Thesis (Ph.D.)--University of Washington, 2023The immune system’s threat detection hinges on T cells’ ability to perceive differences in the quality and quantity of peptide-major histocompatibility complex (pMHC) antigens. Our T cells are tolerant to the vast majority of antigens they sense, as these pMHCs are derived from our own endogenous proteome and represent “self”. In the case of a viral infection, virus-derived pMHC antigens are perceived by T cells as a threat and elicit an effector response to clear the infection. This fundamental perception of “self” vs “foreign” is based on antigen quality. Self pMHCs have a relatively low affinity for the T cell antigen receptor (TCR), while foreign pMHCs have relatively high TCR binding affinities. The current paradigm states that this affinity discrimination is enabled by a signaling network downstream of the TCR that performs kinetic proofreading during antigen sensing, whereby only long-lasting TCR binding events resulting from interactions with high affinity foreign pMHCs pass a series of proofreading reactions and further propagate the signal. However, numerous in vitro and in vivo studies have shown that T cells do not respond uniformly to variations in the quality and quantity of foreign antigens, but rather tailor their response to each unique threat. This suggests an unresolved signal transmission mechanism within the TCR signaling network that regulates T cell responses in a tunable, and not simply all-or-none fashion. The Erk and NFAT signaling pathways connect TCR engagement to gene regulation, and thus their combined signaling dynamics may transmit information about pMHC inputs. We formed this hypothesis from two main bodies of evidence. First, signaling pathway dynamics, including dynamics of the Erk pathway, are known to transmit extracellular signal information in many other contexts to enable input-specific cellular responses. Second, NFAT and the Erk-activated transcription factor AP-1 work cooperatively to regulate gene expression in activated T cells, and disruption of their cooperativity leads to distinct phenotypes. Therefore, it is plausible that signaling activity of these two pathways, in combination, over the course of initial activation can tune gene expression in T cells to generate antigen-specific responses. To test this idea, we developed a dual reporter mouse strain and a quantitative imaging assay that together enable simultaneous monitoring of Erk and NFAT dynamics in live T cells over day-long timescales as they respond to varying pMHC inputs. We found that both pathways initially activate uniformly across various pMHC inputs but diverge over longer (9+ hrs) timescales, enabling independent encoding of pMHC affinity and dose. Next, we combined signaling perturbations and variable pMHC stimulation with RNA sequencing and mathematical modeling to uncover multiple temporal and combinatorial mechanisms for decoding these late signaling dynamics to generate pMHC-specific transcriptional responses. Our results underscore the importance of long timescale signaling dynamics in T cell antigen perception and establish a framework for understanding T cell responses under diverse contexts. Our findings, while only scratching the surface of the complexity and sophistication of T cell signaling and transcriptional regulation, point to a strategy for improving T cell-based therapies by engineering precise temporal control over signaling inputs