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Method Article

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

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DOI:

10.3791/54421

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September 18th, 2016

 ,  ,  , 
1APC Microbiome Institute, University College Cork, 2Division of Gastroenterology and Hepatology, University Hospital Basel, 3Institute of Microbiology and Biotechnology, University of Ulm

In This Article

Summary

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Abstract

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.

This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

Introduction

The intestine is the largest surface of the body that is exposed to the external environment. Vast arrays of resident microbes colonize the human intestine to form the intestinal microbiota (or microflora). This is estimated to consist of up to 100 trillion microbial cells and constitutes one of the most densely populated bacterial habitats known in biology1-3. In the GIT bacteria colonize an intestinal niche where they survive and multiply4. In return, the microbiota endows the host with additional functional features not encoded on its genome1. For example the microbiota stimulates the proliferation of epithelial cells, produces vitamins that hosts cannot produce by themselves, regulates metabolism and protects against pathogens4-6. Given this beneficial relationship, some authors have suggested that humans are "super-organisms" or "holobionts" that are a mix of bacterial and human genes7,8. Given the beneficial impact of the microbiota on the (human) host, the intestinal immune system needs to tolerate commensal microbes to enable their existence in the lumen but also kill the pathogens that invade from luminal side9-11. The intestinal immune system has developed mechanisms to distinguish between harmless and potentially harmful luminal microbes; however these mechanisms are not yet well understood12. Maintaining intestinal integrity requires a tightly regulated immune homeostasis to keep the balance between tolerance and immunity13. An imbalance in immune homeostasis contributes to the induction of intestinal diseases such as inflammatory bowel disease (IBD)3,14.

There are two major types of IBD: Crohn's disease (CD) and ulcerative colitis (UC). Patients with these diseases usually suffer from rectal bleeding, severe diarrhea and abdominal pain15,16. The single cause of IBD is still unknown, but a combination of genetic factors, environmental influences and dysregulated immune responses might be the key event for disease development15.

Animal models for IBD have been used for over 50 years. In the last few decades new IBD model systems have been developed to test the various hypotheses concerning the pathogenesis of IBD17,18. The best-characterized model of chronic colitis is the T-cell transfer model that induces disruption of T-cell homeostasis19,20. This model involves transferring naive T cells from immunocompetent mice into hosts that lack T and B-cells (such as RAG-/- and SCID mice)16,21. The development of disease in this model is monitored for 3-10 weeks by evaluating the presence of diarrhea, reduced physical activity, and loss of body weight. This is so called the wasting syndrome16. Compared to the healthy mice the colonic tissue of transplanted hosts is thicker, shorter and heavier16. Using the T cell transfer model, it is possible to understand how different T cell populations can contribute to the pathogenesis of IBD22. The T cell transfer model does not analyze the interactions between APCs and T cells in the disease process in an antigen-specific manner. It has been shown that an interaction between myeloid cells and lymphoid cells could be responsible for the development of intestinal inflammation23. Although many aspects of IBD have been clarified, the initial events that lead to the disease development still need to be clearly understood.

It has been shown that in the absence of microbiota transfer colitis cannot be established24. Recently, several theories suggest that IBD could be a result of an immune response against commensal bacteria25. Authors have also proposed that commensal bacteria are essential to induce inflammation in the distal intestine26. In germ free (GF) animals the intestinal immune system is generally impaired27,28, but a colonization of these mice with a mixture of specific-pathogen-free bacteria results in the development of the fully-competent intestinal immune system29. Hence, the microbiota seems to be a key element in the pathogenesis of IBD, either as a mechanism that predisposes to or protects against the development of intestinal inflammation30,31. Current theories suggest that IBD is a result of microbial imbalance, called dysbiosis, in genetically predisposed patients32, but it is not clear yet if the dysbiosis is the cause or the consequence of the disease12. Considering the role of microorganisms in the development of IBD, in vitro experiments showed that CD4+ T cells can be activated by APCs pulsed with intestinal bacteria33,34.

Moreover, it has been shown that antigens from different commensal bacterial species, such as E. coli, Bacteroides, Eubacterium and Proteus, are able to activate CD4+ T cells35. This indicates that presentation of bacterial antigens to T cells is of importance for the development of IBD. To reduce the complexity of multiple antigens derived by the microflora in the disease process, an E.coli strain has been created that produces the OVA antigen. Transfer colitis was induced by injecting OVA-specific T cells into RAG-/- animals colonized with OVA-expressing E. coli.

This model is based on recent evidence suggesting that CX3CR1+ MPs, a major cell subset in the colonic lamina propria (cLP)36, are interacting with CD4+ T cells during transfer colitis37. MPs sample the intestinal lumen for particulate antigen, such as bacteria, using their dendrites36, 38,39. Previous studies demonstrated that MPs can also take up soluble antigens, such as OVA, introduced into the intestinal lumen40,41. Given the abundance of CX3CR1+ MPs in the cLP, it is possible that these cells can sample luminal bacteria and interact with CD4 T cells. Confocal imaging of mice transplanted with OVA-specific CD4+ T cells colonized with E. coli CFP-OVA, show that CX3CR1+ MPs are in contact with OT-II CD4+ T cell during the development of antigen-driven colitis. This model enables the study of the antigen presentation process between intestinal APCs and T cells specific only for particular antigen-expressing bacteria in the gut lumen.

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Protocol

Mice were bred and kept under specific pathogen-free (SPF) conditions in the animal facility of Ulm University (Ulm, Germany). All animal experiments were performed according to the guidelines of the local animal use and care committee and the National Animal Welfare Law.

1. Construction of the pCFP-OVA Plasmid

  1. Amplify the full size OVA gene using the primers Ova_SpeI_fw (3'-GACCAACTAGTATGGAATTTTGTTTTGATGTATT-5') and Ova_ClaI_rev (3'- GACCAGATCGATTAAGGGGAAACACATCTGCC-5')37 using plasmid pCI-OVA (ampicillin resistant)42 as a template. Clone the PCR product (i.e. the OVA coding sequence) into a commercially available phage mid-vector using SpeI and ClaI restrictions sites of the primers and vector to yield the specific OVA-plasmid.
  2. Amplify the CFP-coding gene together with the strong constitutive promoter Phyper using plasmid pAD1-cCFP (chloramphenicol resistant) 43 as a template and primers CFP_SacII_fw (3'-GATCGACCGCGGTTCTTGAAGACGAAAGGGCC-5') and CFP_SpeI_rev (3'-GATCGAACTAGTACCACCACCACCACCACCTTTGTAGAGTTCATCCATGC-5')37.
  3. Clone the CFP-coding gene into the specific OVA-plasmid (ampicillin resistant) using the SpeI and SacI restriction sites of the primers and vector37. This introduces a spacer of 6 glycine residues between the CFP-coding from OVA-coding sequence.

2. Construction of E.coli pCFP-OVA

  1. Grow E.coli strain DH10B in 100 ml of Luria Bertani (LB) medium aerobically at 37 °C on a rotary shaker overnight.
  2. Mix 800 µl of bacterial culture with 200 µl of glycerol to create glycerol stock and store at -80 °C.
  3. Add 50 µl of E.coli strain DH10B from glycerol stock in 5 ml of LB medium and grow them aerobically at 37 °C on a rotary shaker overnight.
  4. Transfer the culture in a baffled 1 L Erlenmeyer flask with 250 ml of LB medium at 37 °C in a rotary shake.
  5. Grow bacteria to an optical density at 600 nm (OD600) of 0.5 and then chill the culture on ice for 30 min. Centrifuge the bacteria at 5,000 x g for 10 min at 4 °C.
  6. Resuspend bacteria with 100 ml of ice-cold sterile H2O and centrifuge the bacteria at 5,000 x g for 10 min at 4° C. Resuspend bacteria with 100 ml of ice-cold 10% glycerol and centrifuge the bacteria at 5,000 x g for 10 min at 4 °C. Repeat this last step twice.
  7. After the final wash step, resuspend bacteria in 700 µl of 10% glycerol. Freeze 50 µl aliquots in liquid nitrogen and store at -80 °C.
  8. Thaw a 50 µl aliquot on ice and transfer into a pre-chilled electroporation cuvette (2 mm electrode distance).
  9. Add 5 µl of plasmid DNA (100 ng) to the 50 µl E. coli aliquots. Perform electroporation with a single pulse at 25 µF, 200 ohms (Ω) and 2.5 kV.
  10. Resuspend bacteria in 1 ml pre-warmed SOC medium (5 g/L yeast extract, 2 g/L tryptone, 10 mM sodium chloride, 2.5 mM potassium chloride, 10 mM magnesium chloride, 10 mM magnesium sulfate) and incubate at 37 °C aerobically for 1 hr.
  11. Plate bacteria on LB-agar plates supplemented with ampicillin 100 µg/ml (add 100 µl of a 100 mg/ml ampicillin stock to 100 ml of LB-agar) and incubate overnight aerobically at 37 °C.
  12. Pick single colonies and re-streak them in LB-agar plates supplemented with ampicillin 100 µg/ml. Incubate overnight aerobically at 37 °C.
  13. Pick single colonies and grow them overnight in 10 ml of LB media supplemented with ampicillin 100 µg/ml for plasmid isolation. Use commercially available plasmid mini prep kit and elute plasmid DNA with 30 µl dH2O (distilled water) according to manufacturer's protocol. Verify plasmids by restriction analysis and DNA sequencing37,42,43.
  14. Set up a culture of positive clones of E. coli pCFP-OVA in 100 ml of LB medium supplemented with ampicillin 100 µg/ml. Grow aerobically at 37 °C on a rotary shaker overnight.
  15. Mix 800 µl of bacterial culture with 200 µl of pure glycerol and store at -80 °C.
  16. Centrifuge the rest of the overnight culture at 5,000 x g for 10 min at room temperature (RT) and resuspend the pellet in phosphate buffered saline (PBS). Place 10 µl of the suspension onto glass slide and cover it with a coverslip.
  17. Observe the samples in a standard fluorescence microscopy to confirm the expression fluorescence of CFP-OVA (CFP excitation at 450 nm, emission at 480 nm). Analyze the bacteria starting with magnification 400X.
  18. Centrifuge 1 ml of bacteria from overnight cultures at 5,000 x g for 10 min, RT. Resuspend the pellet in 40 µl of PBS and boil at 95 °C for 10 min.
  19. Centrifuge the samples at 30,000 x g for 10 min and use the supernatants for Western blot analysis37. Use the rabbit-raised anti-OVA polyclonal antibody diluted 1:400.

3. Generation of OT-II/Red Mice

  1. Cross a male/female OT-II mouse with a female/male mouse expressing red fluorescent protein (both strains commercially available) in order to generate the OT-II/Red mice37.

4. Spleen Cell Isolation

  1. Euthanize the OT-II/Red mice by cervical dislocation after anesthesia by inhalation of any halogenated ether (up to 5% for induction) commercially available.
  2. Use scissors to cut the left superior abdominal part of the mouse. Under this abdominal part there is the spleen (oval shape with a dark-red color). Use scissors and tweezers to remove it.
    1. Extract the spleen of the OT-II/Red mice and pass it through a 100 µm cell strainer positioned on a 50 ml tube using the plunger of a 1 ml syringe in order to obtain a single-cell suspension. Wash the strainer twice with 10 ml of PBS supplemented with 1% heat inactivated fetal bovine serum (FBS).
  3. Centrifuge the cell suspension at 390 x g for 5 min at RT. Resuspend the pellet with 5 ml of pre-warmed lysis buffer (144 mM NH4Cl, 17 mM Tris, pH= 7.2) to lyse the red blood cells. Incubate 10 min at 37 °C.
  4. Wash the sample with 25 ml of PBS supplemented with 1% FBS. Centrifuge at 390 x g for 5 min at RT. Resuspend the pellet in 10 ml of PBS supplemented with 1% FBS.
  5. Count the cells using a Neubauer counting chamber. Mix 10 µl of the cells suspension with 10 µl of a 0.4% Trypan Blue solution. Incubate for 1-2 min.
    1. Fill the hemocytometer chamber with 10 µl of stained cells. Count cells under the microscope in four 1 x 1 mm2 squares of one chamber and determine the average number of cells per square. Viable cells appear white, dead cells appear blue. To determine the number of cells/ml multiply cell number by factor of dilution. If 2-4 quadrants are being counted, divide by the number of quadrants.

5. CD4 + CD62 L+ T Cell Enrichment

  1. Enrich spleen cells for CD4+ CD62L+ T cells via magnetic isolation kit using the principles of negative or positive selection. Here, use negative selection to isolate CD4+ T cells and use positive selection to further enrich for the CD62L+ population within the CD4+ T cell pool.
    NOTE: In the negative selection procedure, the cells, which will be not isolated (here: all CD4 negative cells) are labeled with specific antibodies coupled with magnetic microbeads. During the column washing steps the CD4+ T cells will pass through the column so that they can be easily collected. In the positive selection procedure cells to be isolated are labeled with appropriate antibody coupled with magnetic microbeads. During the column washing steps the labeled cells will stay in the column and they can be collected by removing the column from the magnetic field and rinsing it with the buffer.
  2. For the negative selection procedure, resuspend 107 total spleen cells in 40 µl of cold PBS supplemented with 0.5% bovine serum albumin (BSA). Add 10 µl of biotin labeled antibody cocktail supplied with the magnetic isolation kit (specific antibodies binding the non-CD4+ T cells) and incubate 15 min on ice.
    1. Add 30 µl of cold PBS supplemented with 0.5% BSA and 20 µl of anti-biotin microbeads. Incubate 20 min on ice.
    2. Add to the sample 10 ml of PBS supplemented with 0.5% BSA and centrifuge at 390 x g for 5 min. Repeat this step twice. Resuspend the cells in 1 ml of cold PBS supplemented with 0.5% BSA.
    3. Place the column specific for the negative selection procedure in the magnetic field and rinse with 3 ml of cold PBS supplemented with 0.5% BSA onto the column. Add the cell suspension onto the column and wash 3 times with 3 ml of cold PBS supplemented with 0.5% BSA.
    4. Centrifuge cells of the effluent at 390 x g for 5 min. Effluent passing through the column contains unlabeled CD4+ T cells. Resuspend the pellet in 1 ml of PBS supplemented with 0.5% BSA and count the cells.
    5. Count the cells using a Neubauer counting chamber. Mix 10 µl of the cells suspension with 10 µl of a 0.4% Trypan Blue solution. Incubate for 1-2 min.
      1. Fill the hemocytometer chamber with 10 µl of stained cells. Count cells under the microscope in four 1 x 1 mm2 squares of one chamber and determine the average number of cells per square. Viable cells appear white, dead cells appear blue. To determine the number of cells/mL multiply cell number by factor of dilution. If 2-4 quadrants are being counted, divide by the number of quadrants.
  3. For the positive selection procedure, add 10 µl of CD62L microbeads per 107 pre-enriched CD4+ T cell fraction. Incubate 15 min on ice.
    1. Add to the sample 10 ml of PBS supplemented with 0.5% BSA and centrifuge at 390 x g for 5 min. Repeat this step twice. Resuspend in 500 µl of cold PBS supplemented with 0.5% BSA.
    2. Place the specific column for the negative selection procedure in the magnetic field and rinse with 500 µl of cold PBS supplemented with 0.5% BSA onto the column. Add cell suspension onto the column and wash 3 times with 500 µl of cold PBS supplemented with 0.5% BSA.
    3. Remove the column from the magnetic field and place it on a suitable collection tube. Add 1 ml of PBS supplemented with 0.5% BSA onto the column and flush out CD4+ CD62L+ T cells retained in the column by firmly pushing the plunger into the column.
      NOTE: The isolated CD4+ CD62L+ T cells are now ready for downstream applications, such as in vivo experiments. According to the manufacturer's instruction from the CD4+ CD62L+ T cells isolation kit, the microbeads are not toxic. Due to the small size (around 50 nm), they do not activate cells and they will not saturate cell surface epitopes. Therefore the isolated cells can be transferred into immunodeficient mice to induce colitis37,44,45.
    4. Count the cells using a Neubauer counting chamber. Mix 10 µl of the cells suspension with 10 µl of a 0.4% Trypan Blue solution. Incubate for 1-2 min.
      1. Fill the hemocytometer chamber with 10 µl of stained cells. Count cells under the microscope in four 1 x 1 mm2 squares of one chamber and determine the average number of cells per square. Viable cells appear white, dead cells appear blue. To determine the number of cells/ml multiply cell number by factor of dilution. If 2 - 4 quadrants are being counted, divide by the number of quadrants.

6. Induction of Antigen-driven Colitis

  1. Inject intraperitoneally 3 x 105 OT-II/Red CD4+ CD62L+ T cells into CX3CR1GFP/+ x RAG-/- mice. For induction of antigen-specific colitis, administer E. coli pCFP-OVA every second day at a dose of 1 x 108 colony-forming unit (CFU) in 100 µl of PBS per animal by gavage or oral instillation behind the incisors.
  2. Monitor twice weekly the body weight of transplanted mice and their clinical condition (presence of blood in the stool, stool consistency and activity of mice).

7. Tissue Samples for Histopathological Examination

  1. Take tissue samples from the large intestine of mice, fix in neutral-buffered formalin (10%) and embed in paraffin as previously described36,46.
  2. Section the paraffin samples on a microtome, mount on slides, and perform the H&E stain47,48.
  3. Categorize the histology of the large intestine as published previously47,48: normal (score 0); mild colitis (score 1), moderate colitis (score 2) and severe colitis (score 3).

8. Isolation of Colonic Lamina Propria Cells

  1. Euthanize transplanted mice by cervical dislocation after anesthesia by inhalation of any halogenated ether (up to 5% for induction) commercially available.
  2. Place the animal down with the belly facing up. Use forceps to grab the skin anteriorly to the urethral opening. Use scissors to cut the skin along the ventral midline from the groin to the chest. Pull the skin back on the sides.
  3. Cut through the transparent peritoneal muscle wall and opening up the body cavity. Identify the colon that goes from the anus to the caecum49. Cut the 2 extremities and free the tissue from any fat.
  4. Open the colon longitudinally using a dissection scissors. Use a tweezers to shake the colon tissue in a petri dish containing 25 ml of PBS supplemented with 1% FBS to remove debris and mucous. Cut the colon into 5 - 8 mm pieces.
  5. Place the colon segments in a 50 ml tube containing 20 ml of the Wash Buffer. The Wash buffer contains 500 ml DPBS, 10mM Hepes and 5mM ethylenediaminetetraacetic acid (EDTA).
  6. Vortex the tube for 30 sec at maximal speed.
  7. Incubate the tube with the colon segments in a water bath at 37 °C with 200 rpm shaking for 10 min.
  8. After the incubation, vortex the tube for 30 sec at maximal speed.
  9. Discard the supernatants and place the tissue samples in a new 50 ml tube containing 20 ml of the Wash buffer. Repeat steps 6-9 three times
  10. Discard the supernatants and place the tissue samples in a petri dish containing 25 ml of PBS. Use a tweezers to shake gently the tissue samples in the petri dish for few seconds to remove residual epithelial cells. Repeat this step three times.
  11. Cut colonic tissues into 2 x 2 mm2 pieces and digest them by incubation in 10 ml of Roswell Park Memorial Institute (RPMI) medium with 0.5 mg/ml collagenase type VIII from Clostridium histolyticum and 10 U/ml of DNaseI.
  12. Vortex the tube for 30 sec at maximal speed.
  13. Incubate the colon fragments in a water bath at 37 °C with 200 rpm shaking for 35 min and vortex the tube for 30 sec at maximal speed every 5 min.
  14. At the end of the incubation vortex the tube for 30 sec at maximal speed.
  15. Collect the digested fragments by passing them through a 70 µm cell strainer positioned on a new 50 ml tube.
  16. Wash the sample by adding 20 ml of DPBS and centrifuge at 400 g for 5 min. Then resuspend the cells in 1 ml of DPBS.
  17. Count the cells using a Neubauer counting chamber. Mix 10 µl of the cells suspension with 10 µl of a 0.4% Trypan Blue solution. Incubate for 1-2 min.
    1. Fill the hemocytometer chamber with 10 µl of stained cells. Count cells under the microscope in four 1 x 1 mm2 squares of one chamber and determine the average number of cells per square. Viable cells appear white, dead cells appear blue. To determine the number of cells/ml multiply cell number by factor of dilution. If 2-4 quadrants are being counted, divide by the number of quadrants.

9. Extracellular Staining for FCM Analysis

  1. Centrifuge the cLP cells 5 min at 390 x g. Resuspend the cells in 1 ml of FACS A (PBS supplemented with 1% BSA and 0.1% sodium azide)
    NOTE: Sodium azide is highly toxic. It can cause hypotension, hypothermia, headache convulsions or death. Dilute solutions of sodium azide (0.1 to 1.0%) usually do not present extraordinary dangers to the user, but they are eye and skin irritants. Therefore, use sodium azide only in the chemical hood while wearing a laboratory coat and a double pair of disposable nitrile gloves.
  2. Incubate the cells for 15 min at RT with the monoclonal antibody (mAb) 2.4G2 directed against the FcγRIII/II CD16/CD32 (0.5-1 µg mAb/106 cells) diluted in FACS A.
  3. Add 200 µl of FACS A to the cells and centrifuge 5 min at 390 x g. Repeat this step twice. Incubate the cells with 0.5 ng/106 cells of the relevant mAb for 20 min at 4 °C. Dilute the antibodies in FACS A.
  4. Add 200 µl of FACS A to the cells and centrifuge 5 min at 390 x g. Repeat this step twice. Resuspend the samples in 100 µl of FACS A.
  5. Analyze T cells from CX3CR1GFP/+ x RAG-/- mice reconstituted with OT-II/Red CD4 T cells at the flow cytometer37.
  6. Analyze data using commercially available software.

10. Confocal Microscopy Analysis

  1. Analyze the colon of CX3CR1GFP/+ mice reconstituted or not with OT-II/Red CD4+CD62L+ T cell and fed orally every second day with 1 x 108 CFU of E. coli strain DH10B pCFP-OVA.
  2. Remove the colon from the mice using scissors and tweezers. Open it longitudinally using a dissection scissors.
  3. Cut a 5-8 mm piece of colon sample using scissors, put onto glass slides and cover with coverslips.
  4. Analyze the colon samples with a confocal microscope at a magnification of 40/1.30.
  5. Detect the CX3CR1+ MPs, labeled with green fluorescein protein (GFP), using a filter set with excitation at 488 nm and emission at 509 nm.
  6. Detect the CD4+ T cell expressing red protein using a filter set with excitation at 554 nm and emission at 586 nm.
  7. Detect E.coli pCFP-OVA using a filter set with excitation at 450 nm and emission at 480 nm.
  8. Define an area of interest and generate scatter diagrams as previously described50 to carry out the co-localization analysis. Use 2 different formulas: the Overlap Coefficient according to Manders and the Pearson's Coefficient.

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Results

To establish an antigen-driven colitis model an E. coli strain has been constructed that contains a plasmid in which the gene for the CFP is fused to the coding sequence for the chicken ovalbumin protein and the fusion construct is expressed under control of the strong constitutive promoter Phyper (Figure 1A). Fluorescent microscopy shows that the recombinant E. coli pCFP-OVA, but not the parental E. coli DH10B, expr...

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Discussion

As with every other model, the antigen-driven colitis model described above may present few issues that the investigator performing the technique must be aware of. When injecting the OT-II/Red+ CD4+ T CD62L+cells in the hosts, the investigator must be very gentle and careful to insert the needle into the peritoneal cavity. Failure to do so may result in the tearing of the intestine of the mouse which could lead to death, or a subcutaneous administration of cells which will not induce any ...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

JHN is supported by the Swiss National Foundation (SNSF 310030_146290).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
LB Broth, Miller (Luria-Bertani)Difco244620
Rotary ShakeReiss Laborbedarf e. K.Model 3020 GFL
2 mm gap couvettes Peqlab Biotechnologie GmbH71-2020
GlycerolSigma-AldrichG5516-100ML
Gene Pulser Xcell system BioRad Laboratories GmbH1652660
LB Agar, Miller (Luria-Bertani)Difco244510
AmpicillinSigma-AldrichA9393-5G
SOC MediumSigma-AldrichS1797-100ML
High Pure Plasmid Isolation KitRoche11754777001
AgaroseCarl Roth GmbH & Co3810.1
EDTASigma-AldrichE9884-100G
Tris-HClSigma-AldrichT5941
Glacial acetic acidSigma-Aldrich537020 
Gel chamber PEQLAB Biotechnology GmbH40-0708
Loading DyeThermo FisherR0611
GeneRuler 1 kb DNA Ladder Thermo FisherSM0312
Ethidium bromide solutionCarl Roth GmbH & Co. KG2218.3
Photo-documentation system Decon Science Tech GmbHDeVision G 
DNA sequencing MWG-Biotech GmbH
Phosphate buffered saline (PBS)BiochromL182-50
Fluorescent microscope ZeissHBO 100
Mini-PROTEAN Tetra SystemBio-Rad Laboratories GmbH1658005
PageRuler Prestained Protein Ladder Fermentas, St. Leon-Rot, Germany
IstanBlue SolutionExpedeon, Cambridgeshire, United Kingdom
Nitrocellulose membrane Macherey-Nagel GmbH & Co. KG741280
Electro blotter Biometra GmbH846-015-600
Bovine Serum Albumins (BSA)Sigma-AldrichA6003-25G
Anti-Ovalbumin antibody Abcamab181688
Anti-rabbit IgG  HRPSigma-AldrichA0545 
Pierce ECL Plus Western Blotting SubstratePierce Biotechnology, Thermo Fischer Scientific Inc32132
ForeneAbbott2594.00.00
FBSInvitrogen10500-064
Falcon Cell StrainersFischer Scientific 08-771-2
Ammonium chlorideSigma-Aldrich254134-5G
Tris BaseSigma-Aldrich10708976001
CD4+ CD62 L+ T isolation kit Miltenyi Biotec130-093-227 
MACS LS Columns Miltenyi Biotec130-042-401
MACS MS Columns Miltenyi Biotec130-042-201
MidiMACS SeparatorMiltenyi Biotec130-042-302
MiniMACS SeparatorMiltenyi Biotec130-042-102
MACS MultiStandMiltenyi Biotec130-042-303
Feeding Needle 20GSouthPointe Surgical Supply, IncFN-7903
Formalin solution, neutral buffered, 10%Sigma-AldrichHT501128
ParaffinSigma-Aldrich1496904
HematoxylinSigma-AldrichH9627
Eosin YSigma-Aldrich230251 
DithiothreitolSigma-AldrichD9779 
Collagenase type VIIISigma-AldrichC-2139
Roswell Park Memorial Institute (RPMI) mediumAppliChemA2044, 9050
Sodium azideSigma-Aldrich438456
Mouse BD Fc BlockBD Pharmingen553141
FITC-conjugated mAb binding Vß 5.1, 5.2 BD Pharmingen553189
APC-conjugated mAb binding CD4 GK1.5 eBioscience17-0041-83
FACS Calibur BD Biosciences
FCS Express V3 softwareDeNovo
Meta scanning confocal microscope ZeissLSM 710 
Zeiss WorkstationZeissLSM 7
Zeiss ZEM software Zeissv4.2.0.121
Maxisorp immuno plates NUNC, Roskilde442404
Streptavidin conjugated alkaline phosphataseJackson Immuno Research016-050-084
Alkaline phosphatase substrate 4-Nitrophenyl phosphate disodium salt hexahydrateSigma-Aldrich71768-5G
mAb R4-6A2BD Biosciences551216
mAb XMG1.2 BD Biosciences554410
TECAN microplate-ELISA readerTecan
EasyWin softwareTecan
DNase I recombinant4536282001

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T Cell Transfer ModelColonic Lamina PropriaOT II CD4 T CellsCFP OVA Expressing E coliFlow Cytometry AnalysisConfocal Microscopy ImagingCell Isolation ProtocolTissue Digestion Method
Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells
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