Stem-like" TCF1 + CD8 + T cells (T SL ) are necessary for long-term maintenance of T cell responses and the efficacy of immunotherapy but, as tumors contain signals that should drive T-cell terminal-differentiation, how these cells are maintained in tumors remains unclear. In this study, we found that a small number of TCF1 + tumor-specific CD8 + T cells were present in lung tumors throughout their development. Yet, most intratumoral T cells differentiated as tumors progressed, corresponding with an immunologic shift in the tumor microenvironment (TME) from "hot" (T cell-inflamed) to "cold" (non-T cell-inflamed). By contrast, most tumor-specific CD8 + T cells in tumor-draining lymph nodes (dLNs) had functions and gene expression signatures similar to T SL from chronic LCMV infection, and this population was stable over time, despite the changes in the TME. dLN T cells were the developmental precursors of, and were clonally related to, their more differentiated intratumoral counterparts. Our data support the hypothesis that dLN T cells are the developmental precursors of the TCF1 + T cells in tumors which are maintained by continuous migration. Finally, CD8 + T cells similar to T SL were also present in LNs from lung adenocarcinoma patients, suggesting a similar model may be relevant in human disease. Thus, we propose that the dLN T SL reservoir has a critical function in sustaining antitumor T cells during tumor development and protecting them from the terminal differentiation that occurs in the TME.

Inducible expression of neoantigens in mice would enable the study of endogenous antigen-specific naïve T cell responses in disease and infection, but has been difficult to generate because leaky antigen expression in the thymus results in central T cell tolerance. Here, we developed iNversion INduced Joined neoAntigen (NINJA), using RNA splicing, DNA recombination, and three levels of regulation to prevent leakiness and allow tight control over neoantigen expression. We apply NINJA to create tumor cell lines with inducible neoantigen expression, which could be used to study anti-tumor immunity. We also show that the genetic regulation in NINJA mice bypasses central and peripheral tolerance mechanisms and allows for robust endogenous CD8 and CD4 T cells responses upon neoantigen induction in peripheral tissues. NINJA will enable studies of how T cells respond to defined neoantigens in the context of peripheral tolerance, transplantation, autoimmune diseases, and cancer.

Circadian rhythms are driven by daily oscillations of gene expression. An important tool for studying cellular and tissue rhythms is the use of a gene reporter, such as bioluminescence from the reporter gene luciferase controlled by a rhythmically expressed gene of interest. Here we describe methods that allow measurement of bioluminescence from a freely-moving mouse housed in a standard cage. Using a LumiCycle In Vivo (Actimetrics), we determined conditions that allow detection of circadian rhythms of bioluminescence from the PER2 reporter, PER2::LUC, in freely behaving mice. We tested delivery of D-luciferin via a subcutaneous minipump and in the drinking water. Further, we demonstrate that a synthetic luciferase substrate, CycLuc1, can support circadian rhythms of bioluminescence, even when delivered at a lower concentration than D-luciferin. We share our analysis scripts and suggestions for further improvements in this method. This approach will be straightforward to apply to mice with tissue-specific reporters, allowing insights into responses of specific peripheral clocks to perturbations such as environmental or pharmacological manipulations.

Circadian rhythms are driven by daily oscillations of gene expression. An important tool for studying cellular and tissue circadian rhythms is the use of a gene reporter, such as bioluminescence from the reporter gene luciferase controlled by a rhythmically expressed gene of interest. Here we describe methods that allow measurement of circadian bioluminescence from a freely moving mouse housed in a standard cage. Using a LumiCycle In Vivo (Actimetrics), we determined conditions that allow detection of circadian rhythms of bioluminescence from the PER2 reporter, PER2::LUC, in freely behaving mice. The LumiCycle In Vivo applies a background subtraction that corrects for effects of room temperature on photomultiplier tube (PMT) output. We tested delivery of d-luciferin via a subcutaneous minipump and in the drinking water. We demonstrate spikes in bioluminescence associated with drinking bouts. Further, we demonstrate that a synthetic luciferase substrate, CycLuc1, can support circadian rhythms of bioluminescence, even when delivered at a lower concentration than d-luciferin, and can support longer-term studies. A small difference in phase of the PER2::LUC bioluminescence rhythms, with females phase leading males, can be detected with this technique. We share our analysis scripts and suggestions for further improvements in this method. This approach will be straightforward to apply to mice with tissue-specific reporters, allowing insights into responses of specific peripheral clocks to perturbations such as environmental or pharmacological manipulations.

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