Transient antigen-independent TCR clustering and rapid antigen-dependent TCR clustering and internalization in vivo. CMTMR-labeled OT-I–GFP Rag2^−/− cells were transferred into WT recipients and immunized, and LNs were imaged by two-photon microscopy as in Figs. 5 and 6. Data analyzed were combined from images collected using the 510/20- and 525/50-nm filters. For all data with antigen, time = 0 represents the time at which the antigen was added. (a) Bulk dynamics of T cell motility arrest, TCR clustering, and TCR internalization in the presence and absence of antigen. For no antigen, n = 225 combined from four experiments. For + antigen, n = 82 combined from three experiments. (b) Dynamics of TCR clustering and TCR internalization in individual cells in the presence and absence of antigen. Each line corresponds to a single randomly selected representative cell. (c) Frequency of T cell motility arrest, TCR clustering, and TCR internalization. For no antigen, n = 225 combined from four experiments. For + antigen, n = 82 combined from three experiments. Statistical analysis was performed using Fisher’s exact test. (d) Duration of TCR clustering. Only cells with clustered TCR are represented. Each dot represents one cell, and the bars represent the median. For no antigen, n = 21 combined from four experiments. For + antigen, n = 30 combined from three experiments. Statistical analysis was performed using a two-tailed t test with Welch’s correction.