Highlights d TCR signal strength drives dynamic and time-dependent changes in CD4 + T cells d Inhibitory receptor expression recalibrates T cell activation thresholds d PD1 blockade leads to a strong TCR signal signature in T cells (TCR.strong) d TCR.strong can stratify melanoma patient responses to anti-PD1 therapy

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In lymphocytes, Nr4a gene expression is specifically regulated by antigen receptor signalling, making them ideal targets for use as distal T cell receptor (TCR) reporters. Nr4a3-Timer of cell kinetics and activity (Tocky) mice are a groundbreaking tool to report TCR driven Nr4a3 expression using Fluorescent Timer protein (FT). FT undergoes a time-dependent shift in its emission spectrum following translation, allowing for the temporal reporting of transcriptional events. Our recent work suggested that Nr4a1/ Nur77 may be a more sensitive gene to distal TCR signals compared to Nr4a3, so we therefore generated Nur77-Timer-rapidly-expressed-in-lymphocytes (Tempo) mice that express FT under the regulation of Nur77. We validated the ability of Nur77-Tempo mice to report TCR and B cell receptor (BCR) signals and investigated the signals regulating Nur77-FT expression. We found that Nur77-FT was sensitive to low strength TCR signals, and its brightness was graded in response to TCR signal strength. Nur77-FT detected positive selection signals in the thymus, and analysis of FT expression revealed that positive selection signals are often persistent in nature, with most thymic Treg expressing FT Blue. We found that active TCR signals in the spleen are low frequency, but CD69 + lymphoid T cells are enriched for FT Blue +Red + T cells, suggesting frequent TCR signalling. In non-lymphoid tissue, we saw a dissociation of FT protein from CD69 expression, indicating that tissue residency is not associated with tonic TCR signals. Nur77-Tempo mice, therefore, combine the temporal dynamics from the Tocky innovation with increased sensitivity of Nr4a1 to lower TCR signal strengths.

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How T cell receptor (TCR) signal strength modulates T cell function and to what extent this is modified by immune checkpoint blockade (ICB) are key questions in immunology. Using Nr4a3-Tocky mice as a digital read-out of NFAT pathway activity, we identify the rapid quantitative and qualitative changes that occur in CD4+ T cells in response to a range of TCR signalling strengths. We demonstrate that the time and dose dependent programming of distinct co-inhibitory receptors rapidly re-calibrates T cell activation thresholds. By developing a new in vivo model, we analyse the immediate effects of ICB on T cell re-activation. Our findings reveal that anti-PD1 but not anti-Lag3 immunotherapy leads to an increased TCR signal strength. We define a strong TCR signal metric of five genes specifically upregulated by anti-PD1 in T cells (TCR.strong), which can stratify clinical outcomes during anti-PD1 monotherapy in melanoma patients. Our study therefore reveals how analysis of TCR signal strength – and its manipulation – can provide powerful metrics for monitoring outcomes to immunotherapy.