In Vitro Analyses of T Cell Effector Differentiation

Abstract

In vitro culture is an important complement, or substitute, to in vivo approaches in order to study T cell effector differentiation. Here, we describe culture conditions that generate specific effector cell types by exposing naïve T cells to appropriate cytokine signals.

1. Introduction

Effective T cell function is required for protection from invading pathogens. T cell effector differentiation is determined by several signals, notably from the innate immune cells, including (1) stimulation through the T cell receptor (TCR), (2) co-stimulation through the CD28 costimulatory molecule and (3) cytokine exposure that induces acquisition of specific T cell differentiation. Studies exploring how naïve T cells differentiate into fully functional effector cells often require assessing the effector potential of these T cells; protocols for such analyses are presented in this chapter.

During in vivo immune responses, effector T cell differentiation occurs in secondary lymphoid organs or tissues, and is driven by architecturally constrained interactions between T cells, antigen-presenting cells (APC)s and other immune cells. Early stages of an immune response involve small cell numbers of antigen-specific T cells, which may be difficult to identify and purify. Thus, it can be advantageous to reproduce the conditions that promote T cell effector differentiation in vitro. In vitro differentiation allows acquisition of a large number of cells, e.g., for biochemical or gene expression studies. In addition, in vitro studies allow for tighter control of cytokines and other stimuli offered to T cells. These in vitro cultures typically include two distinct stimuli. First, a ligand for the T cell antigen receptor complex is required to promote expression of specific cytokine receptors (e.g., for IL-2) and cell proliferation. This can be a specific peptide antigen if using cells of defined specificity (e.g., carrying a TCR transgene); in many instances however, antibodies against TCR or CD3 are used to mimic antigen stimuli and trigger TCR signaling. In both cases, TCR stimulation needs to be accompanied by engagement of CD28 costimulatory molecules. The second series of ligands is intended to direct cytokine gene expression; it includes cytokines and anti-cytokine antibodies to neutralize the effect of unwanted cytokines. Appropriate combinations of these reagents typically “polarize” T cell differentiation into specific effector fates: for CD4 cells, these generally include Th1 [1], Th2 [1 – 3], Th17, and inducible (i) T regulatory (Treg); and for CD8⁺ cytotoxic T (Tc) cells (see Table 1 and refs. 2, 4, 5). While it is possible to activate highly purified T cells with the latter reagent combinations, using separately purified APCs as “feeder” cells for the differentiating effector T cells results in greater survival for most effector types [6].

Table 1.

Effector differentiation in vitro

Conditioning mix	Cytokines (2x concentrations)	Blocking antibodies (2x concentration)	Cytokines (final concentration)	Blocking antibodies (final concentration)
ThN	IL-2 (20 ng/ml)	-	IL-2 (10 ng/ml)	-
Tc	IL-2 (20 ng/ml)	-	IL-2 (10 ng/ml)	-
Th17	IL-6 (20 ng/ml) TGF-β (5 ng/ml)	Anti-IL-4 (20 μg/ml) Anti-IFNγ (20 μg/ml) Anti-IL-12 (20 μg/ml)	IL-6 (10 ng/ml) TGF-β (2.5 ng/ ml)	Anti-IL-4 (10 μg/ml) Anti-IFNγ (10 μg/ml) Anti-IL-12 (10 μg/ml)
+Th0	IL-2 (20 ng/ml)	Anti-IL-12 (20 μg/ml) Anti-IFNγ (20 μg/ml) Anti-IL-4 (20 μg/ml)	IL-2 (10 ng/ml)	Anti-IL-12 (10 μg/ml) Anti-IFNγ (10 μg/ml) Anti-IL-4 (10 μg/ml)
Th1	IL-2 (20 ng/ml) IL-12 (20 ng/ml)	Anti-IL-4 (20 μg/ml)	IL-2 (10 ng/ml) IL-12 (10 ng/ ml)	Anti-IL-4 (10 μg/ml)
Th2	IL-2 (20 ng/ml) IL-4 (20 ng/ml)	Anti-IL-12 (20 μg/ml) Anti-IFNγ (20 μg/ml)	IL-2 (10 ng/ml) IL-4 (10 ng/ml)	Anti-IL-12 (10 μg/ml) Anti-IFNγ (10 μg/ml)
iTreg	TGF-β (5 ng/ml) IL-2 (20 ng/ml)	Anti-IL-4 (20 μg/ml) Anti-IFNγ (20 μg/ml)	TGF-β (2.5 ng/ ml) IL-2 (10 ng/ml)a	Anti-IL-4 (10 μg/ml) Anti-IFNγ (10 μg/ml)

^aIL-2 is added at 48 h

During in vitro culture, antigenic stimulation (or its surrogate) and cytokines drive cell proliferation, which typically starts within 24–36 h after stimulation and continues for 3 or 4 days. Expression of cytokine genes, and cytokine production (evaluated by ELISA or intracellular cytokine staining) is detected within 3–5 days of stimulation, depending on the type of cytokine. Similar kinetics are observed for fate-determining transcription factors.

Although the choice of the starting T cell population is typically dictated by the specific application, special emphasis must be placed on separating naïve from antigen-experienced (generally referred to as “memory”) cells obtained from peripheral lymphoid organs. In laboratory mice housed under specific pathogen-free conditions, most spleen and lymph node T cells are “naïve.” That is, they are directly derived from thymic precursors without having encountered the antigen that their TCR specifically reacts against and therefore, they do not express effector (e.g., cytokine) genes. A simple conceptual example of a naive cell is a lymphocyte carrying a TCR directed against a virus-derived peptide in a host that has not been in contact with that particular virus. In contrast, upon infection or immunization with the cognate antigen, these newly “antigen-experienced” cells proliferate, and acquire effector properties or differentiate into memory cells. In unmanipulated laboratory mice, cells exhibiting marks of antigen experience are typically reactive against commensal and environmental antigens.

Regardless of whether they are effector or memory, antigen-experienced cells have two properties not shared by naïve cells. They are generally “preprogrammed” to produce specific cytokines (most antigen-experienced cells in a mouse spleen make IFNγ), and they produce these cytokines quickly, within hours of antigen receptor triggering. In contrast, naïve cells typically do not produce effector cytokines until they have undergone multiple rounds of proliferation, and their effector differentiation is heavily influenced by the surrounding cytokine milieu (Table 2). Thus, in activation cultures, the cytokines produced by antigen-experienced cells have the potential to skew, often towards IFNγ production, the effector differentiation of the naïve cells. To avoid this potential bias, it is generally advisable to purify naïve cells for in vitro stimulation cultures. This is all the more necessary when such cells are prepared from inflammatory contexts, in which the frequency of effector or memory cells is much higher. For both CD4⁺ and CD8⁺ mouse T cells, CD44 is the most commonly used marker to distinguish naïve (CD44^lo) from antigen experienced (CD44^hi) subsets.

Table 2.

Effector differentiation in vivo

	Cytokine for differentiation	Transcription factor	Cytokine produced
Th1	IL-12	Tbet	IFNγ
Th2	IL-4	Gata3	IL-4
			IL-5
			IL-13
Th17	IL-6	RORγt	IL-17A
	TGF-β		IL-17F
	IL-1		IL-22
	IL-21
iTreg	TGF-β	Foxp3	IL-10
	Retinoic acid		TGF-β
	IL-2
Tc	IFNγ	Tbet	IFNγ

Here we delineate protocols designed to evaluate the effector potential of naïve T cells exposed to conditions that partially mimic in vivo antigen stimulation. These protocols are designed to differentiate naïve T cells into CD4⁺ iTregs and CD4⁺ and CD8⁺ T effector cells. Because of the versatility of in vitro cultures, the choice of appropriate controls is crucial to establish sound conclusions. We have provided suggestions in Subheading 4 in this regard.

The protocols described below use antibody (anti-CD3)-mediated TCR triggering as a surrogate for antigen stimulation, APCs (either dendritic cells or T cell-depleted splenocytes), and mixes of cytokines and anti-cytokine antibodies appropriate for promoting the differentiation of Th1, Th2, or Th17 CD4⁺ effectors, Tc CD8⁺ effectors, or CD4⁺ iTreg cells. We also provide an alternate protocol that eliminates the APCs.

2. Materials

2.1. Blocking Antibodies

1. Anti-CD3e (without azide, unlabeled), Clone 145–2C11.

2. Anti-CD28 (without azide, unlabeled), Clone 37.51.

3. Anti-IFN-γ (without azide, unlabeled), Clone XMG1.2.

4. Anti-IL-4 (without azide, unlabeled), Clone 11B11.

5. Anti-IL-12 p40/p70 (without azide, unlabeled), Clone C17.8.

2.2. Cytokines

1. Recombinant murine IL-2.

2. Recombinant murine IL-6.

3. Recombinant human TGF-β.

4. Recombinant murine IL-4.

5. Recombinant murine IL-12.

2.3. Beads

1. Mouse pan T (CD90.2) depletion magnetic beads (see Note 1).

2.4. Media Components

1. Fetal calf serum (FCS) (see Note 2).

2. Phosphate buffered saline (PBS, Ca²⁺ Mg²⁺ free) pH 7.4.

3. Iscove’s Modified Dulbecco’s Medium (IMDM) (see Note 3).

4. Culture medium: 10 % FCS, 100 U/ml of penicillin, 0.10 mg/ml of streptomycin, 0.292 mg/ml of l-glutamine, 10 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid) pH range 7.2–7.5 (see Note 4), 55 μM 2-Mercaptoethanol (see Note 5), 1 mM Sodium pyruvate (see Note 6), 1× MEM nonessential amino acids (NEAA) (see Note 7), in RPMI 1640.

5. Digestion medium: 100 U/ml of penicillin, 0.10 mg/ml of streptomycin, 0.292 mg/ml of l-glutamine, 10 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid) pH range 7.2–7.5 (see Note 4), 0.25 mg/ml of deoxyribonuclease I from bovine pancreas ≥85 % protein, and 0.5 mg/ml of Liberase TL (Roche), in RPMI 1640.

6. 500 mM ethylenediamine tetraacetate (EDTA) pH 8.0.

7. Staining medium: PBS (Ca²⁺ Mg²⁺ free) pH 7.4, 0.1 % bovine serum albumin (BSA), 2 mM EDTA pH 8.0.

8. Isolation medium: PBS (Ca²⁺ Mg²⁺ free) pH 7.4, 0.1 % BSA, 2 mM EDTA pH 8.0.

9. Red cell lysis buffer: 8290 mg/l ammonium chloride, 1 g/l potassium bicarbonate, 37 mg/l EDTA pH 8.0.

2.5. Hardware

1. 96-well flat bottom tissue culture treated plates.

2. 96-well round bottom tissue culture treated plates.

3. Cell scraper.

4. 70 μm cell strainers.

5. Nylon tissue filters (pore size 100 μm, cut to 2 or 4 cm²).

6. 1 or 3 ml syringe pestles.

7. 60 mm petri dishes.

8. 15 and 50 ml conical tubes.

9. Magnetic beads compatible with appropriate purified antibodies.

10. Rocker.

11. Two curved forceps.

12. Scissors appropriate for dissection.

13. 37 °C incubator with 5 % CO₂.

14. Vertical magnet holding 15 or 50 ml conical tube (DynaMag 15, 50, or equivalent).

15. 5 ml tuberculin syringe.

2.6. Fluorescent Antibodies for Flow Cell Sorting (See Note 8)

1. Anti-CD4, Clone RM-4.5.

2. Anti-CD44, Clone 1M7.

3. Anti-CD25, Clone 7D4.

4. Anti-CD11c, Clone N418.

5. Anti-MHC Class II (see Note 9).

2.7. Animals (See Note 10)

1. Mice of interest for preparation of T cells.

2. Mice of interest for preparation of dendritic cells or T-depleted splenocytes (see Note 11).

3. Methods

Subheadings 3.1–3.3 describe a protocol to stimulate T cells in the presence of antigen presenting cells (APCs). The protocol involves three distinct steps: (1) prepare a suspension of purified APCs to which antibody and cytokines will be added to form the “conditioning mix”; (2) isolate naïve T cells; and (3) set up cultures by mixing the APC (in the conditioning mix) and T cell suspensions. Addition of antibody and cytokines to APCs, to form conditioning mixes, should be performed last, immediately before setting up the cultures and aliquoted into the culture wells promptly.

Subheading 3.4 presents an alternative protocol for T cell stimulation on antibody-coated plates, without APCs.

3.1. Preparation of APCs

Carry out all procedures at 4 °C unless otherwise specified. Completing the procedures outlined in the section below produces a suspension of APCs in 100 μl per 96 well to which the conditioning mixes are added. Adding the conditioning mixes to the APCs streamlines the procedure.

3.1.1. Isolation of Splenic CD11c⁺ DC

For naïve T cells conditioned in Th0 Th1, Th2, Th17, or Tc conditions: use a ratio of 25–50 T cells for every DC. iTregs Conditioning mix should include a 5–25 ratio of T cells for every DC cultured (see Note 12). The following procedure is for one spleen and volumes can be scaled proportionally as needed. All work should be performed under sterile conditions in a tissue culture cabinet.

1. Place 5 ml of Digestion medium into a 60 mm petri dish. Surgically remove the spleen from a euthanized mouse and place directly into Digestion medium.

2. While holding the spleen with forceps, inject spleen with the Digestion medium using a tuberculin syringe. Then, using scissors, cut spleen into five to ten smaller pieces.

3. Place in 37 °C incubator with 5 % CO₂ for 20 min.

4. Add a final concentration of 5 mM EDTA to the dish and incubate five additional minutes to stop the enzymic activity of the Digestion medium.

5. Transfer spleen pieces and medium to a 50 ml conical tube and pass through a 70 μm cell strainer. Use cell scraper to remove any adherent cells and rinse the petri dish thoroughly with 5 ml Staining medium.

6. Lyse with 1 ml Red cell lysis buffer for 2 min on ice, wash by filling tube to 10 ml with Staining medium.

7. Resuspend in 5 ml Staining medium and count cells.

8. Refer to Chapter 7 on “Isolating T cell subsets” for staining cells with fluorescently labeled antibodies and sorting CD11c⁺ MHC II⁺.

9. Prepare DC suspensions in Culture medium at a concentration of 1–2 × 10³ cells/100 μl/well for Th0, Th1, Th2, Th17, or Tc cultures, or of 2–10 × 10³ cells/100 μl/well for iTreg cultures (see introduction to this section and Fig. 1 for more information). Refrain from aliquoting cells into wells at this point as the Conditioning mix will be added to this master mix. Keep DCs on ice until ready to prepare Conditioning mixes.

10. See Subheading 3.3 for preparing Conditioning mixes.

Fig. 1.

Schematic of procedure

3.1.2. Irradiated T Cell-Depleted Splenocytes

Carry out all procedures at 4 °C unless otherwise specified and under sterile conditions in a tissue culture cabinet. The ideal ratio for this type of APC is 5 for every one T cell.

1. See Chapter 7 on “Isolating T cell subsets” for procedure on isolating a single-cell suspension from a mouse spleen.

2. Immunomagnetic cell separation for depletion of pan T cells. The following protocol is for a 1×10⁸ total splenocyte suspension, and can be scaled up or down as necessary. While this protocol utilizes immunomagnetic beads from Dynabead, users may employ immunomagnetic beads of their choice with the appropriate protocol.

3. Preparation of Dynabeads: Resuspend beads fully (either vortex whole vial or place on a tilt rotator for at least 5 min).

4. Remove 1 × 10⁸ beads and wash in equal volume of Isolation buffer in a fresh tube. Use 1 ml of Isolation buffer to wash if bead volume is under 1 ml.

5. Place tube without lid in DynaMag-15 (or other magnet suitable for bead isolation procedures) for 1 min and discard the supernatant.

6. Remove tube from magnet and resuspend beads in the original starting volume of Isolation buffer.

7. Preparation of pan T cell-depleted splenocytes: Spin down 1 × 10⁸ splenocytes in a separate tube and resuspend in 1 ml of Isolation buffer.

8. Add 1 × 10⁸ pan T cell CD90.2 Dynabeads to cells and incubate for 30 min at 4 °C.

9. Place tube containing cell and bead mixture without lid onto a magnet for 2 min.

10. The T cells will bind to the beads and be removed from the supernatant. Move T-depleted supernatant in a fresh tube, wash with Culture medium, spin down and resuspend in Culture medium to count.

11. Irradiate T-depleted splenocytes with 3,000 RADS (see Note 13 on irradiator use).

12. Make a master mix of 2.5 × 10⁵ T cell-depleted splenocytes in 100 μl Culture medium per well (see introduction to this section and Fig. 1 for more information). Refrain from aliquoting cells into wells at this point as the Conditioning mix will be added to this master mix. Keep DCs on ice until ready to aliquot for culture.

13. See Subheading 3.3 for preparing Conditioning mixes.

3.2. T Cell Preparation

Carry out all procedures at 4 °C unless otherwise specified.

1. Isolate T cells from spleens and lymph nodes using either flow cytometry sorting or alternatively, an immunomagnetic bead selection approach (see Chapter 7 on “Isolating T cell subsets”, see Note 14 on caveats to using bead selection over flow sorting.)

2. Resuspend T cells at 5.0 × 10⁴/100 μl in Culture medium. Keep this on ice until ready to aliquot for culture.

3.3. Conditioning Mixes

To the APCs prepared in Subheading 3.1, add the following reagents to prepare the corresponding 2× Conditioning mixes. These should be prepared immediately before setting up cultures and kept on ice until ready to aliquot for culture. Note that all concentrations given below refer to the concentration in the conditioning mix, therefore twice as high as in the final culture (see Table 1).

3.3.1. ThN or CD8⁺ Tc Conditioning Mix

1. Add: 2 μg/ml anti-CD3, 6 μg/ml anti-CD28, 20 ng/ml IL-2.

3.3.2. Th17 Conditioning Mix

Substituting IMDM for RPMI increases the frequency of IL-17 producing cells (see Note 7). The Th17 Conditioning mix does not contain IL-2.

1. Add: 2 μg/ml anti-CD3, 6 μg/ml anti-CD28, 20 ng/ml IL-6, 5.0 ng/ml TGF-β, 20 μg/ml anti-IL-4, 20 μg/ml anti-IFN-γ, 20 μg/ml anti-IL-12.

3.3.3. Th0 Conditioning Mix

1. Add: 2 μg/ml anti-CD3, 6 μg/ml anti-CD28, 20 ng/ml IL-2, 20 μg/ml anti-IL-12, 20 μg/ml anti-IFNγ, 20 μg/ml anti-IL-4.

3.3.4. Th1 Conditioning Mix

1. Add: 2 μg/ml anti-CD3, 6 μg/ml anti-CD28, 20 ng/ml IL-2, 20 ng/ml IL-12, 20 μg/ml anti-IL-4.

3.3.5. Th2 Conditioning Mix

1. Add: 2 μg/ml anti-CD3, 6 μg/ml anti-CD28, 20 ng/ml IL-2, 20 ng/ml IL-4, 20 μg/ml anti-IL-12, 20 μg/ml anti-IFNγ (see Note 15).

3.3.6. iTreg Conditioning Mix

1. Add: 2 μg/ml anti-CD3, 5.0 ng/ml TGF-β, 20 μg/ml antiIL-4, 20 μg/ml anti-IFNγ (see Note 16). (Retinoic acid can be added to the culture to enhance the induction of Foxp3, see Note 17.)

2. At 48 h, add 10 ng/ml IL-2 (final concentration) and incubate for an additional 24 h. This is earlier than the day 3 culture split recommended for non-iTreg cultures (see Subheading 3.5).

3.3.7. Culture Set Up

1. Add 100 μl/well of each APC-containing conditioning mix to wells in a 96 well round bottom tissue culture plate.

2. Add 100 μl T cell suspension (5 × 10⁴ cells) to each APC-containing well. The final volume should now be 200 μl per well.

3. Place in a 37 °C incubator with 5 % CO₂ for 3 days (see Subheading 3.5) (see Note 18).

3.4. Alternate Protocol: Plate-Bound Anti-CD3/CD28 Stimulation

Plate-bound anti-CD3/CD28 antibody stimulation is useful when examining direct effects on T cells without the complications of a different cell population. Note that anti-CD28 should be omitted for iTreg conditions (see Note 16). T cell preparation in this alternate protocol is performed as in Subheading 3.2. If using this protocol, additional anti-CD3/CD28 in the Conditioning mix is not recommended, as the plate-bound anti-CD3/CD28 is sufficient.

3.4.1. Preparation of Anti-CD3 and Anti-CD28 Coated Plates

1. Make a solution of 1 μg/ml of anti-CD3 antibody and 1 μg/ml anti-CD28 antibody in room temperature PBS.

2. Immediately aliquot 100 μl into each well.

3. Place at 4 °C overnight or 1 h at 37 °C.

4. Wash plate two times with PBS by gently adding to the side of the wells and aspirating off. Wash last time in Culture medium taking care to not let the wells dry out. If necessary, leave the last Culture medium wash in the well until the T cells are isolated and the Conditioning mixes are prepared (see Subheadings 3.2 and 3.3).

3.4.2. Preparation of APC-Free Conditioning Mixes

Prepare 2× cell-free conditioning mixes for each stimulation condition by supplementing APC-free Culture medium with cytokines and anti-cytokine antibodies (but not soluble anti-CD3 and soluble anti-CD28) using concentrations indicated in Subheading 3.3 above

(see also Table 1).

3.4.3. Culture Set Up

1. Per well in a 96 well fl at bottom tissue culture plate, add 100 μl/well of APC-free conditioning mix and 100 μl T cell suspension (5 × 10⁴ cells, prepared as in Subheading 3.2), for a final volume of 200 μl per well.

2. Place in a 37 °C incubator with 5 % CO ₂ for 72 h (see Subheading 3.5).

3.5. Split the Culture at Day 3

1. Incubate for 72 h and then split each 200 μl well into two 100 μl wells and add 100 μl of Culture medium to each well.

2. Incubate an additional 24 h.

3. Harvest cells for downstream applications.

4. Notes

1. Syngeneic mouse strains express either CD90.1 or CD90.2 allelic isoforms. Beads exist against each isoform and should be chosen according to the mouse strain used to prepare APCs.

2. Fetal calf serum should be heat inactivated by incubation at 56 °C for 50 min. Serum from different sources and even different lots can vary in efficacy. Each lot should be tested as it may differentially support T generation and cytokine production. Companies usually offer samples for this purpose.

3. Using IMDM in Th17 culture increases the efficacy of Th17 differentiation. This medium contains natural agonists for aryl hydrocarbon receptor that support differentiation of Th17 cells [7].

4. HEPES extends media stability outside of a CO₂ incubator.

5. 2-Mercaptoethanol reduces oxygen radicals in Culture medium. This chemical is not stable in solution and should be added immediately before use.

6. While not essential for cell growth, Sodium pyruvate is beneficial for most effector differentiation and especially recommended for iTreg induction cultures.

7. NEAA are not required but promote cell growth and viability.

8. The fluorochrome choice for antibodies for fl ow cytometric sorting of populations should be carefully considered during planning. These antibodies can remain on cells and interfere with secondary staining post culture.

9. Choose an antibody that will recognize MHC-II isoforms expressed in the mouse strain used as a source of CD11c⁺ DCs.

10. Animals must be housed, handled, and used according to applicable guidelines and after securing required authorizations.

11. APCs (either dendritic cells or T cell-depleted splenocytes) should come from animals that are sex and strain matched to the experimental T cells.

12. Optimal ratio of T cells to APC (i.e., DCs or T-depleted spleen) should be determined for each specific situation. The ratios suggested here are starting points that may need to be optimized.

13. Secure required authorization training before irradiator use, and follow applicable guidelines.

14. Most bead isolation kits for CD4⁺ or CD8⁺ T do not remove CD44 ^hi (memory or effector cells). Upon antigen stimulation (anti-CD3 exposure in culture), cytokines produced by CD44^hi cells can bias cell differentiation independently from those included in the conditioning medium. It is therefore preferable to exclude CD44^hi cells from in vitro cultures.

15. Overgrowing the cells will result in a loss of IL-4 production. Split them earlier if they are turning yellow, or start from lower cell numbers.

16. iTreg conditions should include polarizing cytokines and blocking antibodies, but no anti-CD28.

17. Retinoic Acid enhances induction of de novo Foxp3-expressing cells [8–10].

18. When plating cells, avoid wells at the edge of the plate—these wells will experience more evaporation. To the surrounding wells add equal volumes of your final culture volume of sterile PBS or water to help reduce evaporation over the course of your cultures.