Women who carry a pregnancy to term have long been known to have a reduced lifetime risk of breast cancer, yet the mechanisms underlying this protection remain poorly understood. While parity-induced differentiation and hormonal remodeling of mammary epithelium have been considered primary drivers of breast cancer protection, the contribution of immune adaptation has remained largely unexplored. In a recent issue of Nature Immunology, Hussain and colleagues identify pregnancy-induced tissue-resident memory-like (TRM-like) CD8⁺ T cells as key mediators of parity-associated breast cancer protection. The authors show that pregnancy establishes a nurturing niche in the mammary gland that supports the expansion of TRM-like cells through epithelial-derived IL-15 and TGF-β, and that depletion of these cells abolishes the cancer-protective effect of parity. Moreover, using therapeutic agents to enhance IL-2Rβ signaling was sufficient to induce expansion of breast cancer-protective TRM-like cells in nulliparous mice, raising the possibility that pregnancy's protective imprint could be pharmacologically induced. Given the established link between TRM cells and immune checkpoint blockade responsiveness in breast cancer, these findings also raise the possibility that pregnancy-induced TRM-like populations could be leveraged to improve immunotherapy outcomes. By linking physiological developmental tissue remodeling to durable local immunosurveillance, this study expands current concepts of tissue-resident immunity and positions pregnancy as an immune-educating event with lasting consequences for breast cancer susceptibility.

Metastatic progression depends upon the ability of disseminated tumor cells to evade immune surveillance. MHC molecule expression facilitates T cell recognition and activation to permit the eradication of metastatic tumor cells. We identified nuclear corepressor 2 (NCOR2) as a key epigenetic regulator of MHC class I molecule expression on breast tumor cells. Patients with triple negative breast cancers (TNBC) that expressed high levels of NCOR2 also exhibited reduced metastasis free survival and decreased MHC class I expression, and the metastatic lesions in patients with TNBC had high nuclear NCOR2 and reduced CD8 T cell levels and activity. Genetically and experimentally reducing NCOR2 expression in tumor cells permitted interferon gamma upregulation of MHC class I, and potentiated CD8 T cell activity and induction of apoptosis to repress metastatic progression of disseminated breast cancer cells. These studies provide evidence to support NCOR2 as a targetable epigenetic regulator of metastasis towards which therapies could be developed to reduce patient mortality.

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. We modeled heterogeneous tumors comprised of "hot" and "cold" tumor populations (giving rise to T cell-rich and T cell-poor tumors, respectively) and introduced fluorescent labels to enable precise spatial tracking. We found the cold tumor cell population exerted a "dominant cold" effect in mixed tumors. Strikingly, spatial analysis revealed that the tumor cells themselves created distinct local microenvironments within heterogeneous tumors: regions occupied by cold tumor cells showed pronounced immunosuppression, harboring increased CD206Hi macrophages and diminished local T cell function. This inferior T cell activity in cold regions persisted even after immunotherapy and mechanistically was mediated by CX3CL1 produced by the cold tumor cells. An immune cold tumor population within a heterogeneous tumor thus impairs tumor immunity on both a tumor-wide and a highly localized spatial scale.

Breast cancer (BRCA) is the second leading cause of cancer-related mortality in women. While cancer immunotherapy shows promise in treating some solid tumors, its effectiveness in BRCA remains limited. High levels of CD8+ T cells, which are key targets in immunotherapy, correlate with positive responses in BRCA, especially in aggressive cases like triple-negative BRCA. However, prolonged exposure to the tumor microenvironment (TME) drives CD8+ T cells to terminal functional exhaustion, reducing immunotherapy efficacy. The mechanisms by which the TME induces this dysfunction in T cells are not fully understood. BRCA tumors are densely infiltrated by tumor-associated macrophages (TAMs), which are linked to poorer patient outcomes. In our previous work, we observed a spatiotemporal co-dependency between exhausted T cells (TEX) and TAMs, wherein TEX attract monocytes into the TME, shaping their differentiation into TAMs. TAMs, in turn, engage in long-lasting interactions with T cells to promote exhaustion. Nonetheless, the molecular mechanism by which TAMs interact with T cells in the BRCA TME to drive exhaustion remains unclear. We propose that TAMs and TEX communicate through chemokine signaling pathways, suppressing anti-tumor immunity. This research aims to uncover new strategies for reprogramming TAMs to reduce CD8+ T cell exhaustion, ultimately enhancing the immunotherapy outcomes for BRCA patients. Conrad Prebys Recruit Award Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Genetic engineering of T cells in mouse models is essential for investigating immune mechanisms. We aimed to develop an approach to manipulate T cells in vivo using an evolved adeno-associated virus (AAV) capsid named Ark313. Delivery of a transient transgene expression cassette was feasible using Ark313, and this serotype outperformed natural serotypes. A single intravenous injection of a Cre recombinase-expressing Ark313 in the Ai9 fluorescent reporter mouse model achieved permanent genetic modifications of T cells. Ark313 facilitated in vivo gene editing in both tissue-resident and splenic T cells and validation of immunotherapy targets in solid tumor models. Ark313 delivered large DNA donor templates to T cells in vivo and integrated transgenes in primary CD4+ and CD8+ T cells, including naive T cells. Ark313-mediated transgene delivery presents an efficient approach to target mouse T cells in vivo and a resource for the interrogation of T cell biology and for immunotherapy applications.

Tumor-associated macrophages (TAMs) are frequently categorized as being 'M1' or 'M2' polarized, even as substantial data challenges this binary modeling of macrophage cell state. One molecule consistently referenced as a delineator of a putative immunosuppressive 'M2' state is the surface protein CD206. We thus made a novel conditional CD206 (Mrc1) knock-in mouse to specifically visualize and/or deplete CD206+ 'M2-like' TAMs and assess their correspondence with pro-tumoral immunity. Early, but not late depletion of CD206+ macrophages and monocytes (here, 'Mono/Macs') 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, and able to differentially attract activated CD8 T cells. In contrast, a population of stress-responsive TAMs ("Hypoxic" or Spp1+) and immature monocytes, which lack CD206 expression and become prominent following early depletion, expressed markedly diminished levels of CXCL9. Those NK and CD8 T cells which enter CD206-depleted tumors express vastly reduced levels of the corresponding receptor Cxcr3, the cDC1-attracting chemokine Xcl1 and cDC1 growth factor Flt3l transcripts. Consistent with the loss of this critical network, early CD206+ TAM depletion decreased tumor control by antigen specific CD8 T cells in mice. Likewise, in humans, the CD206Replete, but not the CD206Depleted Mono/Mac gene signature correlated robustly with CD8 T cell, NK cell and stimulatory cDC1 gene signatures and transcriptomic signatures skewed towards CD206Replete Mono/Macs associated with better survival. Together, these findings negate the unqualified classification of CD206+ 'M2-like' macrophages as immunosuppressive by illuminating contexts for their role in organizing a critical tumor-reactive archetype of immunity.

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-β. 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.

Abstract Cancer immunotherapy (including immune checkpoint blockade (ICB)) has shown great promise in the treatment of some solid cancers, but responses in breast cancer patients are limited. While breast cancer is generally considered poorly immunogenic, the abundance of CD8+ T cells correlates with clinical response. CD8+ T cells are critical mediators of anti-tumor immunity and the main target for ICB. However, the onset of terminal functional T cell exhaustion poses a major challenge. Although T cell exhaustion has been linked to persistent antigen exposure, it remains unclear how the immune composition of the breast tumor microenvironment (TME) contributes to this dysfunctional T cell state. Breast cancers are heavily infiltrated with tumor-associated macrophages (TAMs) and their abundance correlates with T cell exhaustion and poor prognosis. Previously, we discovered a spatiotemporal co-dependency between exhausted T cells (TEX) and TAMs in mouse models of melanoma and breast cancer. We showed that TEX actively recruit monocytes to the TME and shape their differentiation trajectory into TAMs. Reciprocally, these TAMs ‘capture’ T cells in long-lived synaptic interactions that contribute to functional T cell exhaustion. Our current work is focused on studying the TEX-derived chemokines that regulate these TAM – T cell interactions in the breast TME with the ultimate goal to alleviate immune evasion and improve responsiveness of breast cancer to immunotherapy. Citation Format: Meenakshi Sudhakaran, Sofia Lombardi, Sophie Ayma, Matthew F. Krummel, Kelly Kersten. Reciprocal macrophage - T cell interactions regulate anti-tumor immunity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B035.

CD206 is a common marker of a putative immunosuppressive “M2” state in tumor-associated macrophages (TAMs). We made a novel conditional CD206 (Mrc1) knock-in mouse to specifically visualize and/or deplete CD206+ TAMs. Early depletion of CD206+ macrophages and monocytes (Mono/Macs) led to the indirect loss of conventional type I dendritic cells (cDC1), CD8 T cells, and NK cells in tumors. CD206+ TAMs robustly expressed CXCL9, contrasting with stress-responsive Spp1-expressing TAMs and immature monocytes, which became prominent with early depletion. CD206+ TAMs differentially attracted activated CD8 T cells, and the NK and CD8 T cells in CD206-depleted tumors were deficient in Cxcr3 and cDC1-supportive Xcl1 and Flt3l expressions. Disrupting this key antitumor axis decreased tumor control by antigen-specific T cells in mice. In human cancers, a CD206Replete, but not a CD206Depleted Mono/Mac gene signature correlated robustly with CD8 T cell, cDC1, and NK signatures and was associated with better survival. These findings negate the unqualified classification of CD206+ “M2-like” macrophages as immunosuppressive.

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.

ABSTRACTThe 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.

Background Intratumoral heterogeneity—defined as genetic and cellular diversity within a tumor—is linked to the failure of immunotherapy in multiple cancer types.1 2 The reasons for this are not well understood. Recent multi-region studies have found that as many as two-thirds of patient tumors contain both distinct 'hot' and 'cold' tumor regions, defined by high and low T cell infiltrates, respectively.3 We developed a novel system to reproducibly model tumor heterogeneity in mice, and employed this system to ask how ITH shapes the efficacy of immunotherapy. Methods We introduced fluorescent tags into each 'immune hot' and 'immune cold' squamous cell skin carcinoma cell lines, and mixed them together to establish heterogeneous tumors in which tumor populations could be precisely spatially tracked. We analyzed immune cells in the vicinity of each hot and cold tumor cell populations by microdissection and flow cytometry as well as by spatial single cell RNAseq. We subsequently treated mice with a combination of anti-PD-1 blockade + CD40 agonist or control antibodies. We analyzed the response to therapy in each hot and cold regions of heterogeneous tumors, as well as overall tumor growth. Results In untreated heterogeneous tumors, we find local tumor cells direct the formation distinct immune microenvironments. Cold tumor cells establish neighborhoods of immunosuppression, characterized by high macrophage infiltration, few inflammatory monocytes and neutrophils, and limited T cell abundance and function. We identified CX3CL1 as a candidate mediator of immunosuppression in cold tumor regions. When CX3CL1 was overexpressed in hot tumor cells, it drove an increase in suppressive CD206hi macrophages and reduction of monocytes and neutrophils. When heterogeneous tumors were treated with a combination of anti-PD-1 and CD40 agonist antibodies, we observed an influx of T cells post-treatment but only a modest slowing of tumor growth. Microdissection of tumors 6 days after treatment revealed that dysfunctional immune spatial organization persisted following therapy: T cells continued to exhibit limited abundance and function near cold tumor cells. Conclusions We conclude that tumor cells create an architectural blueprint for the abundance and functional activity of tumor-infiltrating immune cells, which shapes both the pre-treatment immune microenvironment and responses to immunotherapy. Treatment with a combinatorial checkpoint inhibition regimen, despite leading to an influx in T cells, failed to eliminate spatial pockets of immunosuppression near cold tumor cells, and was ultimately ineffective at inducing tumor regression. Mechanistically, we identify CX3CL1 as a mediator of intratumoral accumulation of immunosuppressive macrophages. References McGranahan N, Furness AJS, Rosenthal R, et al. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade. Science. 2016;351:1463–1469. Liu D, Schilling B, Liu D, et al. Integrative molecular and clinical modeling of clinical outcomes to PD1 blockade in patients with metastatic melanoma. Nat. Med. 2019;25:1916–1927. Abduljabbar K, Ahmed Raza SE, Rosenthal R, et al. Geospatial immune variability illuminates differential evolution of lung adenocarcinoma. Nat. Med. 2020;26:1054–1062.

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 IFN γ + 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 CD206 Hi 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.

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.

Intratumoral heterogeneity (ITH) – cellular and molecular diversity within a tumor – is linked to failure of immunotherapy in multiple cancer types. A high degree of ITH is associated with poor infiltration of T cells into the tumor and resistance to immune checkpoint blockade (ICB) therapy. To determine how distinct tumor populations within heterogeneous tumors shape the immune microenvironment and how this impacts therapy response, we modeled heterogeneous tumors composed of an RFP-tagged immunosuppressive tumor population and a YFP-tagged pro-inflammatory tumor population. The resulting tumors contained a patchwork of distinct regions with YFP +cells, RFP +cells, or a mixture of YFP +and RFP +cells. Analysis of the immune infiltrates in each region revealed a higher frequency of total CD4 T cells, Th1 cells and IFNg +CD8 T cells in YFP regions compared to RFP regions, whereas macrophages exhibited the opposite pattern. PD-1 blockade and CD40 agonist combinatorial antibody therapy induced an increase in Th1 abundance in RFP regions, but the treatment did not clear the tumors. Together, these results reveal that distinct regional immune infiltration pattens within the tumor are driven by the local tumor cells present in each region, and that the treatment-induced global improvement of Th1 infiltration alone is not sufficient to induce tumor clearance. Moreover, we identified Cx3cl1 as a driver of a dominant immunosuppressive tumor microenvironment, marked by an increase in immunosuppressive CD206 +macrophages and decrease in anti-tumoral neutrophils and monocytes. These results suggest that an immunosuppressive tumor population drives immunotherapy resistance of heterogeneous tumors and Cx3cl1 as a new therapeutic target. Supported by grants from Parker Institute for Cancer Immunotherapy (Project Grant)

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 micropar-ticles 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 metasta-tic lung.

Tumor progression is accompanied by fibrosis, which is associated with diminished anti-tumor immune infiltrate. Here, we demonstrate that tumor infiltrating myeloid cells respond to the stiffened fibrotic tumor microenvironment (TME) by initiating a TGF-beta (TGFβ)-directed, collagen biosynthesis program. A collateral effect of this programming is an untenable metabolic milieu for productive CD8 T cell anti-tumor responses, as collagen-synthesizing macrophages consume environmental arginine, synthesize proline, and secrete ornithine that compromises CD8+ T cell function. Thus, a stiff and fibrotic TME may impede anti-tumor immunity not only by direct physical exclusion of CD8+ T cells, but also via secondary effects of a myeloid mechano-metabolic programming we identified that creates an inhospitable metabolic milieu for CD8+ T cells.

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 (T-ex) 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 T-ex 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.