Fig. 1. T cell activation triggers TCR internalization and TCR release.

Naïve CD3⁺ T cells were stained with anti-TCRβ-APC after (a) or before (b) stimulation with soluble (sAb) or plate-immobilized (iAb) anti-CD3/CD28 antibodies for 3 h. The mean fluorescence intensity (MFI) was measured by flow cytometry. Data represent the mean ± SEM of three independent experiments. c CD3⁺ T cells stained with the CellMask™ Green Plasma Membrane Stain (PMS) and anti-TCRβ-Alexa594 for 1 h at 4 °C were stimulated as in (b). White arrows indicate internalized or released TCRβ clusters. These results were independently repeated three times. d CD8⁺ T cells from an OTI mouse were stained as in (b) and placed on a lipid bilayer presenting pOVA_257–264/H-2K^b/ICAM-1 (0–500 molecules/μm²) for 3 h. Green arrows indicate internalized or released TCRβ⁺ clusters or particles. e Schematic diagram of in vivo experimental setting for antigen-dependent TCRβ release. Wild-type mice were i.p. administered with either 100 μg of pOVA_257-264 or _323-339. After 48 h, TCRβ-Alexa488 and CTV stained naïve OTII CD4⁺ T cells were i.v. injected. TCRβ intensity of CTV⁺ T cells from draining lymph node and spleen was determined by flow cytometry after 4 h. f Schematic model of TCR internalization and release. The model hypothesizes that not all TCRs are internalized (i), but some are released (ii) during T–APC interaction. Statistics were performed using unpaired two-tailed t test (e) or one-way ANOVA with post hoc Tukey’s multiple comparisons test (a, b, d). Source data are provided as a Source Data file.