T cells survey antigen-presenting dendritic cells (DCs) by migrating through DC networks, arresting and maintaining contact with DCs for several hours after encountering high-potency complexes of peptide and major histocompatibility complex (pMHC), leading to T cell activation. The effects of low-potency pMHC complexes on T cells in vivo, however, are unknown, as is the mechanism controlling T cell arrest. Here we evaluated T cell responses in vivo to high-, medium- and low-potency pMHC complexes and found that regardless of potency, pMHC complexes induced upregulation of CD69, anergy and retention of T cells in lymph nodes. However, only high-potency pMHC complexes expressed by DCs induced calcium-dependent T cell deceleration and calcineurin-dependent anergy. The pMHC complexes of lower potency instead induced T cell anergy by a biochemically distinct process that did not affect T cell dynamics.
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We thank R. Steinman and R. Schwartz for discussions; H.A. Zebroski for peptide synthesis; E. Besmer, R. Masilamani and E. Market for manuscript preparation; and T. Starr for technical assistance. TCR-transgenic mice expressing the Vβ3Vα11 were provided by L. Denzin (Memorial Sloan Kettering Cancer Institute). Supported by Fondation de Recherche Medicale (D.S.), the Rothchild Foundation (G.S.), the Cancer Research Institute (T.O.C.), a Bernard Levine Fellowship (R.V.), the Medical Scientist Training Program (GM07739 to R.L.L.), the Schering Foundation (T.S.), the National Institutes of Health (M.C.N. and M.L.D.) and the Howard Hughes Medical Institute (M.C.N.).
The authors declare no competing financial interests.
Supplementary Figures 1–9 and Supplementary Methods (PDF 835 kb)
Two-photon visualization of T cell motility in a representative lymph node of a CD11c-EYFP mouse injected 6-9 hours earlier with the high-potency construct α-DEC-L98A. Shown is a maximum intensity projection of a 50 μm-thick volume. Antigen-specific AND T cells which express ECFP (pseudo-colored red) move more slowly than non-specific T cells (EGFP, pseudo-colored orange), and are more frequently decelerated on dendritic cells (EYFP, pseudo-colored green). The sequence is repeated with tracks of specific ECFP-AND cells indicated in cyan, and tracks of nonspecific EGFP-T cells shown in magenta. Time elapsed is in seconds. (MOV 8153 kb)
A representative lymph node of a mouse similarly treated with the Y97K peptide, visualized as in Supplementary Movie 1. Antigen-specific T cells move as fast as non-specific T cells, and interact similarly with dendritic cells. (MOV 7943 kb)
A representative lymph node of a mouse similarly treated with the low-potency peptide T102L, visualized as in Supplementary Movie 1. Antigen-specific T cells move as fast as non-specific T cells, and interact similarly with dendritic cells. (MOV 9353 kb)
Calcium signaling and migration on glass supported bilayers. In vitro activated AND T cells were labeled with fura-2 and were imaged every 15 seconds. The images on the left are pseudo-colored ratio images representing fura-2 ratios, such that purple corresponds to a ratio of 0.2 and red corresponds to a ratio of 1.0. The images on the right are generated using Interference Reflection Microscopy (IRM) and the dark areas represent cells making contact with the bilayer substrate. The bilayer in this sequence contains 10 molecules/μm2 of L98A loaded GPI–I-Ek and 300 molecules /μm2 of GPI–ICAM-1. High calcium fluxes are observed as soon as cells make an IRM and that calcium levels are sustained for long periods of time. This movie is a companion to figure 5A. Only the cells making IRMs are quantified. Scale bar equals 10 μm. (MOV 4546 kb)
Calcium signaling and migration on glass supported bilayers. This movie was acquired using similar conditions as in movie 3, except that the bilayer contains 10 molecules/μm2 of Y97K loaded GPI–I-Ek and 300 molecules /μm2 of GPI–ICAM-1. Only the cells making IRMs are quantified. Scale bar equals 10 μm. (MOV 4750 kb)
Calcium signaling and migration on glass supported bilayers. This movie was acquired using similar conditions as in movie 3, except that the bilayer contains 10 molecules/μm2 of T102L loaded GPI–I-Ek and 300 molecules /μm2 of GPI–ICAM-1. Only the cells making IRMs are quantified. Scale bar equals 10 μm. (MOV 4512 kb)
Calcium signaling and migration on glass supported bilayers. This movie was acquired using similar conditions as in movie 3, except that the bilayer contains 50 molecules/μm2 of unloaded GPI–I-Ek and 300 molecules /μm2 of GPI–ICAM-1. Only the cells making IRMs are quantified. Scale bar equals 10 μm. (MOV 6740 kb)
Confocal visualization of Ca2+ response in a representative lymph node of a B10BR mouse injected 5 hours earlier with the high-potency construct α-DEC-L98A. Intravital images were acquired between 0 and 2h after cell transfer. Shown is a maximum intensity projection of a 20 μm-thick volume. Blood flow is visualized by Alexa-647 70Kd dextran administration (pseudo-colored blue). Antigen-specific AND T cells labeled with Cell Tracker Orange (CMRA) (pseudo-colored red). Cells were co-labeled with Fluo-4, a calcium sensitive dye (pseudo-colored green). Increases in Fluo-4 fluorescence indicate high Ca2+ levels. Shown here, high Fluo-4 fluorescence intensity occurs as AND T cells exit the blood vessel, appears as yellow/orange when overlapping with the cytoplasmic dye. (AVI 2470 kb)
Confocal visualization of Ca2+ response in a representative lymph node of a B10BR mouse injected 5 hours earlier with the medium-potency construct α-DEC-Y97K. As described in Supplementary Movie 8. (AVI 3897 kb)
Confocal visualization of Ca2+ response in a representative lymph node of a B10BR mouse injected 5 hours earlier with the low-potency construct α-DEC-T102L. As described in Supplementary Movie 8. (AVI 3939 kb)
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Skokos, D., Shakhar, G., Varma, R. et al. Peptide-MHC potency governs dynamic interactions between T cells and dendritic cells in lymph nodes. Nat Immunol 8, 835–844 (2007). https://doi.org/10.1038/ni1490
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