TCR internalization does not require cSMAC formation. Naive or activated OT-I–GFP T cells were incubated with DCs pulsed with SL8 peptide and imaged by epifluorescence microscopy. Data were pooled from two experiments. For naive T cells, n > 60 cells per condition. For activated T cells, n > 30 cells per condition. P-values were determined using Fisher’s exact test. (a) TCR dynamics. The percentages of T cells in contact with DCs (contact), with a cSMAC, or with internalized TCR are indicated. Only T cells that contacted a DC during the time lapse are included. (b) Duration of cSMAC persistence. Only T cells that formed a cSMAC are included. Each dot represents one cell, and the bars represent the median. (c and d) Naive T cell–DC interactions. Contrast (top) and maximum intensity z projection of OT-I–GFP fluorescence (bottom) imaged by epifluorescence microscopy are shown. The dotted line indicates the DC border, and the arrowheads indicate internalized TCR vesicles. Bars, 10 µm. (c) The T cell contacts the antigen-bearing DC (1,000 ng/ml SL8), stops crawling and adopts a rounded morphology, forms a cSMAC, and then internalizes vesicles of TCR through the cSMAC. See Video 2 for full time lapse. (d) The T cell contacts the antigen-bearing DC (10 ng/ml SL8), forms microclusters at the T cell–DC interface, and then rapidly internalizes the TCR without forming a cSMAC. See Video 3 for full time lapse. (e) Frequency of TCR behaviors. (f) Frequency of cSMAC-independent TCR internalization. Only cells that internalized their TCR are included; those that did not form a cSMAC are designated cSMAC independent.