Unleashing Type-2 Dendritic Cells to Drive Protective Antitumor CD4+ T Cell Immunity.

Differentiation of proinflammatory CD4+ conventional T cells (Tconv) 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+ Tconv, but then fail to support antitumor CD4+ Tconv differentiation. Regulatory T cell (Treg) depletion enhanced their capacity to elicit strong CD4+ Tconv responses and ensuing antitumor protection. Analogous cDC2 populations were identified in patients, and as in mice, their abundance relative to Treg predicts protective ICOS+ PD-1lo CD4+ Tconv phenotypes and survival. Further, in melanoma patients with low Treg abundance, intratumoral cDC2 density alone correlates with abundant CD4+ Tconv and with responsiveness to anti-PD-1 therapy. Together, this highlights a pathway that restrains cDC2 and whose reversal enhances CD4+ Tconv abundance and controls tumor growth.

ZipSeq: barcoding for real-time mapping of single cell transcriptomes.

Spatial transcriptomics seeks to integrate single cell transcriptomic data within the three-dimensional space of multicellular biology. Current methods to correlate a cell's position with its transcriptome in living tissues have various limitations. We developed an approach, called 'ZipSeq', that uses patterned illumination and photocaged oligonucleotides to serially print barcodes ('zipcodes') onto live cells in intact tissues, in real time and with an on-the-fly selection of patterns. Using ZipSeq, we mapped gene expression in three settings: in vitro wound healing, live lymph node sections and a live tumor microenvironment. In all cases, we discovered new gene expression patterns associated with histological structures. In the tumor microenvironment, this demonstrated a trajectory of myeloid and T cell differentiation from the periphery inward. A combinatorial variation of ZipSeq efficiently scales in the number of regions defined, providing a pathway for complete mapping of live tissues, subsequent to real-time imaging or perturbation.

IL-17-producing γδ T cells and neutrophils conspire to promote breast cancer metastasis.

Metastatic disease remains the primary cause of death for patients with breast cancer. The different steps of the metastatic cascade rely on reciprocal interactions between cancer cells and their microenvironment. Within this local microenvironment and in distant organs, immune cells and their mediators are known to facilitate metastasis formation. However, the precise contribution of tumour-induced systemic inflammation to metastasis and the mechanisms regulating systemic inflammation are poorly understood. Here we show that tumours maximize their chance of metastasizing by evoking a systemic inflammatory cascade in mouse models of spontaneous breast cancer metastasis. We mechanistically demonstrate that interleukin (IL)-1ß elicits IL-17 expression from gamma delta (γδ) T cells, resulting in systemic, granulocyte colony-stimulating factor (G-CSF)-dependent expansion and polarization of neutrophils in mice bearing mammary tumours. Tumour-induced neutrophils acquire the ability to suppress cytotoxic T lymphocytes carrying the CD8 antigen, which limit the establishment of metastases. Neutralization of IL-17 or G-CSF and absence of γδ T cells prevents neutrophil accumulation and downregulates the T-cell-suppressive phenotype of neutrophils. Moreover, the absence of γδ T cells or neutrophils profoundly reduces pulmonary and lymph node metastases without influencing primary tumour progression. Our data indicate that targeting this novel cancer-cell-initiated domino effect within the immune system--the γδ T cell/IL-17/neutrophil axis--represents a new strategy to inhibit metastatic disease.

Tumor-associated macrophages restrict CD8+ T cell function through collagen deposition and metabolic reprogramming of the breast cancer microenvironment.

Tumor progression is accompanied by fibrosis, a condition of excessive extracellular matrix accumulation, which is associated with diminished antitumor immune infiltration. Here we demonstrate that tumor-associated macrophages (TAMs) respond to the stiffened fibrotic tumor microenvironment (TME) by initiating a collagen biosynthesis program directed by transforming growth factor-ß. A collateral effect of this programming is an untenable metabolic milieu for productive CD8+ T cell antitumor responses, as collagen-synthesizing macrophages consume environmental arginine, synthesize proline and secrete ornithine that compromises CD8+ T cell function in female breast cancer. Thus, a stiff and fibrotic TME may impede antitumor immunity not only by direct physical exclusion of CD8+ T cells but also through secondary effects of a mechano-metabolic programming of TAMs, which creates an inhospitable metabolic milieu for CD8+ T cells to respond to anticancer immunotherapies.

Critical role of CD206+ macrophages in organizing anti-tumor immunity.

Tumor-associated macrophages (TAMs) are frequently and simplistically categorized as immunosuppressive, and one molecule prominently used to highlight their so-called 'M2' state is the surface protein CD206. However, direct evidence of the impact of macrophages remains impaired by the lack of sufficiently penetrant and specific tools to manipulate them in vivo. We thus made a novel conditional CD206 knock-in mouse to specifically visualize and/or deplete these TAMs. Early depletion of CD206+ macrophages and monocytes (here, 'MonoMacs') strikingly led to an indirect loss of a key anti-tumor network of NK cells, conventional type I dendritic cells (cDC1) and CD8 T cells. Among myeloid cells, we found that the CD206+ TAMs are the primary producers of CXCL9, the well-established chemoattractant for CXCR3-expressing NK and CD8 T cells. In contrast, a population of stress-responsive TAMs ("Hypoxic" or Spp1+) and immature monocytes, which remain following depletion, expressed vastly diminished levels of CXCL9. We confirmed that the missing NK and CD8 T cells are the primary producers of the cDC1-attracting chemokine Xcl1 and cDC1 growth factor Flt3l. Consistent with the loss of this critical network, CD206+ TAM depletion decreased tumor control in mice. Likewise, in humans, the CD206+ MonoMac signature correlated robustly with stimulatory cDC1 signature genes. Together, these findings negate the classification of CD206+ macrophages as immunosuppressive and instead illuminate the role of this majority of TAMs in organizing a critical tumor-reactive archetype of immunity.

Uptake of tumor-derived microparticles induces metabolic reprogramming of macrophages in the early metastatic lung.

Pre-metastatic niche formation is a critical step during the metastatic spread of cancer. One way by which primary tumors prime host cells at future metastatic sites is through the shedding of tumor-derived microparticles as a consequence of vascular sheer flow. However, it remains unclear how the uptake of such particles by resident immune cells affects their phenotype and function. Here, we show that ingestion of tumor-derived microparticles by macrophages induces a rapid metabolic and phenotypic switch that is characterized by enhanced mitochondrial mass and function, increased oxidative phosphorylation, and upregulation of adhesion molecules, resulting in reduced motility in the early metastatic lung. This reprogramming event is dependent on signaling through the mTORC1, but not the mTORC2, pathway and is induced by uptake of tumor-derived microparticles. Together, these data support a mechanism by which uptake of tumor-derived microparticles induces reprogramming of macrophages to shape their fate and function in the early metastatic lung.

Tumor cell heterogeneity drives spatial organization of the intratumoral immune response in squamous cell skin carcinoma.

Intratumoral heterogeneity (ITH)-defined as genetic and cellular diversity within a tumor-is linked to failure of immunotherapy and an inferior anti-tumor immune response. The underlying mechanism of this association is unknown. To address this question, we modeled heterogeneous tumors comprised of a pro-inflammatory ("hot") and an immunosuppressive ("cold") tumor population, labeled with YFP and RFP tags respectively to enable precise spatial tracking. The resulting mixed-population tumors exhibited distinct regions comprised of YFP+ (hot) cells, RFP+ (cold) cells, or a mixture. We found that tumor regions occupied by hot tumor cells (YFP+) harbored more total T cells and a higher frequency of Th1 cells and IFNγ+ CD8 T cells compared to regions occupied by cold tumor cells (RFP+), whereas immunosuppressive macrophages showed the opposite spatial pattern. We identified the chemokine CX3CL1, produced at higher levels by our cold tumors, as a mediator of intratumoral macrophage accumulation, particularly immunosuppressive CD206Hi macrophages. Furthermore, we examined the response of heterogeneous tumors to a therapeutic combination of PD-1 blockade and CD40 agonist on a region-by-region basis. While the combination successfully increases Th1 abundance in "cold" tumor regions, it fails to bring overall T cell activity to the same level as seen in "hot" regions. The presence of the "cold" cells thus ultimately leads to a failure of the therapy to induce tumor rejection. Collectively, our results demonstrate that the organization of heterogeneous tumor cells has a profound impact on directing the spatial organization and function of tumor-infiltrating immune cells as well as on responses to immunotherapy.

Neoadjuvant immune checkpoint blockade triggers persistent and systemic Treg activation which blunts therapeutic efficacy against metastatic spread of breast tumors.

The clinical successes of immune checkpoint blockade (ICB) in advanced cancer patients have recently spurred the clinical implementation of ICB in the neoadjuvant and perioperative setting. However, how neoadjuvant ICB therapy affects the systemic immune landscape and metastatic spread remains to be established. Tumors promote both local and systemic expansion of regulatory T cells (Tregs), which are key orchestrators of tumor-induced immunosuppression, contributing to immune evasion, tumor progression and metastasis. Tregs express inhibitory immune checkpoint molecules and thus may be unintended targets for ICB therapy counteracting its efficacy. Using ICB-refractory models of spontaneous primary and metastatic breast cancer that recapitulate the poor ICB response of breast cancer patients, we observed that combined anti-PD-1 and anti-CTLA-4 therapy inadvertently promotes proliferation and activation of Tregs in the tumor, tumor-draining lymph node and circulation. Also in breast cancer patients, Treg levels were elevated upon ICB. Depletion of Tregs during neoadjuvant ICB in tumor-bearing mice not only reshaped the intratumoral immune landscape into a state favorable for ICB response but also induced profound and persistent alterations in systemic immunity, characterized by elevated CD8+ T cells and NK cells and durable T cell activation that was maintained after treatment cessation. While depletion of Tregs in combination with neoadjuvant ICB did not inhibit primary tumor growth, it prolonged metastasis-related survival driven predominantly by CD8+ T cells. This study demonstrates that neoadjuvant ICB therapy of breast cancer can be empowered by simultaneous targeting of Tregs, extending metastasis-related survival, independent of a primary tumor response.

Lessons for the Next Generation of Scientists from the Second Annual Arthur and Sandra Irving Cancer Immunology Symposium.

The Arthur and Sandra Irving Cancer Immunology Symposium has been created as a platform for established cancer immunologists to mentor trainees and young investigators as they launch their research career in the field. By sharing their different paths to success, the senior faculty mentors provide an invaluable resource to support the development of the next generation of leaders in the cancer immunology community. This Commentary describes some of the key topics that were discussed during the 2022 symposium: scientific and career trajectory, leadership, mentoring, collaborations, and publishing. For each of these topics, established investigators discussed the elements that facilitate success in these areas as well as mistakes that can hinder progress. Herein, we outline the critical points raised in these discussions for establishing a successful independent research career. These points are highly relevant for the broader scientific community.

IL-5-producing CD4+ T cells and eosinophils cooperate to enhance response to immune checkpoint blockade in breast cancer.

Immune checkpoint blockade (ICB) has heralded a new era in cancer therapy. Research into the mechanisms underlying response to ICB has predominantly focused on T cells; however, effective immune responses require tightly regulated crosstalk between innate and adaptive immune cells. Here, we combine unbiased analysis of blood and tumors from metastatic breast cancer patients treated with ICB with mechanistic studies in mouse models of breast cancer. We observe an increase in systemic and intratumoral eosinophils in patients and mice responding to ICB treatment. Mechanistically, ICB increased IL-5 production by CD4+ T cells, stimulating elevated eosinophil production from the bone marrow, leading to systemic eosinophil expansion. Additional induction of IL-33 by ICB-cisplatin combination or recombinant IL-33 promotes intratumoral eosinophil infiltration and eosinophil-dependent CD8+ T cell activation to enhance ICB response. This work demonstrates the critical role of eosinophils in ICB response and provides proof-of-principle for eosinophil engagement to enhance ICB efficacy.

Spatiotemporal co-dependency between macrophages and exhausted CD8+ T cells in cancer.

T cell exhaustion is a major impediment to antitumor immunity. However, it remains elusive how other immune cells in the tumor microenvironment (TME) contribute to this dysfunctional state. Here, we show that the biology of tumor-associated macrophages (TAMs) and exhausted T cells (Tex) in the TME is extensively linked. We demonstrate that in vivo depletion of TAMs reduces exhaustion programs in tumor-infiltrating CD8+ T cells and reinvigorates their effector potential. Reciprocally, transcriptional and epigenetic profiling reveals that Tex express factors that actively recruit monocytes to the TME and shape their differentiation. Using lattice light sheet microscopy, we show that TAM and CD8+ T cells engage in unique, long-lasting, antigen-specific synaptic interactions that fail to activate T cells but prime them for exhaustion, which is then accelerated in hypoxic conditions. Spatially resolved sequencing supports a spatiotemporal self-enforcing positive feedback circuit that is aligned to protect rather than destroy a tumor.

MYC promotes immune-suppression in triple-negative breast cancer via inhibition of interferon signaling.

The limited efficacy of immune checkpoint inhibitor treatment in triple-negative breast cancer (TNBC) patients is attributed to sparse or unresponsive tumor-infiltrating lymphocytes, but the mechanisms that lead to a therapy resistant tumor immune microenvironment are incompletely known. Here we show a strong correlation between MYC expression and loss of immune signatures in human TNBC. In mouse models of TNBC proficient or deficient of breast cancer type 1 susceptibility gene (BRCA1), MYC overexpression dramatically decreases lymphocyte infiltration in tumors, along with immune signature remodelling. MYC-mediated suppression of inflammatory signalling induced by BRCA1/2 inactivation is confirmed in human TNBC cell lines. Moreover, MYC overexpression prevents the recruitment and activation of lymphocytes in both human and mouse TNBC co-culture models. Chromatin-immunoprecipitation-sequencing reveals that MYC, together with its co-repressor MIZ1, directly binds promoters of multiple interferon-signalling genes, resulting in their downregulation. MYC overexpression thus counters tumor growth inhibition by a Stimulator of Interferon Genes (STING) agonist via suppressing induction of interferon signalling. Together, our data reveal that MYC suppresses innate immunity and facilitates tumor immune escape, explaining the poor immunogenicity of MYC-overexpressing TNBCs.

Response of metastatic mouse invasive lobular carcinoma to mTOR inhibition is partly mediated by the adaptive immune system.

Effective treatment of invasive lobular carcinoma (ILC) of the breast is hampered by late detection, invasive growth, distant metastasis, and poor response to chemotherapy. Phosphoinositide 3-kinase (PI3K) signaling, one of the major druggable oncogenic signaling networks, is frequently activated in ILC. We investigated treatment response and resistance to AZD8055, an inhibitor of mammalian target of rapamycin (mTOR), in the K14-cre;Cdh1Flox/Flox;Trp53Flox/Flox (KEP) mouse model of metastatic ILC. Inhibition of mTOR signaling blocked the growth of primary KEP tumors as well as the progression of metastatic disease. However, primary tumors and distant metastases eventually acquired resistance after long-term AZD8055 treatment, despite continued effective suppression of mTOR signaling in cancer cells. Interestingly, therapeutic responses were associated with increased expression of genes related to antigen presentation. Consistent with this observation, increased numbers of tumor-infiltrating major histocompatibility complex class II-positive (MHCII+) immune cells were observed in treatment-responsive KEP tumors. Acquisition of treatment resistance was associated with loss of MHCII+ cells and reduced expression of genes related to the adaptive immune system. The therapeutic efficacy of mTOR inhibition was reduced in Rag1-/- mice lacking mature T and B lymphocytes, compared to immunocompetent mice. Furthermore, therapy responsiveness could be partially rescued by transplanting AZD8055-resistant KEP tumors into treatment-naïve immunocompetent hosts. Collectively, these data indicate that the PI3K signaling pathway is an attractive therapeutic target in invasive lobular carcinoma, and that part of the therapeutic effect of mTOR inhibition is mediated by the adaptive immune system.

Therapeutic targeting of macrophages enhances chemotherapy efficacy by unleashing type I interferon response.

Recent studies have revealed a role for macrophages and neutrophils in limiting chemotherapy efficacy; however, the mechanisms underlying the therapeutic benefit of myeloid-targeting agents in combination with chemotherapy are incompletely understood. Here, we show that targeting tumour-associated macrophages by colony-stimulating factor-1 receptor (CSF-1R) blockade in the K14cre;Cdh1F/F;Trp53F/F transgenic mouse model for breast cancer stimulates intratumoural type I interferon (IFN) signalling, which enhances the anticancer efficacy of platinum-based chemotherapeutics. Notably, anti-CSF-1R treatment also increased intratumoural expression of type I IFN-stimulated genes in patients with cancer, confirming that CSF-1R blockade is a powerful strategy to trigger an intratumoural type I IFN response. By inducing an inflamed, type I IFN-enriched tumour microenvironment and by further targeting immunosuppressive neutrophils during cisplatin therapy, antitumour immunity was activated in this poorly immunogenic breast cancer mouse model. These data illustrate the importance of breaching multiple layers of immunosuppression during cytotoxic therapy to successfully engage antitumour immunity in breast cancer.

Understanding the tumor immune microenvironment (TIME) for effective therapy.

The clinical successes in immunotherapy have been both astounding and at the same time unsatisfactory. Countless patients with varied tumor types have seen pronounced clinical response with immunotherapeutic intervention; however, many more patients have experienced minimal or no clinical benefit when provided the same treatment. As technology has advanced, so has the understanding of the complexity and diversity of the immune context of the tumor microenvironment and its influence on response to therapy. It has been possible to identify different subclasses of immune environment that have an influence on tumor initiation and response and therapy; by parsing the unique classes and subclasses of tumor immune microenvironment (TIME) that exist within a patient's tumor, the ability to predict and guide immunotherapeutic responsiveness will improve, and new therapeutic targets will be revealed.

Assessment of PD-L1 expression across breast cancer molecular subtypes, in relation to mutation rate, BRCA1-like status, tumor-infiltrating immune cells and survival.

To better understand the expression pattern of programmed death-ligand 1 (PD-L1) expression in different breast cancer types, we characterized PD-L1 expression in tumor and tumor-infiltrating immune cells, in relation to mutation rate, BRCA1-like status and survival. We analyzed 410 primary treatment-naive breast tumors comprising 162 estrogen receptor-positive (ER+) and HER2-, 101 HER2+ and 147 triple-negative (TN) cancers. Pathologists quantified tumor-infiltrating lymphocytes (TILs) and PD-L1 expression in tumor cells and TILs using whole slides and tissue microarray. Mutation rate was assessed by DNA sequencing, BRCA1-like status using multiplex ligation-dependent probe amplification, and immune landscape by multiplex image analyses of CD4, CD68, CD8, FOXP3, cytokeratin, and PD-L1. Half of PD-L1 scores evaluated by tissue microarray were false negatives compared to whole slide evaluations. We observed at least 1% of PD-L1-positive (PD-L1+) cells in 53.1% of ER+HER2-, 73.3% of HER2+, and 84.4% of TN tumors. PD-L1 expression was higher in ductal compared to lobular carcinomas, also within ER+HER2- tumors (p = 0.04). High PD-L1+ TILs score (> 50%) was independently associated with better outcome in TN tumors (HR = 0.27; 95%CI = 0.10-0.69). Within TN tumors, PD-L1 and TIL scores showed a modest but significant positive association with the number of silent mutations, but no association with BRCA1-like status. Multiplex image analyses indicated that PD-L1 is expressed on multiple immune cells (CD68+ macrophages, CD4+, FOXP3+, and CD8+ T cells) in the breast tumor microenvironment, independent of the PD-L1 status of the tumor cells. We found no evidence that levels of PD-L1+ TILs in TN breast cancer are driven by high mutation rate or BRCA1-like status.

Genetically engineered mouse models in oncology research and cancer medicine.

Genetically engineered mouse models (GEMMs) have contributed significantly to the field of cancer research. In contrast to cancer cell inoculation models, GEMMs develop de novo tumors in a natural immune-proficient microenvironment. Tumors arising in advanced GEMMs closely mimic the histopathological and molecular features of their human counterparts, display genetic heterogeneity, and are able to spontaneously progress toward metastatic disease. As such, GEMMs are generally superior to cancer cell inoculation models, which show no or limited heterogeneity and are often metastatic from the start. Given that GEMMs capture both tumor cell-intrinsic and cell-extrinsic factors that drive de novo tumor initiation and progression toward metastatic disease, these models are indispensable for preclinical research. GEMMs have successfully been used to validate candidate cancer genes and drug targets, assess therapy efficacy, dissect the impact of the tumor microenvironment, and evaluate mechanisms of drug resistance. In vivo validation of candidate cancer genes and therapeutic targets is further accelerated by recent advances in genetic engineering that enable fast-track generation and fine-tuning of GEMMs to more closely resemble human patients. In addition, aligning preclinical tumor intervention studies in advanced GEMMs with clinical studies in patients is expected to accelerate the development of novel therapeutic strategies and their translation into the clinic.

Mammary tumor-derived CCL2 enhances pro-metastatic systemic inflammation through upregulation of IL1ß in tumor-associated macrophages.

Patients with primary solid malignancies frequently exhibit signs of systemic inflammation. Notably, elevated levels of neutrophils and their associated soluble mediators are regularly observed in cancer patients, and correlate with reduced survival and increased metastasis formation. Recently, we demonstrated a mechanistic link between mammary tumor-induced IL17-producing γδ T cells, systemic expansion of immunosuppressive neutrophils and metastasis formation in a genetically engineered mouse model for invasive breast cancer. How tumors orchestrate this systemic inflammatory cascade to facilitate dissemination remains unclear. Here we show that activation of this cascade relies on CCL2-mediated induction of IL1ß in tumor-associated macrophages. In line with these findings, expression of CCL2 positively correlates with IL1Β and macrophage markers in human breast tumors. We demonstrate that blockade of CCL2 in mammary tumor-bearing mice results in reduced IL17 production by γδ T cells, decreased neutrophil expansion and enhanced CD8+ T cell activity. These results highlight a new role for CCL2 in facilitating the breast cancer-induced pro-metastatic systemic inflammatory γδ T cell - IL17 - neutrophil axis.

Exploiting the Immunomodulatory Properties of Chemotherapeutic Drugs to Improve the Success of Cancer Immunotherapy.

Cancer immunotherapy is gaining momentum in the clinic. The current challenge is to understand why a proportion of cancer patients do not respond to cancer immunotherapy, and how this can be translated into the rational design of combinatorial cancer immunotherapy strategies aimed at maximizing success of immunotherapy. Here, we discuss how tumors orchestrate an immunosuppressive microenvironment, which contributes to their escape from immune attack. Relieving the immunosuppressive networks in cancer patients is an attractive strategy to extend the clinical success of cancer immunotherapy. Since the clinical availability of drugs specifically targeting immunosuppressive cells or mediators is still limited, an alternative strategy is to use conventional chemotherapy drugs with immunomodulatory properties to improve cancer immunotherapy. We summarize the preclinical and clinical studies that illustrate how the anti-tumor T cell response can be enhanced by chemotherapy-induced relief of immunosuppressive networks. Treatment strategies aimed at combining chemotherapy-induced relief of immunosuppression and T cell-boosting checkpoint inhibitors provide an attractive and clinically feasible approach to overcome intrinsic and acquired resistance to cancer immunotherapy, and to extend the clinical success of cancer immunotherapy.

Adaptive Immune Regulation of Mammary Postnatal Organogenesis.

Postnatal organogenesis occurs in an immune competent environment and is tightly controlled by interplay between positive and negative regulators. Innate immune cells have beneficial roles in postnatal tissue remodeling, but roles for the adaptive immune system are currently unexplored. Here we show that adaptive immune responses participate in the normal postnatal development of a non-lymphoid epithelial tissue. Since the mammary gland (MG) is the only organ developing predominantly after birth, we utilized it as a powerful system to study adaptive immune regulation of organogenesis. We found that antigen-mediated interactions between mammary antigen-presenting cells and interferon-γ (IFNγ)-producing CD4+ T helper 1 cells participate in MG postnatal organogenesis as negative regulators, locally orchestrating epithelial rearrangement. IFNγ then affects luminal lineage differentiation. This function of adaptive immune responses, regulating normal development, changes the paradigm for studying players of postnatal organogenesis and provides insights into immune surveillance and cancer transformation.