Characterizing Signals Associated with the pre-TCR during T Cell Development

Abstract

Developing T cells are subjected to a gauntlet of tests in the thymus to ensure a safe and effective T cell receptor (TCR) repertoire. β-selection, a checkpoint early in T cell development, tests the functionality of rearranged TCRβ chains. Cells with functional TCRβ chains receive pre-TCR signals, which promote survival, proliferation, differentiation, and maturation of the TCR. While the signals associated with the mature TCR have been carefully studied, those associated with the pre-TCR have not been well described. Thus, we sought to characterize the pre-TCR signals that occur during early T cell development. We first evaluated the role of pre-TCR signaling across early developmental stages. Sorted T cell progenitor populations were co-cultured with OP9-DL4 cells in the presence or absence of BAY61-3606, an inhibitor of the pre-TCR signaling-mediator, Syk. The effect on survival and developmental progression was tracked by flow cytometry over several days. Our data indicate that Syk-dependent pre-TCR signaling is important for cell survival and differentiation beyond the β-selection checkpoint. However, the dependence on Syk-mediated pre-TCR signals decreases with development and is not necessary by the immature single-positive stage. We subsequently characterized the duration and magnitude of pre-TCR signals. Fetal pre-β-selection thymocytes were loaded with a calcium-indicator dye and subjected to time-lapse imaging; calcium flux was analyzed over time in individual cells. Preliminary data indicate that pre-TCR signaling events in a polyclonal population are infrequent and characterized by low, sustained levels of intracellular calcium. Together, our findings describe the pre-TCR signals associated with early T cell development.