Immunology

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Authors: Verena Semmling, Christoph A. Thaiss, Veronika Lukacs-Kornek, Irmgard Förster, Percy A Knolle & Christian Kurts

Introduction

Dendritic cells (DCs) attract naïve cytotoxic CD8+ T lymphocytes (CTL) by chemokines, in order to activate them for immune defense against viruses and tumors. Here we describe a protocol for quantifying such recruitment towards individual DCs in secondary lymphatics by standard immunofluorescence histology. We used reporter mice expressing green fluorescent protein under a chemokine promoter and adoptively transferred into them CTL labeled with a red fluorochrome. Tissue sections from these animals were stained with a third color identifying anatomical regions, in our case the splenic B cell zone, which encircles the T cell zone in which DCs and CTL are located. The number of CTL in that zone and their location adjacent to chemokine-producing CTL can be determined by computer-assisted digital analysis.

Reagents

  1. Alpha murine B220-PE antibody (e-bioscience, Cat. No. 12-0452)
  2. Far Red DDAO-SE (Invitrogen, Cat. No. C34553)
  3. Magnetic beads (Alpha murine CD8 MicroBeads) (Miltenyi, Cat. No. 130-049-401)
  4. Mounting medium (Immu-Mount) (Thermo Scientific, Cat. No. 9990402)
  5. Tissue-Tek® (Satura, Cat. No. 4141)
  6. Blocking buffer – Dissolve 1% BSA (PAA, Cat. No. K45-001) in PBS (wt/vol). Store at 4°C.
  7. MACS buffer – Dissolve 0,1% BSA and 2 mM EDTA (Roth, Cat. No. 8043.2) in PBS (wt/vol). Store at 4°C.
  8. Paraformaldehyde – Dissolve 40 mg of PFA (Sigma, Cat. No. 6148) in 200 ml PBS and incubate at 60°C until PFA is completely solved. Set pH to 7.4. Store at -20°C and further dilute to 4% (wt/vol) in PBS before usage. CAUTION PFA is toxic. Avoid direct contact.

Equipment

  1. Cell F software (Olympus)
  2. Cover slip (Menzel Gläser)
  3. Dako Pen (Dako Cytomation GmbH)
  4. MACS columns and separator (Miltenyi)
  5. Microtome/Cryostat (Leica)
  6. Microscope (IX71, Olympus), 10x objective

Procedure

Isolation and labeling of CTL (day 1)

  • 1. Isolate spleens from CTL-donor mice and generate a single cell suspension by extruding the spleen through a steel sieve with the plunger of a 2 ml syringe and filtering through a Nylon sieve (100 µm).
  • 2. Centrifuge cell suspension at 300 g for 6 mins, 4°C.
  • 3. Resuspend cells in 600 µl of MACS buffer, add 20 µl of alpha murine CD8 MicroBeads per spleen and incubate for 15 mins at 4°C.
  • 4. Centrifuge at 300 g for 6 mins, 4°C.
  • 5. In the meantime, embed MACS columns (1 column per 2-3 spleens) in magnetic field and equilibrate with 3 ml of MACS buffer.
  • 6. Resuspend cells in 3 ml of cold MACS buffer, then pipette filtered cell suspension on column and wash with 3 ml MACS buffer.
  • 7. Remove MACS columns from magnetic field and rinse with 4 ml MACS buffer. Capture passage (= positive fraction of cells) in a 15 ml tube.
  • 8. Centrifuge at 300 g for 6 mins, 4°C.
  • 9. Resuspend cells in 5 ml of PBS in a 15 ml tube and add 2.5 µl of FarRed staining solution into the lid of the tube, and invert tube carefully. Incubate for 20 mins at 37 °C.
    • CRITICAL STEP Due to potential cytotoxicity of the staining reagent, do not exceed incubation time of 20 mins.
  • 10. Stop staining reaction by adding 7 ml of ice-cold PBS and centrifuge at 300 g for 6 mins, 4 °C.
  • 11. Resuspend cells in PBS and adjust the cell number to 12.5×106 cells per ml of PBS. Inject 200 µl (2.5×106 cells) intravenously into mice of favored genetic background.

Administration of antigen and adjuvant (day 2)

  • 12. Inject antigen with or without adjuvant intravenously into mice 16 hrs after adoptive T cell transfer. Injection of antigen alone should be included as a control.

Preparation of cryosections (day 2)

  • 13. After 10 hrs, isolate spleens from injected mice, rinse them briefly in ice-cold PBS, and embed them directly in Tissue-Tek®.
  • 14. Store at -20°C over night.
    • PAUSE POINT Tissue blocks can be stored at -20°C for up to a week or for longer time after transfer to -80°C.

Immunofluorescence staining and quantification of cell numbers (day 3)

  • 15. Prepare 5 µm cryosections from frozen tissue blocks by using a microtome/cryostat at a working temperature of -18°C.
    • CRITICAL STEP Discard at least two sections between individual slides to avoid staining of the same cells in different sections.
  • 16. Transfer sections on microscope slides for further treatment.
    • PAUSE POINT Dry sections for several hours.
    • CAUTION Avoid extended exposure to light.
  • 17. Fix sections with iced acetone for 10 mins, then air-dry them for some minutes.
    • CRITICAL STEP Do not exceed fixation time of 10 mins. Otherwise, tissue integrity might be lost. The fixation might be performed at 4°C.
  • 18. Apply blocking solution (1% BSA in PBS) to samples for 1 h at room temperature.
  • 19. Stain sections with alpha murine B220-PE antibody for 1 h at room temperature, then wash twice with PBS.
  • 20. Cover sections with mounting medium (Immu-Mount) and cover slides.
    • PAUSE POINT Slices can be stored at room temperature for up to one week.
  • 21. View sections and capture images with a fluorescence microscope. Determine number of labeled cells with appropriate software. We used an Olympus IX71, employing a 10x objective. T cell numbers were counted (utilizing the “Touch count” mode) and areas analyzed (“Closed polygon”) with Cell F software (Olympus). Furthermore, if distinct labels for multiple cell types are used, intercellular contacts can be enumerated.

Anticipated Results

Resulting immunofluorescence images should allow for enumeration of fluorescently labeled T cells per unit area of splenic DC/T cell zone. Fig. 1 shows recruitment of FarRed-labeled CTLs to the splenic DC-T cell zone which is encircled by blue B220+ B cell follicles. After challenge with the glycolipid alpha-galactosyl ceramide, NKT cells license DCs and CCL17 is induced, thereby facilitating recruitment of CTL in heterozygous CCL17-eGFP knock-in mice4 (Fig 1 left). In the absence of a functional CCL17 allele (in homozygous CCL17-eGFP knock-in mice), the recruitment of CTL to the spleen is substantially reduced and DC-T cell contacts are not observed (Fig 1 right).

References

  1. von Andrian UH & Mempel TR. Homing and cellular traffic in lymph nodes. Nat Rev Immunol. 3, 867-78 (2003).
  2. Castellino, F. et al. Chemokines enhance immunity by guiding naive CD8+ T cells to sites of CD4+ T cell-dendritic cell interaction. Nature 440, 890-895 (2006).
  3. Semmling, V. et al. Alternative cross-priming through CCL17/CCR4-mediated CTL attraction towards NKT cell-licensed dendritic cells. Nat Immunol. DOI: 10.1038/ni.1848 (2010).
  4. Alferink, J. et al. Compartmentalized production of CCL17 in vivo: strong inducibility in peripheral dendritic cells contrasts selective absence from the spleen. J Exp Med. 197, 585-99 (2003).

Acknowledgements

We thank the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 704) for supporting our work and acknowledge the central animal facilities of the University clinic of Bonn.

Figures

Fig 1

Immunofluorescence staining of spleen cryosections from CCL17-eGFP reporter mice (left) or CCL17-deficient mice (right) injected with 2.5×106 far-red fluorochrome labeled OT-I cells on day -1 and then injected with OVA + aGC on day 0. Spleens were isolated 10hrs after injection of antigen. White bars represent 200 um. Blue staining indicates B220+ cells, eGFP+ DCs appear in green, and OT-I cells are shown in red.

Associated Publications

Alternative cross-priming through CCL17-CCR4-mediated attraction of CTLs toward NKT cell–licensed DCs, Verena Semmling, Veronika Lukacs-Kornek, Christoph A Thaiss, Thomas Quast, Katharina Hochheiser, Ulf Panzer, Jamie Rossjohn, Patrick Perlmutter, Jia Cao, Dale I Godfrey, Paul B Savage, Percy A Knolle, Waldemar Kolanus, Irmgard Förster, and Christian Kurts, Nature Immunology 11 (4) 313 - 320 28/02/2010 doi:10.1038/ni.1848

Author information

Verena Semmling, Christoph A. Thaiss, Veronika Lukacs-Kornek, Percy A Knolle & Christian Kurts, Institutes of Molecular Medicine and Experimental Immunology, Friedrich-Wilhelms-Universität, Bonn, Germany

Irmgard Förster, Molecular Immunology, Institut für Umweltmedizinische Forschung an der Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany

Source: Protocol Exchange (2010) doi:10.1038/nprot.2010.52. Originally published online 5 March 2010.

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