Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Summary

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

Abstract

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGFβ and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.

Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44^low, CD62L^high) and resting (CD25^-, CD69^-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Introduction

Tregs are crucial to maintain immune tolerance and to dampen overshooting immune responses. Tregs suppress bystander T cell activation. Consequently, ablation of Tregs leads to fatal autoimmunity and self-destruction driven by activated T cells¹. Tregs develop in the thymus during negative selection of CD4 single-positive precursors, but they can also differentiate in the periphery from naive CD4 T cells upon low-dose antigen stimulation with suboptimal co-stimulation ^1,2. Thymic Tregs seem to suppress tissue autoimmunity against self-antigens, whereas peripheral Tregs have been implicated in providing tolerance in the gut or lung. These induced Tregs potently prevent T cell activation after recognition of foreign antigens in the mucosa, including environmental antigens from food and air, commensal bacteria, and allergens ^3,4. In addition, Tregs are crucial to establish maternal tolerance to fetal peptides ⁵ and to prevent graft-versus-host disease ⁶. At the same time, Tregs also mediate unwanted effects by attenuating immune surveillance of tumor cells ^7,8. The hallmark feature of Tregs is the expression of the subset-specifying transcription factor Foxp3, a fork-head domain-containing transcription factor that is necessary and sufficient to confer Treg function ^9,10. Some signaling pathways that can induce Foxp3 expression are known. However, the molecular processes that control, regulate, or modulate Treg differentiation in response to T cell receptor triggering are less well understood.

Tregs can very effectively be induced in vitro through the stimulation of naive CD4 T cells with anti-CD3 and anti-CD28 antibodies in the presence of TGFβ and IL-2 ¹¹. As the emerging Tregs are functional in vivo, the manipulation of molecules that promote Treg differentiation bears enormous potential for future therapies, for example, the treatment of asthma, Crohns disease, and transplantation ^11,12. Conversely, therapeutic modulation of molecules to block Treg differentiation may provide benefit in future approaches of combined treatment of tumor patients.

In vitro differentiation assays have been instrumental for the description of molecular changes that are associated with T cell subset differentiation. At the moment, experimental attempts to search or screen for gene products that control T cell differentiation are hampered by the fact that the most common methods of ectopic gene expression fail in naive T cells. For example, electroporation and retroviral transduction are only effective in activated T cells. In contrast to initial expectations, lentiviral transduction, which is typically effective in resting cells, requires pre-activation of naive T cells by cytokines¹³. Furthermore, the transfer of cDNA or mRNA during electroporation involves depolarization of the plasma membrane, which itself confers features of T cell activation and may even mobilize Ca²⁺ signaling and activate NFAT proteins (unpublished observation and ref. ¹⁴). Similarly, for retroviral transduction, the naive T cells have to be activated for 18 - 40 hr. During this time, the breakdown of the nuclear membrane in the course of cell division occurs and allows for the subsequent genomic integration of the retroviral vector ¹⁵. These methods are therefore not able to address the early molecular regulation of initial T cell encounter with antigen, which is the decisive phase of helper T cell differentiation.

Adenoviral transduction is known to confer transient ectopic gene expression in a number of human cell types that express the human Coxsackie adenovirus receptor (CAR). It proceeds without requirement for cell activation or cell-cycle progression. The surface expression of CAR is essential for efficient virus attachment and internalization, and transgenic expression of the truncated version CARΔ1 under a T cell-specific promoter was found to render mouse thymocytes and T cells susceptible to adenoviral infection ¹⁶. Importantly, the transgene does not alter thymocyte development or in vitro differentiation of naive CD4 T cells into different subsets (data not shown; ref. ¹⁷). Adenovirus-mediated transduction of T cells was previously used for overexpression ^17,18 and knock-down approaches ^19,20. The transgenic T cells can be purified from commercially available DO11.10 tg; CARΔ1 tg (Taconic, Inc. and ref. ¹⁷). Importantly, adenoviral transduction allows high expression of a gene of interest in naive T cells without inducing obvious signs of activation. The T cells remain naive (CD44^low, CD62L^high) and resting (CD25^-, CD69^-) after infection and can be activated and differentiated into Treg similar to non-infected cells.

Production of recombinant adenoviruses can be achieved after transfection of HEK293A cells with adenoviral plasmids (Figure 1). These plasmids typically contain the human type 5 adenovirus genome with E1 and E3 genes deleted to render recombinant adenoviruses replication-incompetent ²¹. HEK293A cells complement replication deficiency as they have been immortalized through stable integration of sheared adenovirus ²². Since adenoviral vectors are large (~40 kb) and consequently not well suited for traditional restriction enzyme-mediated cloning, we employed the Gateway system. The gene of interest is initially cloned into a smaller entry vector, from which it can be easily transferred into the adenoviral destination vector via lambda recombination reaction (LR) ²³. We constructed the pCAGAdDu vector by combining the CAG promoter (chicken actin promoter and CMV enhancer) with an expression cassette containing LR sites flanking the procaryotic ccdB selection marker ²⁴ . This expression cassette is fused to an internal ribosome entry site (IRES) element that allows coexpression of the eukaryotic infection marker enhanced green fluorescent protein (eGFP), which is fused to a sequence containing the bovine growth hormone poly(A)-signal. We chose the CAG cis-regulatory sequences, since the prototypic CMV promoter was found to be highly activation-dependent and therefore unfavorable for gene expression in naive T cells.

Here, we provide a protocol for efficient in vitro Treg differentiation and a method to transduce naive CD4 T cells without activation (Figure 2). The method enables ectopic gene expression or knock down preceding CD4 T cell differentiation at the naive state. It allows testing the effect of an overexpressed gene of interest during early signaling events upon initial TCR stimulation until T cell subset commitment. Our validation experiments also provide the basis to establish similar adenovirus application in the differentiation of other T cell subsets such as Th1, Th2, Th9, Th17, Th22, or Tfh cells.

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Protocol

1. Cloning of a Gene of Interest into an Entry Vector

1. Clone the gene of interest into an entry vector. PCR amplification of the gene followed by blunt-end ligation into a topoisomerase-coupled vector (e.g. pENTR/D-TOPO) or restriction enzyme-mediated cloning may be used for this procedure.

2. Transferring the Gene of Interest into the pCAGAdDu Destination Vector

1. Transfer the gene of interest from the entry vector into the destination vector by LR recombination (e.g. Gateway LR Clonase II Enzyme Mix). This will create the adenoviral expression vector (Figure 1).
2. Linearize 10 μg of the adenoviral expression vector in a PacI restriction digest, precipitate the DNA and resuspend it in water at a concentration of 3 μg per 100 μl. Linearization liberates the viral inverted repeats (ITR), which are required for replication and encapsidation of the viral DNA into virus particles.

3. Generation of the Primary Virus Lysate

1. Seed 1 x 10⁵ HEK293A cells in 2 ml cell culture media (DMEM, 10% FBS, 5% PenStrep) in one well of a 6-well plate and incubate the cells for 6 - 14 hr at 37 °C in a 10% CO₂ incubator to allow them to adhere. Cells should then be at approximately 50% confluency.
2. Lipofection: Transfer 6 μl of jetPEI reagent into 94 μl of 50 μM NaCl, vortex briefly. Add the mixed solution to 100 μl linearized adenovirus vector while vortexing and incubate this transfection mix for 15 - 30 min at room temperature.

Perform all following steps under the appropriate biosafety conditions for adenovirus infection!

3. Dispense the solution dropwise on the HEK293A cell-containing well and incubate the cells at 37 °C and 10% CO₂. When using a fluorescence marker, evaluate the transfection efficiency visually after 12 - 36 hr using an inverted fluorescence microscope. Add 0.5 ml of fresh medium every 3 days.
4. Check every 2 - 3 days with a light or fluorescence microscope for cytopathic effects (CPE), which are areas with enlarged and rounded cells that start to detach. This is indicative of efficient virus generation. Upon occurrence of broader zones of CPE (Figure 3), it will take 24 - 72 hr before all cells are infected.
5. When all cells show signs of CPE, but before an overall detachment of cells occurs, detach the cells by gentle pipetting and transfer cells with supernatant (SN, 3 - 5 ml) to a 15 ml polystyrene tube.
6. Freeze the cells in the SN on dry ice for 15 - 20 min and thaw them quickly at 37 °C afterwards to rupture the cells. Repeat this freeze-and-thaw-cycle (F/TC) two more times. Keep the primary virus lysate on ice for usage within a day or freeze it at -80 °C for long-term storage. Any additional F/TC will reduce the virus titer by 30 - 50%.

4. Virus Amplification

1. Seed and grow HEK293A cells to 90% confluence on a 14 cm tissue culture dish.
2. Infect the cells with half of the primary virus lysate (1.5 - 2.5 ml) and incubate the cells for at least 36 hr. Nearly all cells should be infected then, which can be determined by fluorescence microscopy. For the re-amplification of an already amplified (i.e. more concentrated) virus stock, infect HEK293A at a multiplicity of infection (MOI) of 50 (see 6.3).
3. Cells should be harvested when all of them show CPE but before detachment. With efficient virus production this state is reached within 48 hr after infection. (If it takes up to one week, consider another round of amplification by increasing the amount of adenovirus stock used for the infection described in 4.2.)
4. Detach the cells by gentle pipetting and transfer cells and SN to a 50 ml polystyrene tube. Spin cells down at 300 x g for 10 min at 4 °C.
5. Remove the SN and resuspend the pellet in a suitable volume of medium or SN (ca. 1 ml).
6. Perform 3 F/TC to disrupt the cells and centrifuge at 800 x g for 15 min at 4 °C. Take off the SN that contains the virus particles (i.e. the concentrated virus lysate), and aliquot the virus lysate to store it at -80 °C.

5. Virus Titer Determination

1. Seed 10⁵ A549 cells per well into 5 wells of a 12-well plate in 1 ml medium and let cells adhere for 6 hr.
2. Use 1 μl of concentrated adenovirus (thawed on ice) to perform a serial dilution in medium (1:5,000, 1:10,000, 1:50,000, 1:100,000) and add 10 μl per well. Leave one well uninfected to adjust the gating in flow cytometry.
3. After 36 hr, take off the SN, wash with PBS and detach cells (e.g. by trypsinization). For biohazard precautions, it is recommended to fix cells in 100 μl 4% paraformaldehyde in PBS for 10 min at room temperature and wash with PBS one time.
4. Perform a FACS analysis of infection marker expression. Plot 'μl viral lysate applied' against the absolute number of infected cells (Figure 4). Determine the linear range of infection and calculate the titer per ml of undiluted virus from the standard curve over the linear range using x = 1,000 μl.

6. T Cell Infection

1. Isolate naive/resting CD4 T cells from DO11.10 tg; CARΔ1 tg mice using MACS (Naive CD4+ T Cell Isolation Kit II) or FACS sorting (CD4+ CD25- CD62L+ CD44-).
2. For small-scale experiments, pipette an appropriate volume of viral lysate to achieve an MOI of 50 into one well of a 96-well round bottom plate.
3. Add up to 4 x 10⁵ T cells in a final infection volume of 50 μl in T cell medium (RPMI1640, 10% FBS, 5% PenStrep, 5% NaPyruvate, 1x NEAA, 1x MEM Essential vitamin, 1x L-Glutamine, 1:250,000 Mercaptoethanol, 10 mM HEPES).
   Example:
   An MOI of 50 shall be used to infect 3 x 10⁵ T cells; the viral titer is 3 x 10⁹ ml^-1
   Virus volume = MOI x T cell number / viral titer = 50 x (3 x 10⁵) / (3 x 10⁹ ml^-1) = 0.005 ml

Note: for infection of larger cell numbers, scale up using an MOI of 50 in an infection volume of 165 μl per 10⁶ naive T cells in a polystyrene tube with loose cup (up tp 3 ml per tube).

4. Incubate cells for 90 min at 37 °C in a 5% CO₂ incubator.
5. Spin down cells at 300 x g for 5 min at room temperature, take off SN, resuspend in 200 μl PBS. Centrifuge again and take off SN.

(Optional: the cells may be washed again to remove the virus more efficiently)

6. Resuspend cells in 200 μl T cell medium without stimulating antibodies and without IL-2 or other cytokines and rest them for 40 hr at 37 °C in a 5% CO₂ incubator to allow expression of the gene of interest before activation.

7. T Cell Activation and Polarization

1. Pipette a volume of anti-CD3- and anti-CD28-coupled beads that equals the cell number (e.g. 4 x 10⁵) into a small reagent cup, add the 10-fold volume of PBS and put them on a magnet for 2 min. Take off the supernatant and resuspend the beads in 200 μl polarizing medium (for Tregs: T cell medium + 1 ng/ml TGFβ, 100 U/ml IL-2).

Note: Cells can also be activated using tissue culture dishes coated with anti-CD28 and anti-CD3 antibodies, or in a DO11.10 T cell receptor-specific manner using irradiated BALB/c splenocytes pulsed with ovalbumin 323-339 peptide antigen.

2. Centrifuge the rested cells as before, take off the SN and resuspend cells in 200 μl polarizing medium containing anti-CD3- and anti-CD28-antibody-coupled beads. Incubate for 72 hr at 37 °C in a 5% CO₂ incubator without changing medium.

8. T Cell Fixation and Staining for Flow Cytometry

1. Wash cells: Spin down cells at 300 x g for 5 min at room temperature, take off SN, resuspend in 200 μl PBS. Centrifuge again and take off SN. Perform all following washing steps accordingly.
2. Resuspend the cells in 100 μl fixable dead cell staining solution and incubate for 30 min at 4 °C.
3. Wash cells, resuspend in 100 μl PBS, add 100 μl 4% paraformaldehyde in PBS, incubate 15 min at RT.
4. Wash cells, resuspend them in 200 μl ice-cold 70% methanol in PBS and incubate for 30 min on ice.

Note: Cells can be treated from now on without biohazard precaution!

5. Prepare 60 μl master-mix of 60 μl PBS + 10 μg/ml Fc-block (anti-FCR3 to block unspecific binding). Wash cells, resuspend them in 40 μl of PBS + anti-FCR3 and incubate 15 min at RT.
6. Add 20 μl of PBS + anti-FCR3 containing 1 μg PE-coupled anti-Foxp3 antibody, mix well and incubate at 4 °C over night.
7. Wash cells twice in PBS and analyze cells on a flow cytometer.

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Results

Virus Production

For generation of high virus titers, the timing of HEK293A cell harvest in primary virus production or virus amplification is crucial. Representative fluorescent and phase contrast images with visual signs of virus production are shown in Figure 3. The CPE were observed 10 days after transfection of HEK293A cells with a control pCAGAdDu vector without insert. CPE are characterized by the appearance of areas with enlarged and round-up cells that start to detach an...

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Discussion

Virus Generation and Titration

For optimal transfection results, the quality and amount of linearized vector appear most important. We did not observe negative effects on primary lysate production from an initial overgrowth of the culture since the infection will quickly proceed once efficient virus production occurs. However, virus production by HEK293A cells can be affected by long inserts that decrease the efficiency. Some open reading frames were actually found to interfere with virus product...

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Materials

Name	Company	Catalog Number	Comments
pENTR/D-TOPO Cloning Kit	Invitrogen	K240020
Gateway LR Clonase II Enzyme mix	Invitrogen	11791020
PacI	New England Biolabs	R057S
jetPEI	Polyplus-transfection	101-10N
HEK293A Cell Line	Invitrogen	R705-07
A549	ATCC	CCL-185
6-well plates	BD Falcon	353046
14 cm tissue culture dish	Nunc	168381
BALB/cJ-Tg(DO11.10)10Dlo Tg(CARΔ1)1Jdgr	Taconic Farms	Model Nr. 4285
Naive CD4+ T Cell Isolation Kit II	Miltenyi Biotech	130-094-131
DMEM	Invitrogen	41966-052
FBS	PAN Biotech	1502-P110704
PenStrep	Invitrogen	15140-122
RPMI 1640	Lonza	BE12-167F
NaPyruvate	Lonza	BE13-115E
NEAA 100x	Lonza	BE13-114E
L-Glutamine	Invitrogen	25030
HEPES Buffer Solution (1 M)	Invitrogen	15630-056
β-Mercaptoethanol	Sigma-Aldrich	M-7522
MEM Essential vitamin mixture (100x)	Lonza	13-607C
Dynabeads Mouse T-Activator CD3/CD28 for Cell Expansion and Activation	Invitrogen	114-56D
Recombinant Human TGF-beta 1	R&D Systems	240B
Proleukin S (18 x 106IE)	Novartis
LIVE/DEAD Fixable Blue Dead Cell Stain Kit	Invitrogen	L-23105
Mouse BD Fc BlockT	BD Pharmingen	553141
Anti-Mouse/Rat Foxp3 PE	eBioscience	12-5773-82
Mmu-miR-155 TaqMan MicroRNA Assay	Roche Applied Biosystems	4427975
LightCycler 480 Probes Master	Roche Applied Biosystems	04902343001
TaqMan MicroRNA Reverse Transcription Kit	Roche Applied Biosystems	4366596