Abstract SY19-04: Next-generation macrophage and T cell activation reporters to define functional immune states in solid tumors
Authors: Ray Arja , Hu Kenneth , Samad Bushra , Hernandez Grace , Krummel Matthew 
Published: June 2022

Abstract
Although chronic activation is considered a major driver of T cell dysfunction in the tumor microenvironment (TME), there is a virtual absence of methodologies to discern how T cells spatiotemporally progress through activation states into that dysfunction. Furthermore, T cells and other immune populations in the TME communicate extensively with tumor-associated macrophages (TAMs) which are typically simply described as ‘suppressive’, but whose functions are certainly more complex. To address these critical knowledge gaps, we designed two types of novel genetic marking systems to visualize, track and manipulate (i) T cell subsets with distinct activation histories and (ii) specific subsets of macrophages, modulating primarily those in the TME, to study and define these co-evolving immune states.
We generated a novel T cell activation reporter mouse, which is a transcriptional reporter of CD69 expressing the fluorescent protein TFP. By recording transcriptional activity of a locus where the protein expression is under tight, activation-dependent post-transcriptional control, we generated a reporting system to read out contemporary as well as historic T cell triggering. While cell surface CD69 reliably indicates current activation, resting TFP (Cd69 mRNA) expression is downregulated with successive TCR triggering and hence an inverse marker of chronic stimulation. Using multiple tumor models in vivo, tumor slice cultures ex vivo and scRNA-Seq, we thus defined and tracked over time distinct functional states of intratumoral T cells based on their activation histories. These data revealed key insights including the delineation of quiescent versus activated states within progenitor, effector and exhausted T cells and the trajectory of inter-conversion that leads to the emergence of these states in the TME. Probing the inflection points in this differentiation trajectory reveals a particularly interesting subset of CD69+TFP+ cells, representing intratumorally activated effector CD8 T cells prior to their dysfunctional adaptation to chronic stimulation. Dissecting their phenotype led to the discovery of a previously unexplored subset of intratumoral T cells of functional significance. Indeed, these cells were enriched specifically in the context of T cell-mediated tumor control in mice and their phenotypic markers correlated with improved outcomes and survival in human patients.
To complement this system, we generated a second genetic strain of mice that allowed specific depletion of TAMs. The prevailing view of M1 vs. M2 TAMs as ostensibly anti-tumor vs. pro-tumor, although useful, is almost certainly over-simplistic and has lacked further delineation, limited by the quality of available tools to specifically manipulate TAMs in situ. Remarkably, till now, a method to specifically deplete TAMs, without global depletion of all macrophages (e.g CSF1R blockade, Clodronate) did not exist. We alleviated this problem by using this novel mouse strain, in which conditional (lox-Stop-lox flanked) YFP and DTR genes are knocked into the endogenous CD206 locus. Since, within macrophages, CD206 is largely expressed in tumors, an intercross of this allele with a Csf1r-Cre (to specify monocyte-derived lineage) provides a 2-allele gating system to limit and robustly penetrate reporter and depletion function to TAMs. In multiple TAM-rich tumor models, this revealed that TAMs assumed context-dependent divergent roles in anti-tumor immunity. On the one hand, their removal led to a mitigation of T cell exhaustion, consistent with the popular notion that they are immunosuppressive and in need of global elimination. However, our data also reveals that macrophage depletion not only fails to alleviate the tumor burden but also prevents successful T cell-mediated anti-tumor immunity. We linked the latter effect to an obligate role for TAMs in the recruitment and maintenance of cDC1s and CD8 T cells in the TME, respectively. By live imaging of TAMs in vivo using the reporter and subsequent spatially resolved scRNA-Seq, the key TAM subsets and interactional networks involved in these processes were established. Importantly, in responsive tumor models, TAM depletion subverted T cell-mediated tumor control, demonstrating their importance in organizing anti-tumor adaptive immunity.
We thus combine the use of novel engineered mice in vivo, real time imaging and spatial transcriptomics with functional studies to shed new light on immune dysfunction in the TME. Defining the ontology of functional immune states in the TME provides a clear target space to maneuver cancer immunotherapies. Herein, we offer prime examples of dissecting the spatiotemporal biology of CD8 T cells and TAMs in anti-tumor immunity to define such interdependent states. These findings and the newly established models therein pave the way to deepen as well as broaden our understanding of the complex immune landscape in solid tumors.
Citation Format: Arja Ray, Kenneth Hu, Bushra Samad, Grace Hernandez, Matthew Krummel. Next-generation macrophage and T cell activation reporters to define functional immune states in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr SY19-04.