Abstract Introduction Wnt signaling is a fundamental and evolutionarily conserved pathway that governs cell fate, proliferation, and tissue homeostasis. While canonical Wnt/β-catenin signaling has been extensively characterized in stem and stromal cells, its function in lymphocytes remains context dependent and poorly understood. Natural killer (NK) cells provide a powerful system to interrogate this pathway, as they bridge innate and adaptive immunity and undergo antigen-specific clonal expansion and memory formation during antiviral responses such as cytomegalovirus (CMV) infection. Given the capacity of canonical Wnt/β-Catenin signaling to orchestrate core cellular processes, defining how NK cells access and interpret this conserved pathway to shape the transcriptional architecture of antiviral immunity is of particular interest. Methods We used a well-established model of mouse CMV (MCMV) infection, in which Ly49H+ NK cells recognize the viral glycoprotein m157 on infected targets. Molecular, genomic, and epigenomic approaches were applied to dissect how NK cells utilize Wnt signaling during viral infection. Results We identify a cytokine-independent mechanism of NK cell expansion mediated by canonical Wnt signaling. We found that NK cells uniquely express the Wnt ligand receptor Frizzled-5 (Fzd5), which is essential for regulating β-catenin-dependent canonical Wnt signaling. During MCMV, we demonstrate that the Wnt-Fzd5 signaling pathway requires β-catenin as a co-factor and LEF1 as a downstream transcription factor to activate key regulators of cell cycle entry and proliferation. Conclusion These findings establish Fzd5-mediated canonical Wnt signaling and LEF1 activity as critical drivers of the adaptive NK cell response during viral infection, offering new insights into the regulation of innate immunity. Funding Source T32 AI134632-05; F31AI178958 Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)

Natural Killer (NK) cells are innate cytotoxic lymphocytes that possess features of adaptive immunity, including antigen specificity and clonal expansion. NK cells rapidly respond to cytokines released during the innate phase of viral infection and are thought to migrate from circulation into infected organs to execute their early effector functions. However, recent evidence suggests that tissue-resident NK cells are among the first responders to viral infection. In this study, we observe that antigen receptor signaling precedes substantial proinflammatory cytokine signaling in a population of NK cells during mouse cytomegalovirus infection. Early antigen receptor signals epigenetically prime NK cells for optimal expansion during the later adaptive phase of the antiviral response. Mechanistically, receptor signaling increases chromatin accessibility at STAT4-binding genomic sites within differentiating NK cells. To promote adaptive programming of NK cells during infection, activating receptor-dependent epigenetic remodeling antagonizes IL-12 driven terminal maturation, poises NK cells for proliferation via sustained CDK6 expression, and antagonizes early apoptosis of short-lived effector cells via suppression of Bim. Thus, antigen receptor signaling alters an IL-12 dependent fate decision during the innate-to-adaptive transition of antiviral NK cells.

Natural killer (NK) cells possess both innate and adaptive features. Here, we investigated NK cell activation across tissues during cytomegalovirus infection, which generates antigen-specific clonal expansion and long-lived memory responses. Longitudinal tracking and single-cell RNA sequencing of NK cells following infection revealed enhanced activation in the spleen, as well as early formation of a CD69lo precursor population that preferentially gave rise to adaptive NK cells. Splenic NK cells demonstrated heightened tumor necrosis factor alpha (TNF-α) signaling and increased expression of the receptor TNFR2, which coincided with elevated TNF-α production by splenic myeloid cells. TNFR2-deficient NK cells exhibited impaired interferon gamma (IFN-γ) production and expansion. TNFR2 signaling engaged two distinct nuclear factor κB (NF-κB) signaling arms—innate effector NK cell responses required canonical NF-κB signaling, whereas non-canonical NF-κB signaling enforced differentiation of CD69lo adaptive NK cell precursors. Thus, NK cell priming in the spleen during viral infection promotes an innate-to-adaptive transition, providing insight into avenues for generating adaptive NK cell immunity across diverse settings.

Metastasis frequently develops from disseminated cancer cells that remain dormant after the apparently successful treatment of a primary tumour. These cells fluctuate between an immune-evasive quiescent state and a proliferative state liable to immune-mediated elimination1-6. Little is known about the clearing of reawakened metastatic cells and how this process could be therapeutically activated to eliminate residual disease in patients. Here we use models of indolent lung adenocarcinoma metastasis to identify cancer cell-intrinsic determinants of immune reactivity during exit from dormancy. Genetic screens of tumour-intrinsic immune regulators identified the stimulator of interferon genes (STING) pathway as a suppressor of metastatic outbreak. STING activity increases in metastatic progenitors that re-enter the cell cycle and is dampened by hypermethylation of the STING promoter and enhancer in breakthrough metastases or by chromatin repression in cells re-entering dormancy in response to TGFβ. STING expression in cancer cells derived from spontaneous metastases suppresses their outgrowth. Systemic treatment of mice with STING agonists eliminates dormant metastasis and prevents spontaneous outbreaks in a T cell- and natural killer cell-dependent manner-these effects require cancer cell STING function. Thus, STING provides a checkpoint against the progression of dormant metastasis and a therapeutically actionable strategy for the prevention of disease relapse.

Natural killer (NK) cells are circulating innate lymphocytes that are poised to respond rapidly upon encounter with cancer or virally-infected cells as well as proinflammatory cytokines. In addition to their early role in immune responses, it is now appreciated that NK cells can also take on features of adaptive lymphocytes such as antigen-specific clonal expansion and long-lived memory responses, with these processes best characterized following cytomegalovirus (CMV) infection in mouse and human. NK cell receptor recognition of virally-encoded proteins drives CMV-specific NK cells, but the signals that collectively give rise to these adaptive NK cells are still not fully understood. Mouse cytomegalovirus (MCMV) infection is systemic and while NK cells exhibit cytokine-induced activation across multiple tissue sites of infection, we were surprised to find that MCMV-specific NK cell activation and expansion occurs predominantly in the spleen. Transcriptional analysis indicated that canonical and non-canonical NF-kb components may be engaged to a greater extent in NK cells primed in the spleen, and we further identified TNFa signaling as a critical regulator of both innate and adaptive NK cell responses through engagement of distinct downstream signaling arms. Notably, TNFR2-deficient NK cells exhibited hyper-activation and ultimately failed to undergo the strong proliferative burst we observed in the spleen. These findings highlight the central role of the spleen in facilitating the innate-to-adaptive transition NK cells undergo and provide insight into how we can better generate adaptive NK cell immunity across diverse settings. Adriana M. Mujal is supported by the Cancer Research Institute as a Cancer Research Institute/Amgen Fellow.

Supplementary Table from Holistic Characterization of Tumor Monocyte-to-Macrophage Differentiation Integrates Distinct Immune Phenotypes in Kidney Cancer

Abstract The tumor immune microenvironment (TIME) is commonly infiltrated by diverse collections of myeloid cells. Yet, the complexity of myeloid-cell identity and plasticity has challenged efforts to define bona fide populations and determine their connections to T-cell function and their relationship to patient outcome. Here, we have leveraged single-cell RNA-sequencing analysis of several mouse and human tumors and found that monocyte–macrophage diversity is characterized by a combination of conserved lineage states as well as transcriptional programs accessed along the differentiation trajectory. We also found in mouse models that tumor monocyte-to-macrophage progression was profoundly tied to regulatory T cell (Treg) abundance. In human kidney cancer, heterogeneity in macrophage accumulation and myeloid composition corresponded to variance in, not only Treg density, but also the quality of infiltrating CD8+ T cells. In this way, holistic analysis of monocyte-to-macrophage differentiation creates a framework for critically different immune states.

Multiphoton microscopy is a powerful technique for deep in vivo imaging in scattering samples. However, it requires precise, sample-dependent increases in excitation power with depth in order to generate contrast in scattering tissue, while minimizing photobleaching and phototoxicity. We show here how adaptive imaging can optimize illumination power at each point in a 3D volume as a function of the sample's shape, without the need for specialized fluorescent labeling. Our method relies on training a physics-based machine learning model using cells with identical fluorescent labels imaged in situ. We use this technique for in vivo imaging of immune responses in mouse lymph nodes following vaccination. We achieve visualization of physiologically realistic numbers of antigen-specific T cells (~2 orders of magnitude lower than previous studies), and demonstrate changes in the global organization and motility of dendritic cell networks during the early stages of the immune response. We provide a step-by-step tutorial for implementing this technique using exclusively open-source hardware and software.

Natural killer (NK) cells are innate lymphocytes that provide critical host defense against pathogens and cancer. Originally heralded for their early and rapid effector activity, NK cells have been recognized over the last decade for their ability to undergo adaptive immune processes, including antigen-driven clonal expansion and generation of long-lived memory. This review presents an overview of how NK cells lithely partake in both innate and adaptive responses and how this versatility is manifest in human NK cell-mediated immunity.

Multiphoton microscopy is a powerful technique for deep in vivo imaging in scattering samples. However, it requires precise, sample-dependent increases in excitation power with depth in order to maintain signal while minimizing photodamage. We show that cells with identical fluorescent labels imaged in situ can be used to train a physics-based machine learning model that solves this problem. After this training has been performed, the correct illumination power can be predicted and adaptively adjusted at each point in a 3D volume on subsequent samples as a function of the sample’s shape, without the need for specialized fluorescent labelling. We use this technique for in vivo imaging of immune responses in mouse lymph nodes following vaccination, with imaging volumes 2-3 orders of magnitude larger than previously reported. We achieve visualization of physiologically realistic numbers of antigen-specific T cells for the first time, and demonstrate changes in the global organization and motility of dendritic cell networks during the early stages of the immune response.

Natural killer (NK) cells are innate lymphocytes that exhibit adaptive features, such as clonal expansion and memory, during viral infection. Although activating receptor engagement and proinflammatory cytokines are required to drive NK cell clonal expansion, additional stimulatory signals controlling their proliferation remain to be discovered. Here, we describe one such signal that is provided by the adrenergic nervous system, and demonstrate that cell-intrinsic adrenergic signaling is required for optimal adaptive NK cell responses. Early during mouse cytomegalovirus (MCMV) infection, NK cells up-regulated Adrb2 (which encodes the β2-adrenergic receptor), a process dependent on IL-12 and STAT4 signaling. NK cell–specific deletion of Adrb2 resulted in impaired NK cell expansion and memory during MCMV challenge, in part due to a diminished proliferative capacity. As a result, NK cell-intrinsic adrenergic signaling was required for protection against MCMV. Taken together, we propose a novel role for the adrenergic nervous system in regulating circulating lymphocyte responses to viral infection.

Numerous tissue-accommodation functions of immunity offer insights into disease

In order to drive productive tumor-infiltrating lymphocyte (TIL) function, myeloid populations must direct antigens to the lymph node, including to resident antigen-presenting cells (APCs) that have never touched the tumor. It has long been supposed that APCs trade antigens with one another, but the dominant cell biology underlying that remains unknown. We used and assays together with lattice light sheet and multiphoton imaging to show that myeloid cells carry tumor antigen-laden vesicles that they ‘trade’ with one another as they reach distant sites. This accounts for the majority of antigen displayed to T cells and provides tumors with a mechanism to access APCs that differentially direct T cell activation away from memory phenotypes. This work defines efficient cell biology that drives the first steps of TIL generation and represents a new frontier for engineering tumoral immunity.

Differentiation of proinflammatory CD4(+) conventional T cells (T-conv) is critical for productive antitumor responses yet their elicitation remains poorly understood. We comprehensively characterized myeloid cells in tumor draining lymph nodes (tdLN) of mice and identified two subsets of conventional type-2 dendritic cells (cDC2) that traffic from tumor to tdLN and present tumor-derived antigens to CD4(+) T-conv, but then fail to support antitumor CD4(+) T-conv differentiation. Regulatory T cell (T-reg) depletion enhanced their capacity to elicit strong CD4(+) T-conv responses and ensuing antitumor protection. Analogous cDC2 populations were identified in patients, and as in mice, their abundance relative to T-reg predicts protective ICOS+ PD-1(lo) CD4(+) T-conv phenotypes and survival. Further, in melanoma patients with low T-reg abundance, intratumoral cDC2 density alone correlates with abundant CD4(+) T-conv and with responsiveness to anti-PD-1 therapy. Together, this highlights a pathway that restrains cDC2 and whose reversal enhances CD4(+) T-conv abundance and controls tumor growth.

Generation of tumor-infiltrating lymphocytes begins when tumor antigens reach the lymph node (LN) to stimulate T cells, yet we know little of how tumor material is disseminated among the large variety of antigen-presenting dendritic cell (DC) subsets in the LN. Here, we demonstrate that tumor proteins are carried to the LN within discrete vesicles inside DCs and are then transferred among DC subsets. A synapse is formed between interacting DCs and vesicle transfer takes place in the absence of free exosomes. DCs -containing vesicles can uniquely activate T cells, whereas DCs lacking them do not. Understanding this restricted sharing of tumor identity provides substantial room for engineering better anti-tumor immunity.

The tumor microenvironment (TME) of diverse cancer types is often characterized by high levels of infiltrating myeloid cells including monocytes, macrophages, dendritic cells, and granulocytes. These cells perform a variety of functions in the TME, varying from immune suppressive to immune stimulatory roles. In this review, we summarize the different myeloid cell populations in the TME and the intratumoral myeloid targeting approaches that are being clinically investigated, and discuss strategies that identify new myeloid subpopulations within the TME. The TME therapies include agents that modulate the functional activities of myeloid populations, that impact recruitment and survival of myeloid subpopulations, and that functionally reprogram or activate myeloid populations. We discuss the benefits, limitations and potential side effects of these therapeutic approaches.

Dendritic cells (DCs) are a diverse group of specialized antigen-presenting cells with key roles in the initiation and regulation of innate and adaptive immune responses. As such, there is currently much interest in modulating DC function to improve cancer immunotherapy. Many strategies have been developed to target DCs in cancer, such as the administration of antigens with immunomodulators that mobilize and activate endogenous DCs, as well as the generation of DC-based vaccines. A better understanding of the diversity and functions of DC subsets and of how these are shaped by the tumour microenvironment could lead to improved therapies for cancer. Here we will outline how different DC subsets influence immunity and tolerance in cancer settings and discuss the implications for both established cancer treatments and novel immunotherapy strategies.

The immune response to tumors is malleable as we have learned from the various success of immunotherapies across a diversity of tumor types. What are the foundations for a solid immune response, particularly one executed by T cells? Imaging and single-cell approaches provide key insights into immune cell types that can be unleashed on tumors, identifying both their identity and the immune interactions that are to be fostered. We discuss a few of these with particular emphasis on the diversity of the myeloid system and how its component parts can contribute toward antitumor immunity. Citation Format: Adriana Mujal, Kevin Barry, Edward Roberts, Mikhail Binnewies, Megan Ruhland, Vincent Chan, Matthew Frederick Krummel. Imaging- and single cell- based insights into the antitumor immune responses [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr IA05.

SignificanceIFN-γ is an example of a pleiotropic cytokine that plays critical roles in promoting both protective immune responses and immunopathological processes. In response to this cytokine, cells activate a well-identified and conserved JAK/STAT signaling pathway that can still elicit distinct responses, even in the same cell type. How this outcome specificity is achieved remains largely unknown. We have found that IFN-γ regulates CD8 T cell differentiation through noncanonical pathways that are enabled by integrin costimulation of the STAT1 pathway. This leads to cooperative effects between activating cell types that is dictated by proximity. This study demonstrates the importance of cofactors and microenvironment in eliciting specific cytokine functions.