Summary Nr4a receptors are activated by T cell receptor (TCR) signaling and play key roles in T cell differentiation. Which TCR signaling pathways regulate Nr4a receptors and their sensitivities to TCR signal strength and duration remains unclear. Using Nr4a1/Nur77-GFP and Nr4a3 -Timer of cell kinetics and activity (Tocky) mice, we elucidate the signaling pathways governing Nr4a receptor expression. We reveal that Nr4a1 – Nr4a3 are Src family kinase dependent. Moreover, Nr4a2 and Nr4a3 are attenuated by calcineurin inhibitors and bind nuclear factor of activated T cells 1 (NFAT1), highlighting a necessary and sufficient role for NFAT1 in the control of Nr4a2 and Nr4a3, but redundancy for Nr4a1 . Nr4a1- GFP is activated by tonic and cognate signals during T cell development, whereas Nr4a3- Tocky requires cognate peptide:major histocompatibility complex (MHC) interactions for expression. Compared to Nr4a3- Tocky, Nr4a1- GFP is approximately 2- to 3-fold more sensitive to TCR signaling and is detectable by shorter periods of TCR signaling. These findings suggest that TCR signal duration may be an underappreciated aspect influencing the developmental fate of T cells in vivo .

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

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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Nr4a gene family members are activated by T cell receptor (TCR) signalling and play key roles in Regulatory T cell differentiation and have also been implicated in promoting T cell exhaustion in cancer. The precise signalling pathways that govern their transcription, however, remain unclear. Here we utilise Nr4a3-Tocky mice to elucidate the signalling pathways that govern Nr4a1, 2 and 3 expression in response to physiological stimulation of CD4 + and CD8 + T cells. Our findings reveal that Nr4a1-3 are Src family kinase-dependent. Moreover, Nr4a2 and Nr4a3 are abolished by calcineurin inhibitors and bind NFAT1, highlighting a necessary role for NFAT in the control of Nr4a2 and Nr4a3. Interestingly, the NFAT pathway is redundant for Nr4a1 activation, but all three Nr4a members require ERK signalling for optimal expression. Analysis of T cells expressing constitutively active NFAT1 reveals that NFAT1 alone is sufficient to induce expression of Nr4a2 and Nr4a3, but not Nr4a1.These findings further our understanding of Nr4a regulation, highlighting key differences in the sensitivity of Nr4a1 and Nr4a3 to distal TCR signalling pathways.