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

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.

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

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.

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 relation to patient outcome. Here we leverage single-cell RNA-sequencing (scRNA-seq) analysis of several mouse and human tumors and find that monocyte-macrophage diversity is characterized by a combination of conserved lineage states as well as transcriptional programs accessed along the differentiation trajectory. Using mouse models, we also find that tumor monocyte-to-macrophage progression is profoundly tied to regulatory T cell (Treg) abundance. Importantly, 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 in kidney tumors.

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 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., Multiphoton microscopy requires precise increases in excitation power with imaging depth to generate contrast without damaging the sample. Here the authors show how an adaptive illumination function can be learned from the sample’s shape and used for in vivo imaging of whole lymph nodes.

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.

Tumors frequently program CD8 T cell immunity that is ‘exhausted’, or otherwise fails to provide for tumor clearance. This begins when tumor antigens reach the lymph node (LN) to stimulate T cells yet we know little of how tumor material is transported and disseminated amongst the large variety of antigen-presenting dendritic cell (DC) subsets in the LN. Here, in vitro and in vivo assays together with lattice light sheet and multiphoton imaging we demonstrate that tumor proteins are carried to LN within discrete vesicles inside DC and are then transferred amongst DC subsets. A cup-on-cup synapse is formed between interacting DC and directed vesicle transfer takes place routinely in the absence of free exosomes. DC containing these vesicles can uniquely activate T cells whereas DC lacking them do not. Understanding this restricted sharing of tumor identity provides substantial room for engineering better anti-tumor immunity.

Natural killer (NK) cells are critical for protection against viruses. Diaz-Salazar et al. show that adrenergic neurons modulate the adaptive NK cell response in a cell-intrinsic manner. This study identifies the nervous system as a novel axis through which antigen-specific NK cell responses are regulated., 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.

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. S.K.W. is supported by a European Molecular Biology Organization Long- Term Fellowship (grant ALTF 438– 2016) and a CNIC–International Postdoctoral Program Fellowship (grant 17230–2016). F.J.C. is the recipient of a PhD ‘La Caixa’ fellowship. Work in the D.S. laboratory is funded by the CNIC, by the European Research Council (ERC Consolidator Grant 2016 725091), by the European Commission (635122-PROCROP H2020), by the Ministerio de Ciencia, Innovación e Universidades (MCNU), Agencia Estatal de Investigación and Fondo Europeo de Desarrollo Regional (FEDER) (SAF2016-79040-R), by the Comunidad de Madrid (B2017/BMD-3733 Immunothercan- CM), by FIS- Instituto de Salud Carlos III, MCNU and FEDER (RD16/0015/0018-REEM), by Acteria Foundation, by Atresmedia (Constantes y Vitales prize) and by Fundació La Marató de TV3 (201723). The CNIC is supported by the Instituto de Salud Carlos III, the MCNU and the Pro CNIC Foundation, and is a Severo Ochoa Centre of Excellence (SEV-2015-0505). Sí

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 in vitro and in vivo 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)) are critical for productive antitumor responses yet their elicitation remains poorly understood. We exhaustively 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 which restrains cDC2, and whose reversal enhances CD4(+) T(conv) abundance and controls tumor growth.

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.

T cell proliferation is initiated by T cell antigen receptor (TCR) triggering, soluble growth factors or both. In characterizing T cells lacking the septin cytoskeleton, we found that successful cell division has discrete septin-dependent and septin-independent pathways. Septin-deficient T cells failed to complete cytokinesis when prompted by pharmacological activation or cytokines. In contrast, cell division was not dependent on septins when cell-cell contacts, such as those with antigen-presenting cells, provided a niche. This septin-independent pathway was mediated by phosphatidylinositol-3-OH kinase activation through a combination of integrins and costimulatory signals. We were able to differentiate between cytokine- and antigen-driven expansion in vivo and thus show that targeting septins has strong potential to moderate detrimental bystander or homeostatic cytokine-driven proliferation without influencing expansion driven by conventional antigen-presentation.

The strength of self-peptide–major histocompatibility complex (MHC) recognition dictates naïve CD8+ T cell homeostasis, but its effect on foreign antigen reactivity is controversial. As CD5 expression correlates with self-recognition, we studied CD5lo and CD5hi naïve CD8+ T cells. Gene expression characteristics suggested CD5hi cells were better poised for reactivity and differentiation compared to the CD5lo population, and we found that the CD5hi pool exhibited more efficient clonal recruitment and expansion, as well as enhanced reactivity to inflammatory cues, during recognition of foreign antigen. Yet foreign peptide–MHC recognition was similar for both subsets. Thus, CD8+ T cells with higher self-reactivity dominate the immune response against foreign antigens, with implications for T cell repertoire diversity and autoimmunity.