Pre-TCR signaling intensity shapes the TCRβ repertoire

Signaling via pre-TCR is essential for initial T cell differentiation. How the signal is triggered and whether its alteration impacts on lymphocyte repertoires is nevertheless debated. We show that a mutation in the transmembrane domain of the CD3ζ chain impairs murine thymocyte development at the double negative (DN) stage coinciding with reduced levels of steady-state pre-TCR signaling. Single particle tracking and TIRF microscopy on mouse primary DN3/DN4 thymocytes shows that wild-type pre-TCR form particles with a broad range of fluorescent intensities whereas mutant complexes are less intensely labeled and move faster, suggesting reduced pre-TCR nanoclustering. Notably, the mutant early-pre-TCR-selected-DP population shows less TCRβ repertoire diversity. We thus propose that the pre-TCR forms stable nanoclusters, the size of which determines signaling efficacy. This signaling does not merely promote maturation of any thymocyte expressing a rearranged TCRβ chain, but shapes the repertoire of DP thymocytes that can audition for positive and negative selection.