Antigen perception in T cells by long-term signaling dynamics

Introduction::
The immune system can mount tailored responses to different pathogenic threats while sparing healthy cells and tissues in the body. This remarkable selectivity stems from T cells’ ability to obtain information about the challenge from peptide antigens presented on major histocompatibility complex ligands (pMHCs). The affinity of the pMHC ligand for the T cell receptor (TCR) conveys information about the dissimilarity of the antigen to self, whereas pMHC dose conveys complementary information about the severity of the perceived threat.




Cells can convey information about their environment through the dynamics of signaling responses; as such, T cells may encode information about pMHC inputs through dynamics of the signaling pathways downstream of the T cell receptor (TCR), namely the Erk/MAP kinase pathway and the Calcium/NFAT signaling pathway. However, despite mounting evidence that signaling dynamics is critical for immune threat discrimination, this hypothesis has remained unexplored in T cells, in part because it is challenging to measure signaling dynamics in living, primary T cells over the extended timescales of T cell activation. All studies, to our knowledge, have imaged signaling activities over very short timescales (~30 mins), effectively probing only the beginning of the signaling response.




Materials and Methods::
To test this hypothesis, we have developed a dual-color reporter mouse strain and a novel long-term live-cell imaging assay that enables us to measure the signaling dynamics of the Erk/MAP kinase and the NFAT signaling pathways in living primary T cells of day-long timescales. Here, we use this experimental system to measure Erk and NFAT signaling dynamics in response to pMHCs of differing doses and affinities. To further understand how pMHC-specific Erk and NFAT signaling dynamics are decoded to generate distinct functional responses, we perform bulk and single-cell RNA-sequencing measurements of cells stimulated with different pMHC inputs, or treated with different inhibitors of the Erk and NFAT pathways. We then use this data in conjunction with mathematical to identify the candidate cis-regulatory mechanisms underlying pMHC-specific gene regulatory programs.




Results, Conclusions, and Discussions::
We find that both Erk and NFAT pathways initially activate uniformly, but show pMHC-specific pulsatile signaling dynamics over longer (9+ hrs) timescales. Importantly, variations in pMHC dose elicited distinct effects compared to variations in pMHC affinity, thus allowing these two signaling pathways to independently convey information about pMHC affinity and dose. We further found that these late signaling dynamics are decoded via multiple temporal and combinatorial mechanisms to generate pMHC-specific transcriptional responses. Our findings underscore the importance of long timescale signaling dynamics in antigen perception, and establish a quantitative framework for understanding T cell responses under diverse contexts.
























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