Tertiary lymphoid structures harbour stem-like tumour-specific T cells

Integrative single cell analysis of CD8+ T-cells across early and advanced oral cancers reveals signatures of anti-tumour activity

Tumour-targeting CD8⁺ T cells drive responses to every major form of cancer immunotherapy. Identifying them, however, remains an unsolved problem in solid tumours. The antigens they recognize are rarely defined and almost never shared between patients. We profiled 51,459 CD8+ T cells by paired single-cell RNA and T-cell receptor sequencing across 28 samples from 17 HPV-negative oral cancers spanning primary tumours, draining lymph nodes, metastases, and pembrolizumab-treated recurrences. We found that clonotypes that were expanded and shared across anatomical sites and timepoints were enriched within tumours and progressively selected over disease evolution and checkpoint blockade. Designating these shared-expanded clones as putative tumour-targeting cells, we trained a machine learning classifier that identifies them from transcriptome data alone. This 108-feature random forest signature recapitulated programmes of tumour reactivity and generalized to an integrated atlas of 89,318 CD8+ T cells from independent cohorts, showing progressive enrichment from normal to malignant tissue, and localized to tumour-proximal niches in spatial transcriptomics. By demonstrating that clonal behaviour across space and time encodes tumour reactivity in the transcriptome, this work establishes a generalizable framework for mapping tumour-engaged immunity without knowledge of the underlying antigen.

Spatially Organized Tertiary Lymphoid Structures Emerge in Small Cell Lung Cancer and Associate with Improved Survival

Tertiary lymphoid structures (TLS) are ectopic immune aggregates associated with improved prognosis and response to immunotherapy in multiple solid tumors. However, their presence, spatial organization, and functional relevance in small cell lung cancer (SCLC), a malignancy characterized by profound immune evasion, remain poorly understood. Using imaging mass cytometry (IMC) across 320 regions of interest spanning primary lung tumor, tumor-adjacent lung, liver and lymph node metastasis, complemented by Visium HD spatial transcriptomics, we characterized the cellular architecture and molecular programs of TLS-like niches in SCLC. TLS-like niches were identified in a subset of SCLC samples, predominantly primary lung tumor tissues and adjacent lung, spanning a continuum from loose lymphoid aggregates to compact follicle-like immune structures. Organized TLS-like niches contained CD20+ B-cell cores, closely associated with CD4+ and CD8A+ T cells, proliferating lymphocytes, HLA-DR+ antigen-presenting compartments, and αSMA+ stromal scaffolds, and were enriched for canonical TLS organizer signals (CXCL13, LTB, FDCSP). Patients with TLS-positive tumors demonstrated improved overall survival, and core TLS-associated transcriptional programs were associated with favorable survival in an independent bulk RNA-seq cohort. To our knowledge, this represents one of the first spatially resolved analyses of TLS-like immune architecture in SCLC, demonstrating that organized lymphoid immunity can emerge in this classically immune-evasive disease and is associated with improved survival.

46 Anti-tumour immune responses in melanoma brain metastases require cDC1 population and the extracranial tumour

Brain metastases (BrM) are associated with poor prognosis, partially due to the specialized immune-microenvironment in the brain. Our previous study demonstrated that intracranial tumours alone are poorly immunogenic, but simultaneous presence of a subcutaneous tumour, mimicking the extracranial disease observed in patients, greatly enhances immune checkpoint blockade efficacy. Here we aim to elucidate the role of conventional dendritic cells type 1 (cDC1) in anti-tumour immune responses in BrM and their dependence on the systemic anti-tumour immunity generated by extracranial tumour. Luciferase-tagged B16-Ova melanoma cells were injected intracranially into C57Bl/6 mice with or without subcutaneous tumours. Intracranial tumour burden was measured by bioluminescence imaging in constitutive and conditional cDC1 deletion models. Intra-tumoral immune cells were analysed by spectral flow-cytometry. A significantly lower intracranial tumour burden was observed in the presence of cDC1s in mice with extracranial tumours, while in the absence of extracranial tumours cDC1 presence induced only a tendency towards reduced intracranial tumour burden. The simultaneous presence of extracranial tumour and cDC1s was associated with significantly higher abundance of natural killer and effector CD8+ T cells in intracranial tumours as compared to mice lacking cDC1s and/or extracranial tumour. Moreover, the presence of extracranial tumour increased the expression of activation markers in intracranial cDC1s, and the abundance of cDC1s and “mature DCs enriched in immunoregulatory molecules” in intracranial tumours. Our data suggest that extracranial and intracranial cDC1 pools are both required for an efficient control of intracranial tumour growth. We hypothesize that extracranial tumour inhibits intracranial tumour growth through extracranial cDC1-mediated priming and expansion of CD8+ T cells which then home to intracranial tumours, by directly enhancing the activation of intracranial cDC1s, or a combination of both

Clinical impact and therapeutic potential of tertiary lymphoid structures in melanoma

Tertiary lymphoid structures (TLS) are ectopic lymphoid aggregates that arise in non-lymphoid tissues under conditions of chronic inflammation, including cancer. Structurally and functionally resembling secondary lymphoid organs, TLS consist of organized T-cell zones, B-cell follicles, dendritic cells, high endothelial venules, and specialized stromal cells, enabling local antigen presentation and adaptive immune activation. In melanoma, the prototypical immunogenic tumor, the presence of TLS within the tumor microenvironment has been associated with enhanced immune surveillance and favorable clinical outcomes. However, emerging evidence indicates that TLS can also harbor immunosuppressive niches, potentially depending on their developmental stage and cellular composition. In this review, we summarize the molecular and cellular mechanisms underlying TLS biogenesis and outline the experimental methodologies currently used for their identification and characterization, with a focus on melanoma. We examine the clinical relevance of TLS in both primary and metastatic melanomas and discuss strategies aimed at inducing functional TLS within tumors. By promoting TLS formation to amplify intratumoral immune responses, these approaches hold promise for overcoming resistance to immunotherapy and improving outcomes in advanced melanoma and other malignancies.

Immunogenic cell death and tertiary lymphoid structures: bridging tumour cell death and immune organization.

PLA2G2D in tumour-draining lymph nodes regulates anti-tumour immunity.

Systemic anti-tumour immunity results from T cell priming in tumour-draining lymph nodes (TDLNs)1-4. Although the suppression of T cells in tumours is well characterized5-8, whether this occurs in TDLNs-and if so, through which mechanisms-remains poorly understood. Here, using imaging mass cytometry of TDLNs from patients with melanoma, we identify a spatial neighbourhood in the TDLN paracortex that is linked to the development of distant metastases. Targeted spatial transcriptomics of cells inside this neighbourhood revealed activated CD8+ T cells engaging with myeloid cells that expressed high levels of the immunosuppressive secretory phospholipase PLA2G2D. PLA2G2D+ myeloid cells were substantially more abundant in TDLNs than they were in primary tumours or metastases. Genetic loss-of-function or antibody-mediated inhibition of PLA2G2D reduced tumour growth markedly, and single-cell transcriptomics in melanoma-bearing mice revealed that expression of Pla2g2d is confined to lymph-node macrophages. Mechanistically, PLA2G2D directly suppressed the early proliferation of T cells in vitro, and inhibiting PLA2G2D resulted in an expansion of tumour-specific T cells in TDLNs, leading to an increase in these T cells in the circulation and subsequently in tumours. Notably, PLA2G2D and PD-1 act as non-redundant immune checkpoints, with combination treatment showing additive or synergistic efficacy in humanized mice treated with human-specific antibodies. Collectively, our in-depth spatial profiling identifies PLA2G2D as a TDLN-centred targetable immune checkpoint for cancer immunotherapy.