Figure 8.
Figure 8. TCR signaling does not require motility arrest. (a–c) CMTMR-labeled OT-I–GFP Rag2−/− cells were transferred into WT or CD11c-YFP recipients and immunized, and LNs were imaged by two-photon microscopy as in Figs. 5 and 6. (a) Contingency of TCR clustering and internalization on motility arrest in vivo. Only cells that clustered or internalized their TCR are represented. For clustering no antigen (Ag), n = 21 combined from four experiments. For clustering + antigen, n = 30. For internalization + antigen, n = 13 combined from three experiments. (b and c) Single z-plane time lapse images of in vivo T cell–DC interactions in the presence of antigen. See Video 8 for the full time lapse. Data are representative of four experiments. (b) Motile in vivo T cell–DC interaction. (c) Arrested in vivo T cell–DC interaction. (d) Representative in vitro time lapse of a naive OT-I–GFP T cell internalizing TCR while crawling along an antigen-bearing DC. The T cell internalizes its TCR through microclusters as it maintains an active leading edge and continually crawls along a DC. Contrast (top) and maximum intensity z projection of OT-I–GFP fluorescence (bottom) are shown. Arrowheads point to the T cell of interest. The white lines define the xy space rendered in the xz rendering below. See Video 9 for the full time lapse. (e–h) Naive OT-I T cells were labeled with Fura-2AM and placed on lipid bilayers presenting pMHC + ICAM-1. Data are representative of two experiments. (e and f) Contrast (grayscale background image) and calcium imaging (pseudocolor overlay) visualized by epifluorescence microscopy. Low–high calcium is represented by a blue–white pseudocolor scale. See Video 10 for the full time lapses. (e) Representative time lapse of calcium signaling while crawling along a lipid bilayer with oscillations in calcium levels and motility. (f) Representative time lapse of a T cell fluxing calcium while arrested. (b, c, e, and f) White lines show cell tracks. (g and h) Quantification of motility with relation to calcium signaling. At each time point, the instantaneous velocity of the cell was correlated with its calcium level. Time points with a Fura-2AM 340-nm/380-nm emission ratio of >1.4× baseline were scored as having elevated calcium. Time points with an instantaneous velocity <2 µm/min were scored as arrested. The dashed line indicates the cutoff value below which cells were termed nonmotile (2 µm/min). n = 1,970 time points from 40 cells pooled from two experiments. Bars: (b and d–f) 10 µm; (c) 5 µm.

TCR signaling does not require motility arrest. (a–c) CMTMR-labeled OT-I–GFP Rag2−/− cells were transferred into WT or CD11c-YFP recipients and immunized, and LNs were imaged by two-photon microscopy as in Figs. 5 and 6. (a) Contingency of TCR clustering and internalization on motility arrest in vivo. Only cells that clustered or internalized their TCR are represented. For clustering no antigen (Ag), n = 21 combined from four experiments. For clustering + antigen, n = 30. For internalization + antigen, n = 13 combined from three experiments. (b and c) Single z-plane time lapse images of in vivo T cell–DC interactions in the presence of antigen. See Video 8 for the full time lapse. Data are representative of four experiments. (b) Motile in vivo T cell–DC interaction. (c) Arrested in vivo T cell–DC interaction. (d) Representative in vitro time lapse of a naive OT-I–GFP T cell internalizing TCR while crawling along an antigen-bearing DC. The T cell internalizes its TCR through microclusters as it maintains an active leading edge and continually crawls along a DC. Contrast (top) and maximum intensity z projection of OT-I–GFP fluorescence (bottom) are shown. Arrowheads point to the T cell of interest. The white lines define the xy space rendered in the xz rendering below. See Video 9 for the full time lapse. (e–h) Naive OT-I T cells were labeled with Fura-2AM and placed on lipid bilayers presenting pMHC + ICAM-1. Data are representative of two experiments. (e and f) Contrast (grayscale background image) and calcium imaging (pseudocolor overlay) visualized by epifluorescence microscopy. Low–high calcium is represented by a blue–white pseudocolor scale. See Video 10 for the full time lapses. (e) Representative time lapse of calcium signaling while crawling along a lipid bilayer with oscillations in calcium levels and motility. (f) Representative time lapse of a T cell fluxing calcium while arrested. (b, c, e, and f) White lines show cell tracks. (g and h) Quantification of motility with relation to calcium signaling. At each time point, the instantaneous velocity of the cell was correlated with its calcium level. Time points with a Fura-2AM 340-nm/380-nm emission ratio of >1.4× baseline were scored as having elevated calcium. Time points with an instantaneous velocity <2 µm/min were scored as arrested. The dashed line indicates the cutoff value below which cells were termed nonmotile (2 µm/min). n = 1,970 time points from 40 cells pooled from two experiments. Bars: (b and d–f) 10 µm; (c) 5 µm.

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