathogens and tolerance to self.

In addition to the Th1, Th2, and inducible regulatory T cell differentiation pathways, naïve T lymphocytes can also be driven by cytokines down the Th17 pathway. Whereas Th1 cells combat intracellular pathogens, Th2 cells eliminate extracellular pathogens, and regulatory T cells (Tregs) minimize inflammatory responses^{1, 16}; Th17 cells play an important role in the elimination of extracellular bacteria and fungi. Th17 cells are generally denoted by expression of the lineage-specific transcription factor RORγT and production of IL-17A, which promotes the activation of macrophages and neutrophils^{1, 7}.

Th17 cells have been implicated in several autoimmune disorders, and their associated rodent models. For example, it has been demonstrated that IL-23 (which is required to sustain the Th17 phenotype), but not IL-12, was the central culprit in experimental autoimmune encephalitis (EAE), the rodent disease model for MS. It has subsequently been shown that reductions in IL-17 production are correlated to EAE prevention^{2, 6, 17}. Moreover, Th17 cells have been associated with other autoimmune disorders including arthritis and systemic lupus erythematosus (SLE)^{10, 16}. IL-23 deficient p19^-/- mice were shown to have very low numbers of Th17 cells, and are resistant to developing not only EAE, but also collagen-induced arthritis, a model for rheumatoid arthritis^{10, 18}. In addition, mice treated with neutralizing IL-17A antibodies after the onset of collagen-induced arthritis were also found to have resolution of joint damage¹⁸. It should be noted that the role of Th17 cells in the progression of autoimmune disease remains to be characterized as recent research has also shown a protective role of Th17 cells in Type 1 diabetes^{9, 11} and intestinal inflammation¹⁴. These studies confirm the importance of Th17 differentiation in autoimmunity.

In vitro Th17 differentiation is a necessary method in T cell research because there are at least two perplexing questions that require further investigation: 1) How exactly does IL-6 regulate the balance between Treg and Th17 differentiation, and 2) what are the exact mechanisms behind IL-17-induced inflammatory disorders? Our method employs CD4+CD25- T cells from the spleens and lymph nodes of the C57BL/6 mouse. It is important to note that although it is possible to induce Th17 differentiation using an impure population, acquiring at least an 80% pure CD4+CD25- T cell population negates any worry of contamination and ensures more successful Th17 differentiation results. In order to achieve proper Th17 differentiation, CD4+CD25- T cells are incubated in the presence of anti-CD3 and anti-CD28, which provide activation signals, 1 and 2, respectively, and IL-6, and TGF-β. Although it has been reported that IL-23 alone can be used to achieve Th17 differentiation, it was later demonstrated that IL-23 is necessary for the stability of the Th17 cell population, but IL-6 and TGF-β are essential for Th17 differentiation^{3, 18, 19}. Murine studies have shown that the IL-23 receptor is expressed on CD4+ T cells only after they have been stimulated with IL-6 and TGF-β^{13, 18}. Also, Th17 cells will successfully develop in the presence of IL-23-blocking antibodies as long as IL-6 and TGF-β are present^{18, 19}. As such, this Th17 differentiation protocol provides the appropriate conditions to successfully induce Th17 differentiation. Development of a better understanding of the mechanisms underlying Th17 differentiation and IL-17 production present the opportunity for the development of better therapeutics aimed at autoimmune disorders¹³.

Protocol

All animal use was conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee.

1. Preparation of Mixes and Media

1. Sterile PBS pH: 7.3 (1 L)
   0.23 g NaH₂PO₄
   1.15 g Na₂HPO₄
   9.0 g NaCl
   Take up volume with DI water. Sterilize with autoclave.
2. Cell Culture Media (100 ml)
   89 ml RPMI
   10 ml 10% FBS
   1 ml Antibiotic Antimycotic (ABAM) 100 μg 50 mM 2-mercaptoethanol (3.5 μg stock 2-mercaptoethanol into 96.5 μg PBS)
3. FACS Buffer (Fluorescent Activating Cell Sorting) (To be used during flow cytometry) 2% FBS in sterile PBS
4. iTh17 Mix (Based on number of samples, determine the volume needed for all mixes and media)
   1.5 μg/ml anti-CD28
   20 ng/ml IL-6
   5 ng/ml TGF-β

2. Cells Will Be Plated in Triplicate under the Following Conditions

1. Plate bound anti-CD3, anti-CD28 (this is the activation control mix).
2. iTH17: plate bound anti-CD3, anti-CD28, IL-6, TGF-β.

3. Plate-bound anti-CD3 (10 μg/ml)

It is recommended that preparation of plate-bound anti-CD3 plates is done at least 4 hr prior to the time cells will be added to the plates.

1. Add 30 μl anti-CD3 to wells (anti-CD3 is diluted in sterile PBS) of a new 96 well U bottom Microtest tissue culture plate, and tap the sides of the plate to ensure uniform coverage of the wells. Incubate at 37 °C for 4 hr, and then refrigerate until it is time to add the mixes and cells to the plate.

4. Mouse Dissection

1. This protocol is based on the use of C57BL/6 mice, 3-8 months of age, and purchased from The Jackson Laboratory (Bar Harbor, ME).
2. Sacrifice mouse using CO₂ asphyxiation, and confirm death with subsequent cervical dislocation.
3. Sterilize mice dissection tools and incision area with 70% ethanol and begin dissection.
4. From the ventral view, grab the skin that is anterior to the urethral opening and begin cutting with scissors up the ventral midline until reaching the chin area. Take precautions not to tear or cut into the lining of the peritoneal wall.
5. Pull back on the skin and pin down to allow comfortable access to lymph nodes during removal.
6. The lymph nodes and spleens collected will all be removed with forceps, and placed in a sterile Petri dish containing 5 ml of autoMACS Running Buffer purchased from Miltenyi Biotec (refer to Figures 2 and 3 for lymph node and spleen removal diagrams).
   1. Remove the axillary lymph nodes that are found near the axilla (armpit) behind the pectoral muscles of each mouse.
   2. Remove the brachial lymph nodes that are located in the connective tissues located near each axilla.
   3. Remove the superficial cervical lymph nodes found in the neck of each mouse.
   4. Remove the inguinal lymph nodes which are located in the hip region at the conjunction of the 3 blood vessels.
   5. To access the mesenteric lymph nodes, cut through the peritoneal lining up the ventral midline. Mesenteric lymph nodes are found in the connective tissue that holds the intestines together. They are generally found in a string of 4-8 nodes, and may appear as a "string of pearls". Make sure to pull out the entire string.
   6. The spleen is located in the abdominal area, behind the stomach and intestines. Remove it by pulling and detaching it from the pancreas.
7. Grind organs under a sterile hood using 2 frosted microscope slides. Place lymph nodes or spleen on the frosted side of one microscope slide, and rub with frosted side of the second slide until disintegrated. Repeat until all lymph nodes and spleen have been ground.

Note: It is recommended to start with the lymph nodes and finish with the spleen, as the blood will make it difficult to see the remaining lymph nodes in the autoMACs buffer.

9. To filter ground organs into a single cell suspension, begin by folding over a piece of 40 μm nylon material several times, and placing in the top of a 15 ml centrifuge tube. The nylon material should be roughly 3 x 3 in.
10. Use a 5 ml syringe and 21 G needle to aspirate the 5 ml of autoMACs Running buffer and ground organs. Dispense slowly into the folded nylon material. Take care to avoid puncturing the material with the needle.

Note: Once single cell suspension is achieved, the solution should appear as a pale, consistent solution with no visible pieces of solid tissue. If solids remain, re-aspirate and dispense through a new folded piece of nylon placed in a new 15 ml centrifuge tube.

11. Always keep cells on ice when not in use.

5. Cell Separation

1. For optimal results, obtain at least an 80% pure CD4+CD25- cell population through autoMACS Pro Cell Separator using the MACS CD4+CD25+ regulatory T cell isolation kit, mouse. The protocol for the negative retention of the CD4+CD25- cell population is obtained through Miltenyi Biotec.

6. Th17 Differentiation

1. Collect the CD4+CD25- cell population after separation with the autoMACS Pro Cell Separator.
2. Count cells diluted in a 1:1000 ratio with trypan blue using a hemocytometer.
3. Once concentration has been determined from cell counts, dilute cell suspension to 1 x 10⁶ cells/ml in cell culture media.
4. Take out 96 well plates with plate bound anti-CD3 after 4 hr.
5. Wash anti-CD3-coated wells with 200 μl of PBS. Repeat 2 times.
   1. To wash add 200 μl of sterile PBS to anti-CD3-coated wells and remove PBS into a waste container.
6. Add 100 μl of the iTh17 mix or activation control mix (see point #2) to the wells in triplicate.
7. Add 100 μl of cells to each well in which either the Th17 mix or the activation control mix has been placed.
8. Incubate cells for at least 72 hr or up to 5 days (Th17 differentiation can be obtained after 72 hr or after 5 days.)
9. Th17 differentiation can be assessed by intracellular staining and flow cytometric analysis, ELISA, or qPCR

7. Cell Activation (Only Necessary for Intracellular Staining)

1. Remove 96 well plates from incubators at the end of either 72 hr or 5 days
   1. Recall that Th17 differentiation can be achieved after either 72 hr or 5 days.
2. For each condition (e.g. activation control or iTh17), transfer cells that are in triplicate into one well of a 24 well cell culture plate. The cells for one condition have now been pooled into one well of the 24 well culture plate, as opposed to being in triplicate in the 96 well U bottom culture plate.
3. The total volume of each well in the 24 well cell culture plate is now 600 μl. Raise the volume of each well to 1 ml with the cell culture media.
4. Add PMA (phorbol myristate acetate) (50 ng/ml), ionomycin (1 μM), and BFA (Brefeldin-A) (10 μg/ml) to each well in the 24 well cell culture plate at the listed concentrations.
5. Incubate at 37 °C for 4 hr.

8. Intracellular Staining

1. Stain cells with the desired extracellular and intracellular markers for flow cytometric analysis. To detect the presence of IL-17, intracellular staining is done with anti-IL-17A antibodies. Recommended extracellular surface markers include CD4, CD8, and CD25.
   1. Use the Intracellular Cytokine Staining Starter Kit-Mouse from BD Bioscience for IL-17 Intracellular staining.
   2. For each sample, pellet cells, remove supernatant, and resuspend cell pellet in 200 μl FACS buffer (2% FBS in PBS).
   3. Transfer resuspended cells to 96 cell flow cytometry plate.
   4. Spin cells down for 5 min at 1,200 rpm and discard supernatant.
   5. Add 200 μl PBS FACS buffer, centrifuge for 5 min at 1,200 rpm, and discard supernatant.
   6. Resuspend cells in 100 μl of FACS buffer and apply 100 μl of extracellular antibody (Ab) mixture (extracellular Ab mixture is made in FACS buffer). Incubate for 15 min at RT, covered with foil.
   7. Repeat step 8.1.4.
   8. Repeat step 8.1.5 2x.
   9. Resuspend cells in 100 μl of BD Cytofix/Cytoperm Buffer. Incubate for 20 min at RT, covered with foil.
   10. Add 100 μl of 1x BD Perm/Wash buffer, centrifuge for 5 min at 1,200 rpm, and discard supernatant. Repeat.
   11. Add 50 μl of intracellular Ab mixture. (Intracellular Ab mixture is made in 1x BD Perm/Wash) Incubate for 15 min at RT, covered with foil.
   12. Repeat step 8.1.10.
   13. Resuspend cells in 200 μl of BD Staining Buffer.
   14. Place resuspended cells into flow cytometry tubes containing 200 μl BD Staining Buffer (final volume is 400 μl).
   15. Store at 4 °C until samples are ready to be read.

9. Flow Cytometric Analysis

1. Gate live cell population.
2. From the live cell population, gate on CD4+CD8- population.
3. From the CD4+CD8- population, gate on IL-17A+ population.
   1. Based on our previous experimental results, 100% of the IL-17A+ population will be CD25+.

*Total absolute cell counts were obtained after pooling the sample triplicates
**Absolute number of CD4+CD25+IL-17A+ cells was determined by multiplying the total number of cells by the live gate percentage and the percentage of total cells bearing the lineage-specific markers, CD4, CD25, and IL-17A, as determined by flow cytometry.

10. qPCR and ELISA

1. Place cells not used for flow cytometric analysis in a 1.5 ml Eppendorf tube and centrifuge at 13,000 rpm for 5-10 min. The cells found in the cell pellet after centrifugation will be used for qPCR analysis. qPCR analysis was performed using PTC-200 Peltier Thermal cycler (Biorad).
2. Collect supernatant from the centrifuged tubes for ELISAs. IL-17A ELISAs were performed using antibody pair TC11-18H10 (capture, catalog number 555068) and TC11-8H4 biotin (detection, catalog number 555067) purchased from BD Biosciences. IL-17A ELISA standards were obtained from eBioscience (catalog number 14-8171-80).
3. After removing the supernatant, resuspend the remaining cells to be used for qPCR in 175 μl RNA lysis buffer (use immediately for RNA extraction, cDNA synthesis, and qPCR, or store at -80 °C for later use).
   1. Refer to Table II for primer sequences for IL-17A and actin (house-keeping gene)

Results

This Th17 differentiation protocol begins with the removal of the spleen and the axillary, brachial, mesenteric, cervical, and inguinal lymph nodes. A representation of the locations of each can be found in Figures 2 and 3. Figures 1 and 5 both provide a visual representation of the methods described in this protocol.

This Th17 protocol focuses on differentiation from the CD4+CD25- T lymphocyte population. It is important to note how efficie...

Discussion

Here we have described the protocol to achieve in vitro Th17 differentiation. The study of Th17 differentiation is important, as differentiation of T lymphocytes into the Th17 subset is critical for the effective elimination of human pathogens¹³. Conversely, IL17 production has been associated with autoimmune disease progression¹³. The method of Th17 differentiation is applicable to many research settings as it can be applied to numerous distinct murine models of immune regulation and autoi...

Materials

Name	Company	Catalog Number	Comments
			Reagent/Material
Sterile Polyestrene Petri Dish	Fisher Scientific	0875713A	60 mm x 15 mm
autoMACS Running Buffer	Miltenyi Biotec	130-091-221
Premium Microscope Slides, Frosted	Fisher Scientific	12-544-3	3" x 1"1 mm
5 ml 21G1 Latex Free Syringe and Needle	BD Biosciences	309632
Corning 15 ml Centrifuge Tubes	Sigma-Aldrich	CLS430791
Nylon 40 microns	Miami Aquaculture	Nylon 40/26
Microtest tissue culture plate 96 well U bottom	BD Biosciences	35-3077
Corning Costar 24 well cell culture plates	Sigma-Aldrich	CL3524
Eppendorf Tubes 1.5 ml	Fisher Scientific	05-408-129
Purified NA/LE Hamster Anti-Mouse CD3e	BD Biosciences	553057
Purified NA/LE Hamster Anti-Mouse CD28	BD Biosciences	553294
Mouse IL-6 Recombinant Protein	eBioscience	14-8061-62
TGFbeta	R&D Systems	240-B-002
Trypan blue solution 0.4 %	Sigma-Aldrich	66H2364
Pac Blue Rat Anti-Mouse CD4	BD Biosciences	558107
APC Rat Anti-Mouse CD8a	BD Biosciences	553035
PE Conjugated Anti-mouse CD25	eBioscience	E01155-516
Alexa Fluor 700 Rat Anti-mouse IL-17	BD Biosciences	560820
Intracellular Cytokine Staining Starter Kit-Mouse	BD Biosciences	51-2041AK (559311)
MACS CD4+CD25+ regulatory T cell isolation kit, mouse	Miltenyi Biotec	130-091-221
ABAM	Cellgro	30-004-CI
RPMI	Corning, Cellgro	10-040-CM
B 2-MercaptoEthanol	MP Biomedical	194834	Hazardous
Phorbol 12-Myristate 13-Acetate (PMA)
Ionomycin Calcium Salt	Sigma-Aldrich	13909-1ML	Hazardous
Brefeldin A (BFA)	MP Biomedicals	159027
ELISA IL-17A Capture mAb	BD Biosciences	555068
ELISA IL-17A Detection mAb	BD Biosciences	555067
ELISA IL-17A Standard	eBioscience	14-8171-80
IL-2 ELISA Kit	BD Biosciences	555148
TMB Substrate Reagent Set	BD Biosciences	555214
			Equipment
autoMACS Pro Cell Separator	Miltenyi Biotec	130-092-545
Sorvall Legend RT+ Centrifuge	ThermoScientific
Napco series 8000 WJ CO2 Incubator	ThermoScientific
PTC-200 Peltier Thermal Cycler	Biorad