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Dynamic imaging of chemokine-dependent CD8+ T cell help for CD8+ T cell responses

Nature Immunology volume 8, pages 921–930 (2007)Cite this article

Abstract

Naive T lymphocytes move efficiently in lymphoid tissues while scanning dendritic cells in search of cognate complexes of peptide in major histocompatibility molecules. However, T cell migration ceases after recognition of cognate antigen. We show here that during the initiation of antigen-specific CD8+ T cell responses, naive CD8+ polyclonal T cells 'preferentially' interacted in an antigen-independent way with mature dendritic cells competent to present antigen to antigen-specific CD8+ T cells. These antigen-independent interactions required expression of the chemokine receptor CCR5 on polyclonal T cells and increased the efficiency of the induction of naive, low-precursor-frequency CD8+ T cell responses. Thus, antigen-specific CD8+ T cells favor the priming of naive CD8+ T cells by promoting the CCR5-dependent recruitment of polyclonal CD8+ T cells to mature dendritic cells.

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Figure 1: Polyclonal CD8+ T cells arrest in the presence of OVA-DCs that establish long-lasting contacts with OVA-specific CD8+ T cells.
Figure 2: Poly–T cell arrest is sensitive to treatment with PTX.
Figure 3: Poly–T cell arrest is CCR5 dependent.
Figure 4: Poly–T cells arrest in vitro selectively on DCs engaged in antigen-specific interactions with CD8+ T cells.
Figure 5: Polyclonal CD8+ T cells interact 'preferentially' and for longer periods with DCs involved in cognate interactions with T cells.
Figure 6: Similar interactions between poly–T cells and antigen-presenting DCs either interacting simultaneously with an OVA–T cell or not.
Figure 7: CD8+ T cell help for CD8+ T cell responses.

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Acknowledgements

We thank C. Reis e Sousa (Cancer Research UK) for C57BL/6 OT-I Rag2−/− TCR-transgenic mice; and P. Guermonprez for discussions. Supported by the Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, Ligue de Lutte Contre le Cancer, Association de la Recherche contre le Cancer (A.B.), Institut Curie, European Community DC-Thera (LSBH-CT-2004-512074; “Dendritic cells for novel immunotherapies”), European Community CancerImmunotherapy (LSHC-CT-2006-518234; “Cancer Immunology and Immunotherapy”), Ecole Normale Supérieure (A.S.) and a Marie Curie Intra-European Fellowship (A.K.N.).

Author information

Author notes

  1. Stéphanie Hugues and Alix Scholer: These authors contributed equally to this work.

Authors and Affiliations

  1. Institut Curie, Centre de Recherche, Paris, F-75248, France

    Stéphanie Hugues, Alix Scholer, Alexandre Boissonnas, Alexander Nussbaum, Sebastian Amigorena & Luc Fetler

  2. Institut National de la Santé et de la Recherche Médical U653, Immunité et Cancer, Institut Curie, Paris, F-75248, France

    Stéphanie Hugues, Alix Scholer, Alexandre Boissonnas, Alexander Nussbaum & Sebastian Amigorena

  3. Institut National de la Santé et de la Recherche Médical U543, Laboratoire d'Immunologie Cellulaire, Hôpital Pitié-Salpétrière, Paris, F-75013, France

    Christophe Combadière

  4. Centre National de la Recherche Scientifique UMR 168, Laboratoire Physico-Chimie Curie, Institut Curie, Paris, F-75248, France

    Luc Fetler

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Contributions

S.H., A.S. and L.F., design, immunobiology and imaging experiments, analysis, interpretation, coordination and writing; A.B., help with experiments and interpretation; A.N., gp33-expressing B16 melanoma cells; C.C., CCR5-deficient mice; and S.A., design, interpretation, coordination and writing.

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Correspondence to Sebastian Amigorena or Luc Fetler.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 (PDF 1191 kb)

Supplementary Movie 1

Polyclonal CD8+ T (poly-T) cells (green, cytoplasmic labelling) and OVA-specific CD8+ T (OVA-T) cells (red) were injected i.v. into mice that were immunized 4 h later with anti-DEC205-OVA conjugates plus anti-CD40. Two-photon imaging was performed 18 h later on draining LNs, after labelling of LN resident DCs (green, membrane labelling). Time of imaging: 30 min. Playback speed: 200×. Size: 175×175μm. (AVI 1833 kb)

Supplementary Movie 2

Polyclonal CD8+ T (poly-T) cells (green, cytoplasmic labelling) were injected i.v. into mice that were immunized 4 h later with anti-DEC205-OVA conjugates plus anti-CD40. 2-photon imaging was performed 18 h later on draining LNs, after labelling of LN resident DC (green, membrane labelling). Time of imaging: 30 min. Playback speed: 200×. Size: 280×280μm. (AVI 1551 kb)

Supplementary Movie 3

Time–lapse video–microscopy (acceleration 52×; 65×65 μm) showing transient interactions between polyclonal CD8+ T cells and bone marrow-derived DC previously incubated with OVA peptide (5μM) and LPS for 18h. (AVI 1050 kb)

Supplementary Movie 4

Time–lapse video-microscopy (acceleration 52×; 65×65 μm) showing long–lasting interactions between naive OVA–specific CD8+ T cells and bone marrow-derived DC previously incubated with OVA peptide (5μM) and LPS for 18h. (AVI 1410 kb)

Supplementary Movie 5

Time-lapse video-microscopy (acceleration 52×; 65×65 μm) showing long-lasting interactions of both polyclonal CD8+ T cells (poly-T, green) and OVA–specific CD8+ T cells (OVA–T, red) with bone marrow-derived DC previously incubated with OVA peptide (5μM) and LPS for 18h. (AVI 1757 kb)

Supplementary Movie 6

Time-lapse video-microscopy (acceleration 52×; 65×65 μm) showing long-lasting interactions of only OVA–specific CD8+ T cells (OVA–T, red) and Pertussis toxin pre-treated polyclonal CD8+ T cells (PTX treated poly–T, green) with bone marrow-derived DC previously incubated with OVA peptide (5μM) and LPS for 18h. (AVI 1757 kb)

Supplementary Movie 7

Time–lapse video-microscopy (acceleration 52×; 65×65 μm) of CFSE-labeled OVA–specific CD8+ T cells (OVA–T, green) and polyclonal CD8+ T cells (poly–T, unlabeled) interacting with OVA–loaded bone marrow derived DC (OVA-DC, unlabeled) versus CMTMR–labeled unloaded bone marrow–derived DC (0-DC, red). (AVI 4387 kb)

Supplementary Movie 8

CMTMR–labeled polyclonal CD8+ T (poly–T) cells (blue) were injected i.v. into mice that were injected s.c. with a mixture (1:1) of GFP+ OVA–pulsed DC (red) and CFP+ unloaded DC (green). Two-photon imaging was performed 18 h later on draining LNs. The numbers of contacts between poly-T cells and OVA-DC (red squares) or 0-DC (green squared) were quantified and incremented each time a new contact appears. Time of imaging: 30 min. Playback speed: 200×. Size: 230×230μm. (AVI 2181 kb)

Supplementary Movie 9

CMTMR–labeled polyclonal CD8+ T (poly–T) cells (blue) and OVA–specific CD8+ T cells (unlabeled) were injected i.v. into mice that were injected s.c. with a mixture (1:1) of GFP+ OVA–pulsed DC (red) and CFP+ unloaded DC (green). Two–photon imaging was performed 18 h later on draining LNs. The numbers of contacts between poly–T cells and OVA–DC (red squares) or 0–DC (green squared) were quantified and incremented each time a new contact appears. Time of imaging: 30 min. Playback speed: 200×. Size: 230×230μm. (AVI 5184 kb)

Supplementary Movie 10

CMTMR–labeled CCR5–deficient polyclonal CD8+ T (poly–T) cells (blue) and OVA–specific CD8+ T cells (unlabeled) were injected i.v. into mice that were injected s.c. with a mixture (1:1) of GFP+ OVA–pulsed DC (red) and CFP+ unloaded DC (green). Two–photon imaging was performed 18 h later on draining LNs. The numbers of contacts between poly–T cells and OVA–DC (red squares) or 0–DC (green squared) were quantified and incremented each time a new contact appears. Time of imaging: 25 min. Playback speed: 200×. Size: 230×230μm. (AVI 1895 kb)

Supplementary Movie 11

CMTMR–labeled polyclonal CD8+ T (poly–T) cells (blue) and GFP+ OVA–specific CD8+ T cells (green) were injected i.v. into mice that were injected s.c. with CFP+ OVA–pulsed DC (red). Two–photon imaging was performed 18 h later on draining lymph nodes. Time of imaging: 15 min. Playback speed: 200×. Size: 110×110μm. (AVI 533 kb)

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Hugues, S., Scholer, A., Boissonnas, A. et al. Dynamic imaging of chemokine-dependent CD8+ T cell help for CD8+ T cell responses. Nat Immunol 8, 921–930 (2007). https://doi.org/10.1038/ni1495

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