This video demonstrates a protocol to enrich thymic epithelial cells (TECs) with density gradient for FACS isolation. It also shows the use of EAK16-II/EAKIIH6 peptides to promote the TEC aggregate formation. The microenvironments of EAK16-II/EAKIIH6 hydrogel provide the 3-D configuration necessary to maintain the survival and function of the TECs.
Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing thymus is crucial to maintain effective T cell-mediated acquired immune response against invading pathogens. However, thymus regeneration and revitalization proved to be challenging, largely due to the difficulties of reproducing the unique 3D microenvironment of the thymic stroma that is critical for the survival and function of thymic epithelial cells (TECs). We developed a novel hydrogel system to promote the formation of TEC aggregates, based on the self-assembling property of the amphiphilic EAK16-II oligopeptides and its histidinylated analogue EAKIIH6. TECs were enriched from isolated thymic cells with density-gradient, sorted with fluorescence-activated cell sorting (FACS), and labeled with anti-epithelial cell adhesion molecule (EpCAM) antibodies that were anchored, together with anti-His IgGs, on the protein A/G adaptor complexes. Formation of cell aggregates was promoted by incubating TECs with EAKIIH6 and EAK16-II oligopeptides, and then by increasing the ionic concentration of the medium to initiate gelation. TEC aggregates embedded in EAK hydrogel can effectively promote the development of functional T cells in vivo when transplanted into the athymic nude mice.
The thymus is the primary lymphoid organ responsible for the generation of a diverse population of pathogen-reactive, self-tolerant T cells that is essential to the function of the acquired immune system. It is a dynamic organ where developing thymocytes, immigrated from the bone marrow as lymphocyte progenitor cells, migrate through the sponge-like three-dimensional (3-D) matrix of the thymic stroma, undergo lineage-specification and differentiation, and eventually emigrate as mature T-cells. The success of this well programmed process depends largely on the cross-talk between the migrating thymocytes and the residential thymic epithelial cells (TECs), the predominant population of the thymic stroma that are essential for establishing and maintaining the integrity of the thymus microenvironment.
Based on their anatomical location and unique function, TECs can be divided into two subsets: the TECs in the cortex (cTECs) that are responsible for selecting self-MHC (major histocompatibility complex) restricted T-cells (positive selection), and the TECs in the medulla (mTECs) that are essential for eliminating autoreactive T-cells (negative selection) 1,2. Many factors (e.g., aging, infection, irradiation, drug treatments) can cause irreversible damages to the thymic epithelium, resulting in compromised adaptive immunity. Despite numerous attempts, restoring the thymic function has been challenging due to the difficulty to reproduce the thymic microenvironment. Notably, thymic three-dimensional (3-D) configuration is critical to the survival and function of TECs, whereas TECs cultured in a 2-D environment rapidly decrease the expression of genes critical for thymopoiesis 3,4.
EAK16-II (AEAEKAKAEAEAKAK) and its C-terminal histidinylated analogue EAKIIH6 (AEAEKAKAEAEAKAKHHHHHH) are low-molecular weight, amphiphilic oligopeptides that are soluble in deionized water, but undergo gelation to form β-fibrils when exposed to ionic strength higher than 20 mM NaCl (normal salt concentration in human body fluid is 154 mM). This environmental responsive property makes them versatile building blocks to form 3D structures. The His-tag on EAKII-H6 provides a docking mechanism, by which the anti-His IgGs/Fc-binding recombinant protein A/G (αH6:pA/G) complexes can serve as an adaptor to anchor protein drugs and other biomolecules (e.g., antibodies) on the hydrogel composite 5-8.
We have previously demonstrated that fluorescent-labeled IgG molecules anchored on the hydrogel can be retained at the injection site for up to 13 days 9. Furthermore, when the αH6:pA/G adaptors anchored with anti-CD4 IgGs were added to the EAK16-II/EAKIIH6 (EAK) hydrogel, CD4+ T cells were specifically captured 10. Using similar technique, we have recently demonstrated that 3-D aggregation of TECs could be promoted in EAK hydrogel with adaptor complexes carrying TEC-specific anti-EpCAM antibodies (αH6:pA/G:αEpCAM). When transplanted underneath the kidney capsules of nude mice, the TEC clusters embedded in EAK hydrogel can effectively support the development of functional T cells in vivo11,12.
Here we illustrate our method to quickly and effectively purify TECs with fluorescence-activated cell sorting (FACS) and generate 3-D TEC aggregates with the EAK hydrogel system.
All the animals used in the experiments were housed in the animal facility at Allegheny-Singer Research Institute under the protocol reviewed and approved by the Institutional Animal Care and Use Committee of the Allegheny Health Network/Allegheny Singer Research Institute.
1. Digesting the Thymus with Collagenase
2. Enrichment of TECs with Discontinuous Density Gradient
3. Isolating TECs with FACS
4. Generation of TEC/EAK Aggregates
Note: Perform reagent preparation and steps involving cell mixing under the laminar hood.
5. Characterization of the TEC/EAK Aggregates
To examine the effectiveness of using the density gradient separation protocol to enrich the CD45- stromal cells, cells harvested from both the interface and the precipitated lymphocyte pellets were stained with anti-CD45 and anti-EpCAM antibodies. Both anti-Ulex Europaeus Agglutinin 1 (UEA1) and anti-MHC Class II antibodies were also included in the staining cocktail to further identify the cTEC and mTEC subsets. As shown in Figure 1, we were able to routinely achieve close to 15-20 times enrichment of the TECs.
To examine the aggregation of the TECs in the EAK hydrogel, TECs were isolated from 4-week old B6.ROSAmT/mG mice, which ubiquitously express the membrane-bound, red fluorescent tdTomato molecules. TEC aggregates, prepared as described above, were cultured for two days in vitro and examined under a confocal microscope (Figure 2).
Figure 1. Flow cytometric analysis of TECs enriched with the density gradient solution. Cells were stained with anti-CD45 and anti-EpCAM antibodies to distinguish TEC population (CD45- EpCAM+). Left panels: Thymic cells after enzyme digestion, before the density gradient procedure. Middle panels: Cells in the top layer and the interface after separation with the density gradient solution. Right panels: Pelleted cells (lymphocyte pellet) after separation with the density gradient solution. Please click here to view a larger version of this figure.
Figure 2. Confocal microscopic images of TECs clusters in EAK hydrogel. Red fluorescent TECs harvested from the B6.ROSAmT/mG mice were isolated with FACS. TECs clusters were embedded in EAK hydrogel, cultured in 96-well plate and were harvested at day 2. Left panel, TECs were labeled with IgG antibody; Right panel, TECs were labeled with anti-EpCAM antibody. Please click here to view a larger version of this figure.
While TECs are the predominant population of the thymic stroma and play essential roles for the structure and function of the thymus glands, they represent only about 0.1-0.5% of the total thymic cellularity. They are also fragile cells as high percentages of cell death are occasionally observed following collagenase digestion, i.e., the treatment to dissociate TECs from the extracellular matrix (ECM). Their rarity (~200,000 per mouse thymus) and fragility made it a challenging task to isolate TECs. The recent utilization of highly purified collagenase in TEC isolation have significantly increased the numbers of live cells following the enzymatic digestion 15-19.
However, the very small number of TECs remains a major challenge for their analysis and isolation, as large quantities of antibodies are needed for cell surface labeling and excessive numbers of events need to be collected by FACS. While antibody-conjugated magnetic bead technology can be used to deplete CD45+ thymocytes, their large quantity renders it economically prohibitive in the long run. We were able to effectively enrich TECs 15-20 times with a simple, one step density gradient centrifugation. The iso-osmotic property of the iodixanol density gradient medium allows the separation of cells based on their shapes, sizes and densities 20-22. It should be noted that the purity and yield of TECs are affected by the percentage of the density gradient medium used. Lower percentage of the density gradient medium (18-20% v/v) will result in higher purity of TECs in the interface but lower yield. In contrast, when higher iodixanol concentration (22% v/v) is used, larger quantity of thymic cells can be collected with lower percentage of TECs. The concentration of iodixanol should be adjusted based on the purpose of the experiment. While the purity of TECs is largely improved with the utility of density gradient solution, it should be emphasized that the cells collected from the top layer and the interface still contain a relatively high fraction of CD45+ cells (Figure 1). Therefore, when only the TEC population is required as in this experiment, cell sorting will be crucial, although this extra procedure may cause some loss of cells and requires more time.
Unlike most of the epithelial cells arranged in a single or multiple layers lining the visceral organs, TECs are organized in 3-D configuration in the thymus stroma, which is essential for their survival and function. The self-assembling EAK hydrogel system facilitates the formation of 3-D TEC aggregates that are mediated by the tri-molecular αH6:pA/G:αEpCAM adaptor complexes. Of note, by fine-tuning the concentrations and ratios of the EAK16-II/EAKIIH6 oligopeptides, the porosity and density of the hydrogel can be adjusted to allow the flow of nutrients and the migration of thymocytes. Indeed, functional T-cells were generated in athymic nude mice transplanted with the TEC/EAK composites.
The drawback in this EAK hydrogel system is that with this current protocol, specific cell composition cannot be localized to a designated area. In a physical environment a thymus is comprised of two distinct compartments, in which specific types of TECs reside, cTECs in the cortex and mTECs in medulla. With our system described here, both cTECs and mTECs are labeled with EpCAM, a general epithelial marker. Therefore it is speculated that the aggregates in the formed hydrogel includes both subtypes of TECs. Although cTECs and mTECs derive from common bipotent TEC progenitors, their roles in the development of T cells are clearly distinctive. Cortex is the first region in the thymus where the immature thymic progenitor cells enter, and the positive selection takes place 23. Then the selected cells move into the medullary region and interact with mTECs, through which the self-reactive cells are eliminated by negative selection 24. Moreover, it has been demonstrated that 25% of the genes are differentially expressed between cTECs and mTECs 25. These findings underline the importance of promoting aggregation of TEC subsets to increase the efficacy of T cell development. One possible approach is to use different antibodies targeting surface molecules specific to either cTECs or mTECs, such as anti-cytokeratin 8 for cTECs, and anti-cytokeratin 5 for mTECs.
From the perspective of future clinical application, the TEC clusters embedded in the biodegradable hydrogel might function as injectable "mini thymus units" to modulate the adaptive immune system that could be important for various medical conditions, such as rejuvenating the adaptive immune system in aged individuals, or inducing donor-specific tolerance in solid organ transplantation.
The authors have nothing to disclose.
This work was supported in part by the National Institutes of Health grants R21 AI113000 (W.S.M) and R01 AI123392 (Y.F.).
1. TEC Isolation | |||
70% Ethanol | Decon Laboratories | 2701(1 Gallon) | Ethanol 200 Proof, deionized water |
Dissecting scissors, straight | Fine Science Tools, Inc. | 91460-11 | |
Graefe forceps, straight | Fine Science Tools, Inc. | 11053-10 | |
Graefe forceps, curved | Fine Science Tools, Inc. | 11052-10 | |
Washing solution | 1X PBS, 0.1% BSA, 2mM EDTA | ||
1x PBS (Phosphate buffered saline) | Gibco | 10010-023 | |
BSA (Bovine serum albumin) | Sigma | A1470-100G | |
EDTA (ethylenediaminetetraacetic acid) | Invitrogen | 15575-038 | |
Digestion solution | 9 mL RPMI-1640, 0.025 mg/mL Liberase TM Research grade, 10 mM HEPES, 0.2 mg/mL DNaseI | ||
RPMI-1640 | Gibco | 11879-020 | |
Liberase TM Research Grade | Roche | 05 401 127 001 | referred as "purified collagenase" |
1M HEPES | Lonza | 17-737E | |
Dnase I | Roche | 10 104 159 001 | |
50mL Centrifuge tube | Corning | 430290 | |
60mm tissue culture dish | Falcon | 353002 | |
1/2cc U-100 Insulin syringe 28G1/2 | Becton Dickinson | 329461 | |
5mL Polystyrene round-bottom tube | Falcon | 352058 | |
5ml glass pipet | Fisher Healthcare | 13-678-27E | Use for rinsing the thymic fragments. Thymic fragments tend to stick to the wall with plastic pipets. |
MACSmix tube rotator | Miltenyi | 130-090-753 | |
100um Cell strainer | Falcon | 352360 | |
Density gradient medium: OptiPrep | Axis-Shield | ||
Name | Company | Catalog Number | Yorumlar |
2. Cell sorting | |||
5mL Polypropylene round-bottom tube | Falcon | 352063 | |
Anti-mouse CD16/CD32 (Fc Block) | BD Biosciences | 553142 | Use as undiluted, 2uL per sample |
Anti-mouse CD45-PercpCy5.5 | eBioscience | 45-0451-80 | Use at 1:150, 10uL per sample |
Anti-mouse CD326 (EpCAM)-PE | eBioscience | 12-5791-82 | Use at 1:100, 10uL per sample |
BD Influx | BD Biosciences | ||
Single cell analysis software | FlowJo | ||
Name | Company | Catalog Number | Yorumlar |
2. EAK gel assembly | |||
Anti-His-Tag | AnaSpec | 29673 | "anti-His-Tag IgG" |
Purified anti-mouse CD326 (EpCAM) | BioLegend | 118202 | "anti-EpCAM IgG" |
Recombinant protein A/G | Pierce Biotechnology | ||
1.5mL Safe-Lock Tubes, Biopur, Sterile | Fisher Healthcare | 05-402-24B | referred as "1.5mL microcentrifuge tube" |
96-well, Tissue culture plate, Round-bottom with low evaporation lid | BD Falcon | 353917 | |
Rocking platform: Nutator Mixer no.1105 | BD Clay Adams | ||
10% sucrose | Sigma | S0389 | Prepare with sterile distilled water |
EAK16-II (AcNH-AEAEAKAKAEAEAKAK-CONH2) | American Peptide Company | custom synthesized, 10mg/mL | |
EAKIIH6 (AcNH-AEAEAKAKAEAEAKAKHHHHHH-CONH2) | American Peptide Company | custom synthesized, 7.5mg/mL | |
Complete medium | RPMI-1640, 10% FBS, 1% Pen/Strep, 1% L-glutamine, 1% NEAA, 5mM HEPES, 50uM 2-Mercaptoethanol | ||
RPMI-1640 | Gibco | 11879-020 | |
FBS (Fetal Bovine Serum) | Atlanta Biologicals | S11150 | Heat inactivated before use |
Pen/Strep | Gibco | 15140-122 | |
L-glutamine 200mM (100x) | Gibco | 25030-081 | |
NEAA (non-essential amino acid) 100x | Gibco | 11140-050 | |
1M HEPES | BioWhittaker | 17-737E | |
2-Mercaptoethanol (100X) | Millipore | ES-007-E | |
Platform shaker: The Belly Dancer | Stovall Life Sciences Inc. | model: USBDbo |