Secondary T cell–T cell synaptic interactions drive the differentiation of protective CD8+ T cells

Abstract

Immunization results in the differentiation of CD8+ T cells, such that they acquire effector abilities and convert into a memory pool. Priming of T cells takes place via an immunological synapse formed with an antigen-presenting cell (APC). By disrupting synaptic stability at different times, we found that the differentiation of CD8+ T cells required cell interactions beyond those made with APCs. We identified a critical differentiation period that required interactions between primed T cells. We found that T cell–T cell synapses had a major role in the generation of protective CD8+ T cell memory. T cell–T cell synapses allowed T cells to polarize critical secretion of interferon-γ (IFN-γ) toward each other. Collective activation and homotypic clustering drove cytokine sharing and acted as regulatory stimuli for T cell differentiation.

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Figure 1: Temporal requirement for CD8+ T cell differentiation.
Figure 2: The generation of central memory precursor cells and the recall response depends on LFA-1-dependent stable interactions during the CDP.
Figure 3: CD8+ T cell differentiation relies mainly on T cell–T cell contacts.
Figure 4: Characterization of T cell–T cell contacts.
Figure 5: OT-I cell differentiation is regulated by cytokine secretion at T cell–T cell contacts.

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Acknowledgements

We thank M. Nussenzeig (Rockefeller University) for CD11c-YFP mice; M. Coles (Medial Research Council, York) for CD2-RFP mice; R. Locksley (University of California at San Francisco) for YETI mice; and personnel of the Biological Imaging Development Center for technical assistance with imaging. Supported by the Juvenile Diabetes Foundation (M.F.K.) and the US National Institutes of Health (R01AI52116 to M.F.K.).

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Contributions

A.G. and M.F.K. designed the experiments and wrote and revised the manuscript; A.G. did the experiments; O.K. did or participated in experiments involving immunization with LCMV and LM-OVA; P.B. analyzed data and generated Matlab scripts; E.O. generated IFN-γ–GFP constructs and did preliminary experiments; and J.H. and M.M. provided P14 mice and participated in LCMV-challenge experiments.

Corresponding authors

Correspondence to Audrey Gérard or Matthew F Krummel.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 (PDF 4301 kb)

Supplementary Video 1

CD8 T cell behavior relative to DCs 2h after DEC-OVA immunization. Representative video of OTI-RFP cell behavior relative to DCs 2h after DEC-OVA immunization. OTI-RFP cells (red) were transferred into CD11c-YFP (green) recipients. Recipients were immunized with DEC-OVA and CD40 Ab in the footpads. 2 hours after immunization, the draining popliteal LN was explanted and visualized by 2-photon microscopy. The video shows a normal shading of an imaging volume of 280 (x) × 280 (y) × 84 μm (z). Elapsed time is displayed in minutes:seconds. (MOV 2815 kb)

Supplementary Video 2

CD8 T cell behavior relative to DCs 10h after DEC-OVA immunization. Representative video of OTI-RFP cell behavior relative to DCs 10h after DEC-OVA immunization. OTI-RFP cells (red) were transferred into CD11c-YFP (green) recipients. Recipients were immunized with DEC-OVA and CD40 Ab in the footpads. 10 hours after immunization, the draining popliteal LN was explanted and visualized by 2-photon microscopy. The video shows a normal shading of an imaging volume of 280 (x) × 280 (y) × 99 μm (z). Elapsed time is displayed in minutes:seconds. (MOV 4958 kb)

Supplementary Video 3

CD8 T cell clustering around DCs 24h after DEC-OVA immunization. Representative video of OTI-RFP cell behavior relative to DCs 24h after DEC-OVA immunization. OTI-RFP cells (red) were transferred into CD11c-YFP (green) recipients. Recipients were immunized with DEC-OVA and CD40 Ab in the footpads. 24 hours after immunization, the draining popliteal LN was explanted and visualized by 2-photon microscopy. The video shows a normal shading of an imaging volume of 280 (x) × 280 (y) × 93 μm (z). Elapsed time is displayed in minutes:seconds. Clusters are indicated by arrows as they occur and have been verified in the 3 dimensions. (MOV 2994 kb)

Supplementary Video 4

CD8 T cell swarming around DCs 72h after DEC-OVA immunization. Representative video of OTI-RFP cell behavior relative to DCs 72h after DEC-OVA immunization. OTI-RFP cells (red) were transferred into CD11c-YFP (green) recipients. Recipients were immunized with DEC-OVA and CD40 Ab in the footpads. 72 hours after immunization, the draining popliteal LN was explanted and visualized by 2-photon microscopy. The video shows a normal shading of an imaging volume of 280 (x) × 280 (y) × 63 μm (z). Elapsed time is displayed in minutes:seconds. (MOV 2297 kb)

Supplementary Video 5

Impairment of ICAM-1-/- CD8 T cell clustering 24h after DEC-OVA immunization. Representative video of WT and ICAM-1-/- OTI cell behavior 24h after DEC-OVA immunization. CFSE labeled-WT OTI cells (green) and CMTMR labeled-ICAM-1-/- OTI cells (red) were ad-mixed and transferred into C57Bl6 recipients. Recipients were immunized with DEC-OVA and CD40 Ab in the footpads. 24 hours after immunization, the draining popliteal LN was explanted and visualized by 2-photon microscopy. The video shows a normal shading of an imaging volume of 280 (x) × 280 (y) × 96 μm (z). Elapsed time is displayed in minutes:seconds. Major WT OTI and ICAM-1-/- OTI clusters are indicated respectively by a green and a red arrow as they occur and have been verified in the 3 dimensions. (MOV 841 kb)

Supplementary Video 6

CD8 T cell clustering around DCs 24h after DEC-OVA immunization is inhibited by LFA1 blocking Ab treatment. Representative video of OTI-RFP cell behavior relative to DCs 24h after DEC-OVA immunization after LFA1 blocking Ab treatment. OTI-RFP cells (red) were transferred into CD11c-YFP (green) recipients. Recipients were immunized with DEC-OVA and CD40 Ab in the footpads. 22 hours after immunization, mice were treated with 150ug LFA1 Ab. 2 hours after treatment, the draining popliteal LN was explanted and visualized by 2-photon microscopy. The video shows a normal shading of an imaging volume of 280 (x) × 280 (y) × 84 μm (z). Elapsed time is displayed in minutes:seconds. Clusters are indicated by arrows as they occur and have been verified in the 3 dimensions, compare to equivalents in Movie S2. (MOV 2524 kb)

Supplementary Video 7

CD8 T cell clustering 24h after Cd11c-DTR BMDCs immunization. Representative video of CFSE-labeled OTI cell behavior relative to Cd11c-DTR BMDCs 24-30h after immunization. CFSE labeled OTI cells (green) were transferred into WT recipients. Recipients were immunized in the flanks with CMTMR-labeled and OVA peptide-pulsed BMDCs (red) generated from Cd11c-DTR mice. Twenty-two to thirty hours after immunization, the draining inguinal LN was explanted and cell clustering was visualized by 2-photon microscopy. The video shows a normal shading of an imaging volume of 280 (x) × 280 (y) × 105 μm (z). Elapsed time is displayed in minutes:seconds. Clusters are indicated by arrows as they occur and have been verified in the 3 dimensions. (MOV 310 kb)

Supplementary Video 8

Presence of CD8 T cell clustering following APC ablation after initial T cell activation. Representative video of CFSE-labeled OTI cell behavior relative to Cd11c-DTR BMDCs 24h after immunization and after BMDCs ablation. CFSE-labeled OTI cells (green) were transferred into WT recipients. Recipients were immunized in the flanks with CMTMR-labeled, OVA peptide-pulsed BMDCs generated from Cd11c-DTR mice. Diphtheria toxin was administered 8 hours post-immunization. Twenty-two to thirty hours after immunization, the draining inguinal LN was explanted and cell clustering was visualized by 2-photon microscopy. The video shows a normal shading of an imaging volume of 280 (x) × 280 (y) × 105 μm (z). Elapsed time is displayed in minutes:seconds. Clusters are indicated by arrows as they occur and have been verified in the 3 dimensions. (MOV 359 kb)

Supplementary Video 9

Localization of vesicle-containing IFNγ-GFP during T cell clustering. Representative video of IFNγ-GFP localization in a T cell during T cell clustering event. T cell blasts were transduced with a plasmid coding for IFNγ-GFP. One to two days after infection, cells were stimulated with PMA and Ionomycin to induce clustering. Two hours after stimulation, cells were imaged using an epifluorescence microscope. Video shows dynamic IFNγ-GFP localization at the site of T-T contact. Images were acquired at a rate of 1 image every 30 seconds. (MOV 739 kb)

Supplementary Video 10

Example of T-T synapse. OTI T-cells were stimulated with PMA and Ionomycin. Twenty-four hours after activation, cells were stained for ICAM-1 (green) and captured IFNγ (red). Movie shows 360-degree rotation of a 10um z-stack of a representative confocal image of a T-T synapse. (MOV 817 kb)

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Gérard, A., Khan, O., Beemiller, P. et al. Secondary T cell–T cell synaptic interactions drive the differentiation of protective CD8+ T cells. Nat Immunol 14, 356–363 (2013). https://doi.org/10.1038/ni.2547

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