This protocol uses the Tg4 Nr4a3-Tocky mouse model to recalibrate T cell activation thresholds and reveals the role that immune checkpoints play in controlling T cell activation. The example approach here uses flow cytometry to characterize quantitative and qualitative changes in splenic CD4+ T cells reactivated in the presence of anti-PD1 immunotherapy. The protocol is optimized for studying anti-PD1 pathway blockade only. The protocol is not compatible with cellular fixation, and T cells should be analyzed immediately after staining. For complete details on the use and execution of this protocol, please refer to Elliot et al. (2021).
© 2022 The Author(s).

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, we characterized early quantitative and qualitative changes that occur in CD4+ T cells in relation to TCR signaling strength. We captured how dose- and time-dependent programming of distinct co-inhibitory receptors rapidly recalibrates T cell activation thresholds and visualized the immediate effects of ICB on T cell re-activation. Our findings reveal that anti-PD1 immunotherapy leads to an increased TCR signal strength. We defined a strong TCR signal metric of five genes upregulated by anti-PD1 in T cells (TCR.strong), which was superior to a canonical T cell activation gene signature in stratifying melanoma patient outcomes to anti-PD1 therapy. Our study therefore reveals how analysis of TCR signal strength-and its manipulation-can provide powerful metrics for monitoring outcomes to immunotherapy.
Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.

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.
Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.

The immune system adapts to constitutive antigens to preserve self-tolerance, which is a major barrier for anti-tumor immunity. Antigen-specific reversal of tolerance constitutes a major goal to spur therapeutic applications. Here, we show that robust, iterative, systemic stimulation targeting tissue-specific antigens in the context of acute infections reverses established CD8+ T cell tolerance to self, including in T cells that survive negative selection. This strategy results in large numbers of circulating and resident memory self-specific CD8+ T cells that are widely distributed and can be co-opted to control established malignancies bearing self-antigen without concomitant autoimmunity. Targeted expansion of both self- and tumor neoantigen-specific T cells acts synergistically to boost anti-tumor immunity and elicits protection against aggressive melanoma. Our findings demonstrate that T cell tolerance can be re-adapted to responsiveness through robust antigenic exposure, generating self-specific CD8+ T cells that can be used for cancer treatment.
Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.