Parks, C.

Abstract TCR mediated antigen recognition results in a signaling cascade leading to T cell activation. Antigen binding to its cognate TCR drives receptor aggregation and conformational changes that trigger signaling from the receptor. Conformational change in the CD3 complex (CD3Δc) is required for robust signaling and productive T cell responses. In contrast, weakly recognized antigens do not induce CD3Δc and the resulting T cell response is poor. Thus, T cell responses to weak antigens may be improved by induction of CD3Δc. We previously described an anti-mouse CD3ɛ monovalent Fab (Mono-7D6-Fab) that induced CD3Δc and synergized with weak antigens to enhance the T cell response, a process we termed co-potentiation. However, Mono-7D6-Fab did not induce classical T cell signaling in the absence of antigenic ligation. Mono-7D6-Fab effectively co-potentiated T cells against murine B16-F10 melanoma cells and reduced tumor burden in vivo in a T cell dependent manner. We exploited a model of human cytomegalovirus (CMV) to generate proof of concept that co-potentiation may enhance T cell antigen recognition in humans. Anti-human CD3ɛ monovalent Fab (Mono-OKT3-Fab) improved the CD8 T cell response to the CMV pp65495–503 peptide as measured by increased T cell proliferation, cytokine production, and the ability to kill target cells. Co-potentiation was dependent on antigen specificity, as 1) the effects were dampened when antigen recognition was impaired and 2) sequencing of the TCRs revealed a restricted number of expanded TCR clones. Our results suggest that Mono-OKT3-Fab may have therapeutic clinical applications to achieve a more robust antigen-specific T cell response in the context of diverse human disease, from cancer to chronic infection.

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.