A role for CD3 conformation in the preTCR/CD3 complex (P4428)

Abstract Mature T cells recognize and respond to antigens through the T cell antigen receptor (TCR). We have established that upon TCR antigenic engagement, the TCR/CD3 complex undergoes a conformational change (CD3dc) of its CD3 epsilon tails to expose a proline-rich sequence and allow downstream signaling to occur. CD3dc has been shown to be required for full TCR/CD3 signaling and function of mature T cells. Pre-TCR assembly with the CD3 complex serves as a developmental checkpoint that ensures the expression of a function variant TCRβ chain in thymocytes. Interestingly, stimulation of the preTCR complex has been found to be ligand-independent; assembly is sufficient. Knowing this, we first determined the stoichiometry of the preTCR/CD3 complex and asked the question, how does assembly impact its ability to undergo conformational change? To answer this question, we employed various novel biochemical methods developed in our laboratory including a flow-cytommetry based immunoprecipitation (IP-FCM) as well as a CD3-pull down assay (CD3-PD). We aim to study the conformation of CD3 in the preTCR/CD3 complex and establish whether or not the assembly of this receptor is enough to induce the active CD3 conformation that supports functional CD3 signaling in mature cells. With our data, we are further able to characterize previously unstudied requirements for CD3 conformational change in the preTCR/CD3 complex with the potential of using this knowledge to manipulate CD3dc in the future.