Faculty Bibliography

Lymphatic transport shapes the homeostatic immune repertoire of lymph nodes (LNs). LN-resident memory T cells (T_RMs) play an important role in site-specific immune memory, yet how LN T_RMs form de novo after viral infection remains unclear. Here, we tracked the anatomical distribution of antiviral CD8⁺ T cells as they seeded skin and LN T_RMs using a model of vaccinia virus-induced skin infection. LN T_RMs localized to the draining LNs (dLNs) of infected skin, and their formation depended on the lymphatic egress of effector CD8⁺ T cells from the skin, already poised for residence. Effector CD8⁺ T cell transit through skin was required to populate LN T_RMs in dLNs, a process reinforced by antigen encounter in skin. Furthermore, LN T_RMs were protective against viral rechallenge in the absence of circulating memory T cells. These data suggest that a subset of tissue-infiltrating CD8⁺ T cells egress from tissues during viral clearance and establish a layer of regional protection in the dLN basin.

Lymphatic transport maintains homeostatic health and is necessary for immune surveillance, and yet lymphatic growth is often associated with solid tumour development and dissemination. Although tumour-associated lymphatic remodelling and growth were initially presumed to simply expand a passive route for regional metastasis, emerging research puts lymphatic vessels and their active transport at the interface of metastasis, tumour-associated inflammation and systemic immune surveillance. Here, we discuss active mechanisms through which lymphatic vessels shape their transport function to influence peripheral tissue immunity and the current understanding of how tumour-associated lymphatic vessels may both augment and disrupt antitumour immune surveillance. We end by looking forward to emerging areas of interest in the field of cancer immunotherapy in which lymphatic vessels and their transport function are likely key players: the formation of tertiary lymphoid structures, immune surveillance in the central nervous system, the microbiome, obesity and ageing. The lessons learnt support a working framework that defines the lymphatic system as a key determinant of both local and systemic inflammatory networks and thereby a crucial player in the response to cancer immunotherapy.

T cell-based immunotherapies have exhibited promising outcomes in tumor control; however, their efficacy is limited in immune-excluded tumors. Cancer-associated fibroblasts (CAFs) play a pivotal role in shaping the tumor microenvironment and modulating immune infiltration. Despite the identification of distinct CAF subtypes using single-cell RNA-sequencing (scRNA-seq), their functional impact on hindering T-cell infiltration remains unclear, particularly in soft-tissue sarcomas (STS) characterized by low response rates to T cell-based therapies. In this study, we characterize the STS microenvironment using murine models (in female mice) with distinct immune composition by scRNA-seq, and identify a subset of CAFs we termed glycolytic cancer-associated fibroblasts (glyCAF). GlyCAF rely on GLUT1-dependent expression of CXCL16 to impede cytotoxic T-cell infiltration into the tumor parenchyma. Targeting glycolysis decreases T-cell restrictive glyCAF accumulation at the tumor margin, thereby enhancing T-cell infiltration and augmenting the efficacy of chemotherapy. These findings highlight avenues for combinatorial therapeutic interventions in sarcomas and possibly other solid tumors. Further investigations and clinical trials are needed to validate these potential strategies and translate them into clinical practice.

Early in solid tumor development, antigens are presented in tumor-draining lymph nodes (tdLNs), a process that is necessary to set up immune surveillance. Recent evidence indicates that tdLNs fuel systemic tumor-specific T cell responses which may halt cancer progression and facilitate future responses to immunotherapy. These protective responses, however, are subject to progressive dysfunction exacerbated by lymph node (LN) metastasis. We discuss emerging preclinical and clinical literature indicating that the tdLN is a crucial reservoir for systemic immunity that can potentiate immune surveillance. We also discuss the impact of LN metastasis and argue that a better understanding of the relationship between LN metastasis and systemic immunity will be necessary to direct regional disease management in the era of immunotherapy.

INTRODUCTION/UNASSIGNED:Quantitative, multiplexed imaging is revealing complex spatial relationships between phenotypically diverse tumor infiltrating leukocyte populations and their prognostic implications. The underlying mechanisms and tissue structures that determine leukocyte distribution within and around tumor nests, however, remain poorly understood. While presumed players in metastatic dissemination, new preclinical data demonstrates that blood and lymphatic vessels (lymphovasculature) also dictate leukocyte trafficking within tumor microenvironments and thereby impact anti-tumor immunity. Here we interrogate these relationships in primary human cutaneous melanoma. METHODS/UNASSIGNED:We established a quantitative, multiplexed imaging platform to simultaneously detect immune infiltrates and tumor-associated vessels in formalin-fixed paraffin embedded patient samples. We performed a discovery, retrospective analysis of 28 treatment-naïve, primary cutaneous melanomas. RESULTS/UNASSIGNED:Here we find that the lymphvasculature and immune infiltrate is heterogenous across patients in treatment naïve, primary melanoma. We categorized five lymphovascular subtypes that differ by functionality and morphology and mapped their localization in and around primary tumors. Interestingly, the localization of specific vessel subtypes, but not overall vessel density, significantly associated with the presence of lymphoid aggregates, regional progression, and intratumoral T cell infiltrates. DISCUSSION/UNASSIGNED:We describe a quantitative platform to enable simultaneous lymphovascular and immune infiltrate analysis and map their spatial relationships in primary melanoma. Our data indicate that tumor-associated vessels exist in different states and that their localization may determine potential for metastasis or immune infiltration. This platform will support future efforts to map tumor-associated lymphovascular evolution across stage, assess its prognostic value, and stratify patients for adjuvant therapy.

Lymph node metastasis in breast cancer depends in part on the acquisition of an IFN-dependent, MHC-II+ state that induces regulatory T cell expansion and local immune suppression (Lei et al. 2023. J. Exp. Med.https://doi.org/10.1084/jem.20221847).

Diffuse large B-cell lymphoma (DLBCL) is a typically immune suppressed lymphoma subtype with poor response to immune checkpoint blockade and CAR T cell therapy. Recent data demonstrated an association between an activated, myofibroblast-like tumor stroma with improved outcome. Based on these findings, Apollonio and colleagues explored the phenotypic, transcriptional, and functional state of fibroblastic reticular cells (FRC) in human and murine DLBCL. This study reveals that DLBCL cells trigger the activation and remodeling of FRCs, leading to a chronic inflammatory state that supports malignant B cell survival. Transcriptional reprogramming of the FRCs may inhibit CD8+ T cell migration and function through changes in homing chemokines, adhesion molecules, and antigen presentation machinery, which together limit the anti-DLBCL immune response. High-dimensional imaging mass cytometry revealed heterogeneous CD8+ T Cell and FRC neighborhoods that associated with different clinical outcomes and ex vivo modeling of the microenvironment indicated an opportunity to target the FRC network for improved T cell motility, infiltration, and effector function. This research broadens our understanding of the complex interactions between the lymph node microarchitecture and anti-tumor immune surveillance, defines structural vulnerabilities in DLBCL, and thereby offers opportunities for combined therapeutic approaches.

T cell position in the tumor microenvironment determines the probability of target encounter and tumor killing. CD8+ T cell exclusion from the tumor parenchyma is associated with poor response to immunotherapy, and yet the biology that underpins this distinct pattern remains unclear. Here we show that the vascular destabilizing factor angiopoietin-2 (ANGPT2) causes compromised vascular integrity in the tumor periphery, leading to impaired T cell infiltration to the tumor core. The spatial regulation of ANGPT2 in whole tumor cross-sections was analyzed in conjunction with T cell distribution, vascular integrity, and response to immunotherapy in syngeneic murine melanoma models. T cell exclusion was associated with ANGPT2 upregulation and elevated vascular leakage at the periphery of human and murine melanomas. Both pharmacological and genetic blockade of ANGPT2 promoted CD8+ T cell infiltration into the tumor core, exerting antitumor effects. Importantly, the reversal of T cell exclusion following ANGPT2 blockade not only enhanced response to anti-PD-1 immune checkpoint blockade therapy in immunogenic, therapy responsive mouse melanomas, but it also rendered non-responsive tumors susceptible to immunotherapy. Therapeutic response after ANGPT2 blockade, driven by improved CD8+ T cell infiltration to the tumor core, coincided with spatial TIE2 signaling activation and increased vascular integrity at the tumor periphery where endothelial expression of adhesion molecules was reduced. These data highlight ANGPT2/TIE2 signaling as a key mediator of T cell exclusion and a promising target to potentiate immune checkpoint blockade efficacy in melanoma.

Antigen-specific CD8⁺ T cell accumulation in tumors is a prerequisite for effective immunotherapy, and yet the mechanisms of lymphocyte transit are not well defined. Here we show that tumor-associated lymphatic vessels control T cell exit from tumors via the chemokine CXCL12, and intratumoral antigen encounter tunes CXCR4 expression by effector CD8⁺ T cells. Only high-affinity antigen downregulates CXCR4 and upregulates the CXCL12 decoy receptor, ACKR3, thereby reducing CXCL12 sensitivity and promoting T cell retention. A diverse repertoire of functional tumor-specific CD8⁺ T cells, therefore, exit the tumor, which limits the pool of CD8⁺ T cells available to exert tumor control. CXCR4 inhibition or loss of lymphatic-specific CXCL12 boosts T cell retention and enhances tumor control. These data indicate that strategies to limit T cell egress might be an approach to boost the quantity and quality of intratumoral T cells and thereby response to immunotherapy.

The tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) is a complex ecosystem that drives tumor progression; however, in-depth single cell characterization of the PDAC TME and its role in response to therapy is lacking. Here, we perform single-cell RNA sequencing on freshly collected human PDAC samples either before or after chemotherapy. Overall, we find a heterogeneous mixture of basal and classical cancer cell subtypes, along with distinct cancer-associated fibroblast and macrophage subpopulations. Strikingly, classical and basal-like cancer cells exhibit similar transcriptional responses to chemotherapy and do not demonstrate a shift towards a basal-like transcriptional program among treated samples. We observe decreased ligand-receptor interactions in treated samples, particularly between TIGIT on CD8 + T cells and its receptor on cancer cells, and identify TIGIT as the major inhibitory checkpoint molecule of CD8 + T cells. Our results suggest that chemotherapy profoundly impacts the PDAC TME and may promote resistance to immunotherapy.