To determine whether density and distribution of tumor-infiltrating lymphocytes (TILs; TIL grade) is an independent predictor of sentinel lymph node (SLN) status and survival in patients with clinically localized primary cutaneous melanoma.From the Melanoma Institute Australia database, 1,865 patients with a single primary melanoma ≥ 0.75 mm in thickness were identified. The associations of clinical and pathologic factors with SLN status, recurrence-free survival (RFS), and melanoma-specific survival (MSS) were analyzed.The majority of patients had either no (TIL grade 0; 35.4%) or few (TIL grade 1; 45.1%) TILs, with a minority showing moderate (TIL grade 2; 16.3%) or marked (TIL grade 3; 3.2%) TILs. Tumor thickness, mitotic rate, and Clark level were inversely correlated with TIL grade (each P <.001). SLN biopsy was performed in 1,138 patients (61.0%) and was positive in 252 (22.1%). There was a significant inverse association between SLN status and TIL grade (SLN positivity rates for each TIL grade: 0, 27.8%; 1, 20.1%; 2, 18.3%; 3, 5.6%; P <.001). Predictors of SLN positivity were decreasing age (P <.001), decreasing TIL grade (P <.001), ulceration (P =.003), increasing tumor thickness (P =.01), satellitosis (P =.03), and increasing mitoses (P =.03). The 5-year MSS and RFS rates were 83% and 76%, respectively (median follow-up, 43 months). Tumor thickness (P <.001), ulceration (P <.001), satellitosis (P <.001), mitotic rate (P =.003), TIL grade (P <.001), and sex (P =.01) were independent predictors of MSS. Patients with TIL grade 3 tumors had 100% survival.TIL grade is an independent predictor of survival and SLN status in patients with melanoma. Patients with a pronounced TIL infiltrate have an excellent prognosis.

The dynamic interactions of cancer cells with their microenvironment consisting of stromal cells (cellular part) and extracellular matrix (ECM) components (non-cellular) is essential to stimulate the heterogeneity of cancer cell, clonal evolution and to increase the multidrug resistance ending in cancer cell progression and metastasis. The reciprocal cell-cell/ECM interaction and tumor cell hijacking of non-malignant cells force stromal cells to lose their function and acquire new phenotypes that promote development and invasion of tumor cells. Understanding the underlying cellular and molecular mechanisms governing these interactions can be used as a novel strategy to indirectly disrupt cancer cell interplay and contribute to the development of efficient and safe therapeutic strategies to fight cancer. Furthermore, the tumor-derived circulating materials can also be used as cancer diagnostic tools to precisely predict and monitor the outcome of therapy. This review evaluates such potentials in various advanced cancer models, with a focus on 3D systems as well as lab-on-chip devices. Video abstract.

Exhausted CD8 T (Tex) cells are a distinct cell lineage that arise during chronic infections and cancers in animal models and humans. Tex cells are characterized by progressive loss of effector functions, high and sustained inhibitory receptor expression, metabolic dysregulation, poor memory recall and homeostatic self-renewal, and distinct transcriptional and epigenetic programs. The ability to reinvigorate Tex cells through inhibitory receptor blockade, such as αPD-1, highlights the therapeutic potential of targeting this population. Emerging insights into the mechanisms of exhaustion are informing immunotherapies for cancer and chronic infections. However, like other immune cells, Tex cells are heterogeneous and include progenitor and terminal subsets with unique characteristics and responses to checkpoint blockade. Here, we review our current understanding of Tex cell biology, including the developmental paths, transcriptional and epigenetic features, and cell intrinsic and extrinsic factors contributing to exhaustion and how this knowledge may inform therapeutic targeting of Tex cells in chronic infections, autoimmunity, and cancer.

Ten years since the immune checkpoint inhibitor ipilimumab was approved for advanced melanoma, it is time to reflect on the lessons learned regarding modulation of the immune system to treat cancer and on novel approaches to further extend the efficacy of current and emerging immunotherapies. Here, we review the studies that led to our current understanding of the melanoma immune microenvironment in humans and the mechanistic work supporting these observations. We discuss how this information is guiding more precise analyses of the mechanisms of action of immune checkpoint blockade and novel immunotherapeutic approaches. Lastly, we review emerging evidence supporting the negative impact of melanoma metabolic adaptation on anti-tumor immunity and discuss how to counteract such mechanisms for more successful use of immunotherapy.© 2022. Springer Nature America, Inc.

Immunotherapy remains more effective for hematologic tumors than for solid tumors. One of the main challenges to immunotherapy of solid tumors is the immunosuppressive microenvironment these tumors generate, which limits the cytotoxic capabilities of immune effector cells (e.g., cytotoxic T and natural killer cells). This microenvironment is characterized by hypoxia, nutrient starvation, accumulated waste products, and acidic pH. Tumor-hijacked cells, such as fibroblasts, macrophages, and T regulatory cells, also contribute to this inhospitable microenvironment for immune cells by secreting immunosuppressive cytokines that suppress the antitumor immune response and lead to immune evasion. Thus, there is a strong interest in developing new drugs and cell formulations that modulate the tumor microenvironment and reduce tumor cell immune evasion. Microphysiological systems (MPSs) are versatile tools that may accelerate the development and evaluation of these therapies, although specific examples showcasing the potential of MPSs remain rare. Advances in microtechnologies have led to the development of sophisticated microfluidic devices used to recapitulate tumor complexity. The resulting models, also known as microphysiological systems (MPSs), are versatile tools with which to decipher the molecular mechanisms driving immune cell antitumor cytotoxicity, immune cell exhaustion, and immune cell exclusion and to evaluate new targeted immunotherapies. Here, we review existing microphysiological platforms to study immuno-oncological applications and discuss challenges and opportunities in the field.© The Author(s) 2023.

The genetic and cellular alterations that define cancer provide the immune system with the means to generate T cell responses that recognize and eradicate cancer cells. However, elimination of cancer by T cells is only one step in the Cancer-Immunity Cycle, which manages the delicate balance between the recognition of nonself and the prevention of autoimmunity. Identification of cancer cell T cell inhibitory signals, including PD-L1, has prompted the development of a new class of cancer immunotherapy that specifically hinders immune effector inhibition, reinvigorating and potentially expanding preexisting anticancer immune responses. The presence of suppressive factors in the tumor microenvironment may explain the limited activity observed with previous immune-based therapies and why these therapies may be more effective in combination with agents that target other steps of the cycle. Emerging clinical data suggest that cancer immunotherapy is likely to become a key part of the clinical management of cancer. Copyright © 2013 Elsevier Inc. All rights reserved.

Despite decades of study, there are still many unanswered questions about metastasis, the process by which a localized cancer becomes a systemic disease. One of these questions is the nature of the tumor cells that give rise to metastases. Although conventional models suggest that metastases are seeded by single cells from the primary tumor, there is growing evidence that seeding requires the collective action of tumor cells traveling together in clusters. Here, we review this evidence, which comes from analysis of both experimental models and patient samples. We present a model of metastatic dissemination that highlights the activities of clusters of tumor cells that retain and require their epithelial properties. Copyright © 2016, American Association for the Advancement of Science.

We previously identified tumor-reactive lymphocytes in the first lymph nodes that drain the primary tumor. In this study, we performed lymphatic mapping to investigate the possibility of finding the first lymph nodes that drain metastases, and of learning whether these lymph nodes contained tumor-reactive lymphocytes suitable for adoptive immunotherapy.Nineteen patients were studied. The primary tumor site was colorectal cancer in seven patients, malignant melanoma in four, ovarian cancer and breast cancer in two, and one each with pancreatic cancer, cholangiocarcinoma, leiomyosarcoma, and squamous cellular cancer of the tongue. By injection of Patent blue dye or radioactive tracers around the metastases, we identified draining lymph nodes from liver metastases (n = 9), intra-abdominal local recurrences (n = 3), and regional lymph node metastases (n = 7). In six patients, a preoperative lymphoscintigraphy was performed.We located the first draining lymph node or nodes from metastases or local recurrences; we named them "metinel nodes." Lymphocytes from the metinel nodes proliferated, showed clonal expansion, and produced interferon gamma (via in vitro expansions on stimulation with tumor homogenate) and interleukins, all of which demonstrate the characteristics of tumor-reactive lymphocytes. Eight of the nineteen patients received immunotherapy on the basis of tumor-reactive T cells derived from the metinel nodes.We demonstrate that it is possible to locate the first lymph nodes draining subcutaneous, lymphatic, and visceral metastases, the so-called metinel nodes. Metinel node-derived lymphocytes may be used to treat disseminated solid cancer, and clinical trials should evaluate the effect of such treatment.

The immune system is a multicentered organ that is characterized by intimate interactions between its cellular components to efficiently ward off invading pathogens. A key constituent of this organ system is the distinct migratory activity of its cellular elements. The lymph node represents a pivotal meeting point of immune cells where adaptive immunity is induced and regulated. Additionally, besides barrier tissues, the lymph node is a critical organ where invading pathogens need to be eliminated in order to prevent systemic distribution of virulent microbes. Here, we explain how the lymph node is structurally and functionally organized to fulfill these two critical functions - pathogen defense and orchestration of adaptive immunity. We will discuss spatio-temporal aspects of cellular immune responses focusing on CD8 T cells and review how and where these cells are activated in the context of viral infections, as well as how viral antigen expression kinetics and different antigen presentation pathways are involved. Finally, we will describe how such responses are regulated and 'helped', and discuss how this relates to intranodal positioning and cellular migration of the various cellular components that are involved in these processes. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Tumors remodel their draining lymph nodes to simultaneously affect both metastatic potential and systemic immune surveillance.

Generation of tumor-infiltrating lymphocytes begins when tumor antigens reach the lymph node (LN) to stimulate T cells, yet we know little of how tumor material is disseminated among the large variety of antigen-presenting dendritic cell (DC) subsets in the LN. Here, we demonstrate that tumor proteins are carried to the LN within discrete vesicles inside DCs and are then transferred among DC subsets. A synapse is formed between interacting DCs and vesicle transfer takes place in the absence of free exosomes. DCs -containing vesicles can uniquely activate T cells, whereas DCs lacking them do not. Understanding this restricted sharing of tumor identity provides substantial room for engineering better anti-tumor immunity.Copyright © 2020 Elsevier Inc. All rights reserved.

Understanding the consequences of immune infiltration of solid tumors is vital to continued development of successful immune therapies. However, little is known about the fate of tumor-experienced leukocytes. Here we used in vivo photoconversion to label tumor-infiltrating cells and demonstrated specific egress and dissemination of effector T-cell subsets from tumors. The identity and functions of tumor-egressing cells, and those retained inside tumors, are of great importance to our understanding of tumor immunity because of the crucial role tumor-infiltrating cells play in primary tumors and their potential to provide antitumor immunity at secondary tumor sites and in secondary lymphoid organs. The results of this study will guide strategies that harness T-cell immunity for anticancer therapies.

In tumors, a subset of CD8 T cells expressing the transcription factor TCF-1 drives the response to immune checkpoint blockade. We examined the mechanisms that maintain these cells in an autochthonous model of lung adenocarcinoma. Longitudinal sampling and single-cell sequencing of tumor-antigen specific TCF-1 CD8 T cells revealed that while intratumoral TCF-1 CD8 T cells acquired dysfunctional features and decreased in number as tumors progressed, TCF-1 CD8 T cell frequency in the tumor draining LN (dLN) remained stable. Two discrete intratumoral TCF-1 CD8 T cell subsets developed over time-a proliferative SlamF6 subset and a non-cycling SlamF6 subset. Blocking dLN egress decreased the frequency of intratumoral SlamF6 TCF-1 CD8 T cells. Conventional type I dendritic cell (cDC1) in dLN decreased in number with tumor progression, and Flt3L+anti-CD40 treatment recovered SlamF6 T cell frequencies and decreased tumor burden. Thus, cDC1s in tumor dLN maintain a reservoir of TCF-1 CD8 T cells and their decrease contributes to failed anti-tumor immunity.Copyright © 2021 Elsevier Inc. All rights reserved.

The mechanisms by which tumors metastasize to sentinel and distant lymph nodes, and beyond, are poorly understood. We developed transgenic mice that overexpress vascular endothelial growth factor-C (VEGF-C) and green fluorescent protein specifically in the skin and studied the effects of chemically-induced skin carcinogenesis in this model. We found that in contrast to VEGF-A, VEGF-C does not increase the growth of primary tumors, but instead induces expansion of lymphatic networks within sentinel lymph nodes, even before the onset of metastasis. Once the metastatic cells arrived at the sentinel lymph nodes, the extent of lymphangiogenesis at these sites increased. Of importance, in mice with metastasis-containing sentinel lymph nodes, tumors that expressed VEGF-C were more likely to metastasize to additional organs, such as distal lymph nodes and lungs. No metastases were observed in distant organs in the absence of lymph node metastases. These findings indicate an important role of VEGF-C-induced lymph node lymphangiogenesis in the promotion of cancer metastasis beyond the sentinel lymph nodes. VEGF-C is therefore a good target to slow or even prevent the onset of metastasis.

Tumours often engage the lymphatic system in order to invade and metastasize. The tumour-draining lymph node may be an immune-privileged site that protects the tumour from host immunity, and lymph flow that drains tumours is often increased, enhancing communication between the tumour and the sentinel node. In addition to increasing the transport of tumour antigens and regulatory cytokines to the lymph node, increased lymph flow in the tumour margin causes mechanical stress-induced changes in stromal cells that stiffen the matrix and alter the immune microenvironment of the tumour. We propose that synergies between lymphatic drainage and flow-induced mechanotransduction in the stroma promote tumour immune escape by appropriating lymphatic mechanisms of peripheral tolerance.

For many solid malignancies, lymph node (LN) involvement represents a harbinger of distant metastatic disease and, therefore, an important prognostic factor. Beyond its utility as a biomarker, whether and how LN metastasis plays an active role in shaping distant metastasis remains an open question. Here, we develop a syngeneic melanoma mouse model of LN metastasis to investigate how tumors spread to LNs and whether LN colonization influences metastasis to distant tissues. We show that an epigenetically instilled tumor-intrinsic interferon response program confers enhanced LN metastatic potential by enabling the evasion of NK cells and promoting LN colonization. LN metastases resist T cell-mediated cytotoxicity, induce antigen-specific regulatory T cells, and generate tumor-specific immune tolerance that subsequently facilitates distant tumor colonization. These effects extend to human cancers and other murine cancer models, implicating a conserved systemic mechanism by which malignancies spread to distant organs.Copyright © 2022 Elsevier Inc. All rights reserved.

Recent advances in cancer immunotherapy - ranging from immune-checkpoint blockade therapy to adoptive cellular therapy and vaccines - have revolutionized cancer treatment paradigms, yet the variability in clinical responses to these agents has motivated intense interest in understanding how the T cell landscape evolves with respect to response to immune intervention. Over the past decade, the advent of multidimensional single-cell technologies has provided the unprecedented ability to dissect the constellation of cell states of lymphocytes within a tumour microenvironment. In particular, the rapidly expanding capacity to definitively link intratumoural phenotypes with the antigen specificity of T cells provided by T cell receptors (TCRs) has now made it possible to focus on investigating the properties of T cells with tumour-specific reactivity. Moreover, the assessment of TCR clonality has enabled a molecular approach to track the trajectories, clonal dynamics and phenotypic changes of antitumour T cells over the course of immunotherapeutic intervention. Here, we review the current knowledge on the cellular states and antigen specificities of antitumour T cells and examine how fine characterization of T cell dynamics in patients has provided meaningful insights into the mechanisms underlying effective cancer immunotherapy. We highlight those T cell subsets associated with productive T cell responses and discuss how diverse immunotherapies might leverage the pre-existing tumour-reactive T cell pool or instruct de novo generation of antitumour specificities. Future studies aimed at elucidating the factors associated with the elicitation of productive antitumour T cell immunity are anticipated to instruct the design of more efficacious treatment strategies.© 2023. Springer Nature Limited.

The emergence of immune checkpoint inhibitors (ICIs), mainly including anti-programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PD-L1) and anti-cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) monoclonal antibodies (mAbs), has shaped therapeutic landscape of some type of cancers. Despite some ICIs have manifested compelling clinical effectiveness in certain tumor types, the majority of patients still showed de novo or adaptive resistance. At present, the overall efficiency of immune checkpoint therapy remains unsatisfactory. Exploring additional immune checkpoint molecules is a hot research topic. Recent studies have identified several new immune checkpoint targets, like lymphocyte activation gene-3 (LAG-3), T cell immunoglobulin and mucin-domain containing-3 (TIM-3), T cell immunoglobulin and ITIM domain (TIGIT), V-domain Ig suppressor of T cell activation (VISTA), and so on. The investigations about these molecules have generated promising results in preclinical studies and/or clinical trials. In this review, we discussed the structure and expression of these newly-characterized immune checkpoints molecules, presented the current progress and understanding of them. Moreover, we summarized the clinical data pertinent to these recent immune checkpoint molecules as well as their application prospects.

Compared with previous standards of care (including chemotherapy, radiotherapy, and surgery), cancer immunotherapy has brought significant improvements for patients in terms of survival and quality of life. Immunotherapy has now firmly established itself as a novel pillar of cancer care, from the metastatic stage to the adjuvant and neoadjuvant settings in numerous cancer types. In this review article, we highlight how the history of cancer immunotherapy paved the way for discoveries that are now part of the standard of care. We also highlight the current pitfalls and limitations of cancer checkpoint immunotherapy and how novel research in the fields of personalized cancer vaccines, autoimmunity, the microbiome, the tumour microenvironment, and metabolomics is aiming to solve those challenges.2020 Multimed Inc.

Research in two fronts has enabled the development of therapies that provide significant benefit to cancer patients. One area stems from a detailed knowledge of mutations that activate or inactivate signaling pathways that drive cancer development. This work triggered the development of targeted therapies that lead to clinical responses in the majority of patients bearing the targeted mutation, although responses are often of limited duration. In the second front are the advances in molecular immunology that unveiled the complexity of the mechanisms regulating cellular immune responses. These developments led to the successful targeting of immune checkpoints to unleash anti-tumor T cell responses, resulting in durable long-lasting responses but only in a fraction of patients. In this Review, we discuss the evolution of research in these two areas and propose that intercrossing them and increasing funding to guide research of combination of agents represent a path forward for the development of curative therapies for the majority of cancer patients.Copyright © 2015 Elsevier Inc. All rights reserved.

Immunotherapy strategies against cancer are emerging as powerful weapons for treatment of this disease. The success of checkpoint inhibitors against metastatic melanoma and adoptive T-cell therapy with chimeric antigen receptor T cells against B-cell-derived leukemias and lymphomas are only two examples of developments that are changing the paradigms of clinical cancer management. These changes are a result of many years of intense research into complex and interrelated cellular and molecular mechanisms controling immune responses. Promising advances come from the discovery of cancer mutation-encoded neoantigens, improvements in vaccine development, progress in delivery of cellular therapies, and impressive achievements in biotechnology. As a result, radical transformation of cancer treatment is taking place in which conventional cancer treatments are being integrated with immunotherapeutic agents. Many clinical trials are in progress testing potential synergistic effects of treatments combining immunotherapy with other therapies. Much remains to be learned about the selection, delivery, and off-target effects of immunotherapy used alone or in combination. The existence of numerous escape mechanisms from the host immune system that human tumors have evolved still is a barrier to success. Efforts to understand the rules of immune cell dysfunction and of cancer-associated local and systemic immune suppression are providing new insights and fuel the enthusiasm for new therapeutic strategies. In the future, it might be possible to tailor immune therapy for each cancer patient. The use of new immune biomarkers and the ability to assess responses to therapy by noninvasive monitoring promise to improve early cancer diagnosis and prognosis. Personalized immunotherapy based on individual genetic, molecular, and immune profiling is a potentially achievable future goal. The current excitement for immunotherapy is justified in view of many existing opportunities for harnessing the immune system to treat cancer.©2016 American Association for Cancer Research.

Antigen-specific CD8 T cells are central to the control of chronic infections and cancer, but persistent antigen stimulation results in T cell exhaustion. Exhausted CD8 T cells have decreased effector function and proliferative capacity, partly caused by overexpression of inhibitory receptors such as programmed cell death (PD)-1. Blockade of the PD-1 pathway has opened a new therapeutic avenue for reinvigorating T cell responses, with positive outcomes especially for patients with cancer. Other strategies to restore function in exhausted CD8 T cells are currently under evaluation-many in combination with PD-1-targeted therapy. Exhausted CD8 T cells comprise heterogeneous cell populations with unique differentiation and functional states. A subset of stem cell-like PD-1 CD8 T cells responsible for the proliferative burst after PD-1 therapy has been recently described. A greater understanding of T cell exhaustion is imperative to establish rational immunotherapeutic interventions.

The development of immune-checkpoint inhibitors (ICIs) has heralded a new era in cancer treatment, enabling the possibility of long-term survival in patients with metastatic disease, and providing new therapeutic indications in earlier-stage settings. As such, characterizing the long-term implications of receiving ICIs has grown in importance. An abundance of evidence exists describing the acute clinical toxicities of these agents, although chronic effects have not been as well catalogued. Nonetheless, emerging evidence indicates that persistent toxicities might be more common than initially suggested. While generally low-grade, these chronic sequelae can affect the endocrine, rheumatological, pulmonary, neurological and other organ systems. Fatal toxicities also comprise a diverse set of clinical manifestations and can occur in 0.4-1.2% of patients. This risk is a particularly relevant consideration in light of the possibility of long-term survival. Finally, the effects of immune-checkpoint blockade on a diverse range of immune processes, including atherosclerosis, heart failure, neuroinflammation, obesity and hypertension, have not been characterized but remain an important area of research with potential relevance to cancer survivors. In this Review, we describe the current evidence for chronic immune toxicities and the long-term implications of these effects for patients receiving ICIs.© 2022. Springer Nature Limited.

T cell dysfunction is a hallmark of many cancers, but the basis for T cell dysfunction and the mechanisms by which antibody blockade of the inhibitory receptor PD-1 (anti-PD-1) reinvigorates T cells are not fully understood. Here we show that such therapy acts on a specific subpopulation of exhausted CD8 tumor-infiltrating lymphocytes (TILs). Dysfunctional CD8 TILs possess canonical epigenetic and transcriptional features of exhaustion that mirror those seen in chronic viral infection. Exhausted CD8 TILs include a subpopulation of 'progenitor exhausted' cells that retain polyfunctionality, persist long term and differentiate into 'terminally exhausted' TILs. Consequently, progenitor exhausted CD8 TILs are better able to control tumor growth than are terminally exhausted T cells. Progenitor exhausted TILs can respond to anti-PD-1 therapy, but terminally exhausted TILs cannot. Patients with melanoma who have a higher percentage of progenitor exhausted cells experience a longer duration of response to checkpoint-blockade therapy. Thus, approaches to expand the population of progenitor exhausted CD8 T cells might be an important component of improving the response to checkpoint blockade.

Checkpoint blockade mediates a proliferative response of tumor-infiltrating CD8 T lymphocytes (TILs). The origin of this response has remained elusive because chronic activation promotes terminal differentiation or exhaustion of tumor-specific T cells. Here we identified a subset of tumor-reactive TILs bearing hallmarks of exhausted cells and central memory cells, including expression of the checkpoint protein PD-1 and the transcription factor Tcf1. Tcf1PD-1 TILs mediated the proliferative response to immunotherapy, generating both Tcf1PD-1 and differentiated Tcf1PD-1 cells. Ablation of Tcf1PD-1 TILs restricted responses to immunotherapy. Tcf1 was not required for the generation of Tcf1PD-1 TILs but was essential for the stem-like functions of these cells. Human TCF1PD-1 cells were detected among tumor-reactive CD8 T cells in the blood of melanoma patients and among TILs of primary melanomas. Thus, immune checkpoint blockade relies not on reversal of T cell exhaustion programs, but on the proliferation of a stem-like TIL subset.Copyright © 2018 Elsevier Inc. All rights reserved.

Neoadjuvant immune checkpoint blockade has shown promising clinical activity. Here, we characterized early kinetics in tumor-infiltrating and circulating immune cells in oral cancer patients treated with neoadjuvant anti-PD-1 or anti-PD-1/CTLA-4 in a clinical trial (NCT02919683). Tumor-infiltrating CD8 T cells that clonally expanded during immunotherapy expressed elevated tissue-resident memory and cytotoxicity programs, which were already active prior to therapy, supporting the capacity for rapid response. Systematic target discovery revealed that treatment-expanded tumor T cell clones in responding patients recognized several self-antigens, including the cancer-specific antigen MAGEA1. Treatment also induced a systemic immune response characterized by expansion of activated T cells enriched for tumor-infiltrating T cell clonotypes, including both pre-existing and emergent clonotypes undetectable prior to therapy. The frequency of activated blood CD8 T cells, notably pre-treatment PD-1-positive KLRG1-negative T cells, was strongly associated with intra-tumoral pathological response. These results demonstrate how neoadjuvant checkpoint blockade induces local and systemic tumor immunity.Copyright © 2022 Elsevier Inc. All rights reserved.

Triple negative breast cancer (TNBC) is a significant clinical problem to which immunotherapeutic strategies have been applied with limited success. Using the syngeneic E0771 TNBC mouse model, this work explores the potential for antitumor CD8 T cell immunity to be primed extratumorally in lymphoid tissues and therapeutically leveraged. CD8 T cell viability and responses within the tumor microenvironment (TME) were found to be severely impaired, effects coincident with local immunosuppression that is recapitulated in lymphoid tissues in late stage disease. Prior to onset of a locally suppressed immune microenvironment, however, CD8 T cell priming within lymph nodes (LN) that depended on tumor lymphatic drainage remained intact. These results demonstrate tumor-draining LNs (TdLN) to be lymphoid tissue niches that support the survival and antigenic priming of CD8 T lymphocytes against lymph-draining antigen. The therapeutic effects of and CD8 T cells response to immune checkpoint blockade were furthermore improved when directed to LNs within the tumor-draining lymphatic basin. Therefore, TdLNs represent a unique potential tumor immunity reservoir in TNBC for which strategies may be developed to improve the effects of ICB immunotherapy.

CD8 T cell responses are critical for anti-tumor immunity. While extensively profiled in the tumor microenvironment, recent studies in mice identified responses in lymph nodes (LNs) as essential; however, the role of LNs in human cancer patients remains unknown. We examined CD8 T cells in human head and neck squamous cell carcinomas, regional LNs, and blood using mass cytometry, single-cell genomics, and multiplexed ion beam imaging. We identified progenitor exhausted CD8 T cells (Tpex) that were abundant in uninvolved LN and clonally related to terminally exhausted cells in the tumor. After anti-PD-L1 immunotherapy, Tpex in uninvolved LNs reduced in frequency but localized near dendritic cells and proliferating intermediate-exhausted CD8 T cells (Tex-int), consistent with activation and differentiation. LN responses coincided with increased circulating Tex-int. In metastatic LNs, these response hallmarks were impaired, with immunosuppressive cellular niches. Our results identify important roles for LNs in anti-tumor immune responses in humans.Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.

Paired single-cell RNA and T cell receptor sequencing (scRNA/TCR-seq) has allowed for enhanced resolution of clonal T cell dynamics in cancer. Here, we report a scRNA/TCR-seq analysis of 187,650 T cells from 31 tissue regions, including tumor, adjacent normal tissues, and lymph nodes (LN), from three patients with non-small cell lung cancer after immune checkpoint blockade (ICB). Regions with viable cancer cells are enriched for exhausted CD8 T cells, regulatory CD4 T cells (Treg), and follicular helper CD4 T cells (TFH). Tracking T cell clonotypes across tissues, combined with neoantigen specificity assays, reveals that TFH and tumor-specific exhausted CD8 T cells are clonally linked to TCF7SELL progenitors in tumor draining LNs, and progressive exhaustion trajectories of CD8 T, Treg, and TFH cells with proximity to the tumor microenvironment. Finally, longitudinal tracking of tumor-specific CD8 and CD4 T cell clones reveals persistence in the peripheral blood for years after ICB therapy.Copyright © 2023 Elsevier Inc. All rights reserved.

Radiotherapy (RT) has been shown to stimulate an antitumor immune response in irradiated tumors as well as unirradiated distant sites (abscopal effect). Previous studies have demonstrated a role for the tumor-draining lymph node (LN) in mediating an anti-programmed death-1 (PD-1)/programmed death ligand-1 (PD-L1) stimulated antitumor immune response. Here, we investigated whether the LN is also important in mediating a RT alone stimulated abscopal response.

Radiation therapy is one of the primary treatment modalities for cancer along with chemotherapy and surgical therapy. The main mechanism of the tumor reduction after irradiation has been considered to be damage to the tumor DNA. However, we found that tumor-specific CTL, which were induced in the draining lymph nodes (DLN) and tumor tissue of tumor-bearing mice, play a crucial role in the inhibition of tumor growth by radiation. Indeed, the therapeutic effect of irradiation was almost completely abolished in tumor-bearing mice by depleting CD8(+) T cells through anti-CD8 monoclonal antibody administration. In mice whose DLN were surgically ablated or genetically defective (Aly/Aly mice), the generation of tetramer(+) tumor-specific CTL at the tumor site was greatly reduced in parallel with the attenuation of the radiation-induced therapeutic effect against the tumor. This indicates that DLN are essential for the activation and accumulation of radiation-induced CTL, which are essential for inhibition of the tumor. A combined therapy of local radiation with Th1 cell therapy augmented the generation of tumor-specific CTL at the tumor site and induced a complete regression of the tumor, although radiation therapy alone did not exhibit such a pronounced therapeutic effect. Thus, we conclude that the combination treatment of local radiation therapy and Th1 cell therapy is a rational strategy to augment antitumor activity mediated by tumor-specific CTL.

In the proper context, radiotherapy can promote antitumor immunity. It is unknown if elective nodal irradiation (ENI), a strategy that irradiates tumor-associated draining lymph nodes (DLN), affects adaptive immune responses and combinatorial efficacy of radiotherapy with immune checkpoint blockade (ICB). We developed a preclinical model to compare stereotactic radiotherapy (Tumor RT) with or without ENI to examine immunologic differences between radiotherapy techniques that spare or irradiate the DLN. Tumor RT was associated with upregulation of an intratumoral T-cell chemoattractant chemokine signature (CXCR3, CCR5-related) that resulted in robust infiltration of antigen-specific CD8 effector T cells as well as FoxP3 regulatory T cells (Tregs). The addition of ENI attenuated chemokine expression, restrained immune infiltration, and adversely affected survival when combined with ICB, especially with anti-CLTA4 therapy. The combination of stereotactic radiotherapy and ICB led to long-term survival in a subset of mice and was associated with favorable CD8 effector-to-Treg ratios and increased intratumoral density of antigen-specific CD8 T cells. Although radiotherapy technique (Tumor RT vs. ENI) affected initial tumor control and survival, the ability to reject tumor upon rechallenge was partially dependent upon the mechanism of action of ICB; as radiotherapy/anti-CTLA4 was superior to radiotherapy/anti-PD-1. Our results highlight that irradiation of the DLN restrains adaptive immune responses through altered chemokine expression and CD8 T-cell trafficking. These data have implications for combining radiotherapy and ICB, long-term survival, and induction of immunologic memory. Clinically, the immunomodulatory effect of the radiotherapy strategy should be considered when combining stereotactic radiotherapy with immunotherapy..©2018 American Association for Cancer Research.

Prostate cancer is poorly responsive to immune checkpoint inhibition, yet a combination with radiotherapy may enhance the immune response. In this study, we combined radiotherapy with immune checkpoint inhibition (iRT) in a castration-resistant prostate cancer (CRPC) preclinical model.Two Myc-CaP tumor grafts were established in each castrated FVB mouse. Anti-PD-1 or anti-PD-L1 antibodies were given and one graft was irradiated 20 Gy in 2 fractions.In CRPC, a significant increase in survival was found for radiation treatment combined with either anti-PD-1 or anti-PD-L1 compared to monotherapy. The median survival for anti-PD-L1 alone was 13 days compared to 30 days for iRT (p = 0.0003), and for anti-PD-1 alone was 21 days compared to 36 days for iRT (p = 0.0009). Additional treatment with anti-CD8 antibody blocked the survival effect. An abscopal treatment effect was observed for iRT in which the unirradiated graft responded similarly to the irradiated graft in the same mouse. At 21 days, the mean graft volume for anti-PD-1 alone was 2094 mm compared to iRT irradiated grafts 726 mm (p = 0.04) and unirradiated grafts 343 mm (p = 0.0066). At 17 days, the mean graft volume for anti-PD-L1 alone was 1754 mm compared to iRT irradiated grafts 284 mm (p = 0.04) and unirradiated grafts 556 mm (p = 0.21). Flow cytometry and immunohistochemistry identified CD8+ immune cell populations altered by combination treatment in grafts harvested at the peak effect of immunotherapy, 2-3 weeks after starting treatment.These data provide preclinical evidence for the use of iRT targeting PD-1 and PD-L1 in the treatment of CRPC. Immune checkpoint inhibition combined with radiotherapy treats CPRC with significant increases in median survival compared to drug alone: 70% longer for anti-PD-1 and 130% for anti-PD-L1, and with an abscopal treatment effect.Castration-resistant prostate cancer in a wild-type mouse model is successfully treated by X-ray radiotherapy combined with PD-1 or PD-L1 immune checkpoint inhibition, demonstrating significantly increased median overall survival and robust local and abscopal treatment responses, in part mediated by CD8 T-cells.

Immune checkpoint blockade (ICB) has changed the therapeutic landscape of oncology but its impact is limited by primary or secondary resistance. ICB resistance has been related to a lack of T cells infiltrating into the tumor. Strategies to overcome this hurdle have so far focused on the tumor microenvironment, but have mostly overlooked the role of tumor-draining lymph nodes (TDLN). Whereas for CTLA-4 blockade TDLN have long since been implicated due to its perceived mechanism-of-action involving T cell priming, only recently has evidence been emerging showing TDLN to be vital for the efficacy of PD-1 blockade as well. TDLN are targeted by developing tumors to create an immune suppressed pre-metastatic niche which can lead to priming of dysfunctional antitumor T cells. In this review, we will discuss the evidence that therapeutic targeting of TDLN may ensure sufficient antitumor T cell activation and subsequent tumor infiltration to facilitate effective ICB. Indeed, waves of tumor-specific, proliferating stem cell-like, or progenitor exhausted T cells, either newly primed or reinvigorated in TDLN, are vital for PD-1 blockade efficacy. Both tumor-derived migratory dendritic cell (DC) subsets and DC subsets residing in TDLN, and an interplay between them, have been implicated in the induction of these T cells, their imprinting for homing and subsequent tumor control. We propose that therapeutic approaches, involving local delivery of immune modulatory agents for optimal access to TDLN, aimed at overcoming hampered DC activation, will enable ICB by promoting T cell recruitment to the tumor, both in early and in advanced stages of cancer.

Lymph nodes draining the primary tumor are essential for the initiation of an effective anti-tumor T-cell immune response. However, cancer-derived immune suppressive factors render the tumor-draining lymph nodes (TDLN) immune compromised, enabling tumors to invade and metastasize. Unraveling the different mechanisms underlying this immune escape will inform therapeutic intervention strategies to halt tumor spread in early clinical stages. Here, we review our findings from translational studies in melanoma, breast, and cervical cancer and discuss clinical opportunities for local immune modulation of TDLN in each of these indications.

Targeted delivery of a nanovaccine loaded with a tumor antigen and adjuvant to the lymph nodes (LNs) is an attractive approach for improving cancer immunotherapy outcomes. However, the application of this technique is restricted by the paucity of suitable tumor-associated antigens (TAAs) and the sophisticated technology required to identify tumor neoantigens. Here, we demonstrate that a self-assembling melittin-lipid nanoparticle (α-melittin-NP) that is not loaded with extra tumor antigens promotes whole tumor antigen release in situ and results in the activation of antigen-presenting cells (APCs) in LNs. Compared with free melittin, α-melittin-NPs markedly enhance LN accumulation and activation of APCs, leading to a 3.6-fold increase in antigen-specific CD8 T cell responses. Furthermore, in a bilateral flank B16F10 tumor model, primary and distant tumor growth are significantly inhibited by α-melittin-NPs, with an inhibition rate of 95% and 92%, respectively. Thus, α-melittin-NPs induce a systemic anti-tumor response serving as an effective LN-targeted whole-cell nanovaccine.

The prognostic role of the extent of lymphadenectomy during surgery for esophageal cancer is uncertain and requires clarification.To clarify whether the number of removed lymph nodes influences mortality following surgery for esophageal cancer.Conducted from January 1, 2000, to January 31, 2014, this was a cohort study of patients who underwent esophagectomy for cancer in 2000-2012 at a high-volume hospital for esophageal cancer surgery, with follow-up until 2014.The main exposure was the number of resected lymph nodes. Secondary exposures were the number of metastatic lymph nodes and positive to negative lymph node ratio.The independent role of the extent of lymphadenectomy in relation to all-cause and disease-specific 5-year mortality was analyzed using Cox proportional hazard regression models, providing hazard ratios (HRs) with 95% CIs. The HRs were adjusted for age, pathological T category, tumor differentiation, margin status, calendar period of surgery, and response to preoperative chemotherapy.Among 606 included patients, 506 (83.5%) had adenocarcinoma of the esophagus, 323 (53%) died within 5 years of surgery, and 235 (39%) died of tumor recurrence. The extent of lymphadenectomy was not statistically significantly associated with all-cause or disease-specific mortality, independent of the categorization of lymphadenectomy or stratification for T category, calendar period, or chemotherapy. Patients in the fourth quartile of the number of removed nodes (21-52 nodes) did not demonstrate a statistically significant reduction in all-cause 5-year mortality compared with those in the lowest quartile (0-10 nodes) (HR, 0.86; 95% CI, 0.63-1.17), particularly not in the most recent calendar period (HR, 0.98; 95% CI, 0.57-1.66 for years 2007-2012). A greater number of metastatic nodes and a higher positive to negative node ratio was associated with increased mortality rates, and these associations showed dose-response associations.This study indicated that the extent of lymphadenectomy during surgery for esophageal cancer might not influence 5-year all-cause or disease-specific survival. These results challenge current clinical guidelines.

To determine the impact of lymphadenectomy (LND) on survival of clinically apparent early-stage malignant ovarian germ cell tumors (MOGCTs).We retrospectively analyzed the survival of patients who were diagnosed with the three most common histopathology types of malignant ovarian germ cell tumors (yolk sac tumor, dysgerminoma and immature teratoma) and with clinical stage I and II disease, and treated at Sun Yat-sen University Cancer Center between 1 January 1970 and 30 September 2018.There were 227 stage I, 28 stage II and one stage IIIA cases after surgery. One hundred and twenty-six patients underwent lymphadenectomy and 130 did not. Only one lymph node metastasis (0.8%) was found in the lymphadenectomy group. Two hundred and twenty-four out of 256 patients (87.5%) received postoperative chemotherapy. There were five relapses (4.0%) in the lymphadenectomy group and four (3.1%) in the non-lymphadenectomy group. Median follow-up was 68 months (range, 1-388). The 10-year disease-free survival rate in the lymphadenectomy group and non-lymphadenectomy group were 88.2 and 96.4%, respectively (P = 0.412); the 10-year overall survival rate in the two groups were 95.7 and 98.2%; respectively (P = 0.798). The results showed that lymphadenectomy did not improve patient survival.Lymphadenectomy may have little impact on survival in patients with clinically apparent early-stage malignant ovarian germ cell tumors and may be omitted in the surgical treatment.© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Lymph node dissection is part of the standard treatment protocol for various cancers, but its role in prostate cancer has been debatable for some time. Pelvic lymphadenectomy has been shown to better help stage prostate cancer patients, but has yet to be definitively proven to be of any benefit for survival. Various templates for lymph node dissections exist, and though some national guidelines have endorsed an extended pelvic node dissection, the choice of template is still controversial. Pelvic lymphadenectomy may lead to a slightly higher rate complications and operative time, and their use must be judiciously applied to patients with a high enough risk of lymph node involvement. We present a comprehensive review of the literature regarding the benefits and harms of lymph node dissection in prostate cancer.Copyright © 2018. Published by Elsevier Inc.

For patients with breast cancer and metastases in the sentinel nodes, axillary dissection has been standard treatment. However, for patients with limited sentinel-node involvement, axillary dissection might be overtreatment. We designed IBCSG trial 23-01 to determine whether no axillary dissection was non-inferior to axillary dissection in patients with one or more micrometastatic (≤2 mm) sentinel nodes and tumour of maximum 5 cm.In this multicentre, randomised, non-inferiority, phase 3 trial, patients were eligible if they had clinically non-palpable axillary lymph node(s) and a primary tumour of 5 cm or less and who, after sentinel-node biopsy, had one or more micrometastatic (≤2 mm) sentinel lymph nodes with no extracapsular extension. Patients were randomly assigned (in a 1:1 ratio) to either undergo axillary dissection or not to undergo axillary dissection. Randomisation was stratified by centre and menopausal status. Treatment assignment was not masked. The primary endpoint was disease-free survival. Non-inferiority was defined as a hazard ratio (HR) of less than 1·25 for no axillary dissection versus axillary dissection. The analysis was by intention to treat. Per protocol, disease and survival information continues to be collected yearly. This trial is registered with ClinicalTrials.gov, NCT00072293.Between April 1, 2001, and Feb 28, 2010, 465 patients were randomly assigned to axillary dissection and 469 to no axillary dissection. After the exclusion of three patients, 464 patients were in the axillary dissection group and 467 patients were in the no axillary dissection group. After a median follow-up of 5·0 (IQR 3·6-7·3) years, we recorded 69 disease-free survival events in the axillary dissection group and 55 events in the no axillary dissection group. Breast-cancer-related events were recorded in 48 patients in the axillary dissection group and 47 in the no axillary dissection group (ten local recurrences in the axillary dissection group and eight in the no axillary dissection group; three and nine contralateral breast cancers; one and five [corrected] regional recurrences; and 34 and 25 distant relapses). Other non-breast cancer events were recorded in 21 patients in the axillary dissection group and eight in the no axillary dissection group (20 and six second non-breast malignancies; and one and two deaths not due to a cancer event). 5-year disease-free survival was 87·8% (95% CI 84·4-91·2) in the group without axillary dissection and 84·4% (80·7-88·1) in the group with axillary dissection (log-rank p=0·16; HR for no axillary dissection vs axillary dissection was 0·78, 95% CI 0·55-1·11, non-inferiority p=0·0042). Patients with reported long-term surgical events (grade 3-4) included one sensory neuropathy (grade 3), three lymphoedema (two grade 3 and one grade 4), and three motor neuropathy (grade 3), all in the group that underwent axillary dissection, and one grade 3 motor neuropathy in the group without axillary dissection. One serious adverse event was reported, a postoperative infection in the axilla in the group with axillary dissection.Axillary dissection could be avoided in patients with early breast cancer and limited sentinel-node involvement, thus eliminating complications of axillary surgery with no adverse effect on survival.None.Copyright © 2013 Elsevier Ltd. All rights reserved.

Sentinel lymph node dissection (SLND) accurately identifies nodal metastasis of early breast cancer, but it is not clear whether further nodal dissection affects survival.To determine the effects of complete axillary lymph node dissection (ALND) on survival of patients with sentinel lymph node (SLN) metastasis of breast cancer.The American College of Surgeons Oncology Group Z0011 trial, a phase 3 noninferiority trial conducted at 115 sites and enrolling patients from May 1999 to December 2004. Patients were women with clinical T1-T2 invasive breast cancer, no palpable adenopathy, and 1 to 2 SLNs containing metastases identified by frozen section, touch preparation, or hematoxylin-eosin staining on permanent section. Targeted enrollment was 1900 women with final analysis after 500 deaths, but the trial closed early because mortality rate was lower than expected.All patients underwent lumpectomy and tangential whole-breast irradiation. Those with SLN metastases identified by SLND were randomized to undergo ALND or no further axillary treatment. Those randomized to ALND underwent dissection of 10 or more nodes. Systemic therapy was at the discretion of the treating physician.Overall survival was the primary end point, with a noninferiority margin of a 1-sided hazard ratio of less than 1.3 indicating that SLND alone is noninferior to ALND. Disease-free survival was a secondary end point.Clinical and tumor characteristics were similar between 445 patients randomized to ALND and 446 randomized to SLND alone. However, the median number of nodes removed was 17 with ALND and 2 with SLND alone. At a median follow-up of 6.3 years (last follow-up, March 4, 2010), 5-year overall survival was 91.8% (95% confidence interval [CI], 89.1%-94.5%) with ALND and 92.5% (95% CI, 90.0%-95.1%) with SLND alone; 5-year disease-free survival was 82.2% (95% CI, 78.3%-86.3%) with ALND and 83.9% (95% CI, 80.2%-87.9%) with SLND alone. The hazard ratio for treatment-related overall survival was 0.79 (90% CI, 0.56-1.11) without adjustment and 0.87 (90% CI, 0.62-1.23) after adjusting for age and adjuvant therapy.Among patients with limited SLN metastatic breast cancer treated with breast conservation and systemic therapy, the use of SLND alone compared with ALND did not result in inferior survival.clinicaltrials.gov Identifier: NCT00003855.

If treatment of the axilla is indicated in patients with breast cancer who have a positive sentinel node, axillary lymph node dissection is the present standard. Although axillary lymph node dissection provides excellent regional control, it is associated with harmful side-effects. We aimed to assess whether axillary radiotherapy provides comparable regional control with fewer side-effects.Patients with T1-2 primary breast cancer and no palpable lymphadenopathy were enrolled in the randomised, multicentre, open-label, phase 3 non-inferiority EORTC 10981-22023 AMAROS trial. Patients were randomly assigned (1:1) by a computer-generated allocation schedule to receive either axillary lymph node dissection or axillary radiotherapy in case of a positive sentinel node, stratified by institution. The primary endpoint was non-inferiority of 5-year axillary recurrence, considered to be not more than 4% for the axillary radiotherapy group compared with an expected 2% in the axillary lymph node dissection group. Analyses were by intention to treat and per protocol. The AMAROS trial is registered with ClinicalTrials.gov, number NCT00014612.Between Feb 19, 2001, and April 29, 2010, 4823 patients were enrolled at 34 centres from nine European countries, of whom 4806 were eligible for randomisation. 2402 patients were randomly assigned to receive axillary lymph node dissection and 2404 to receive axillary radiotherapy. Of the 1425 patients with a positive sentinel node, 744 had been randomly assigned to axillary lymph node dissection and 681 to axillary radiotherapy; these patients constituted the intention-to-treat population. Median follow-up was 6·1 years (IQR 4·1-8·0) for the patients with positive sentinel lymph nodes. In the axillary lymph node dissection group, 220 (33%) of 672 patients who underwent axillary lymph node dissection had additional positive nodes. Axillary recurrence occurred in four of 744 patients in the axillary lymph node dissection group and seven of 681 in the axillary radiotherapy group. 5-year axillary recurrence was 0·43% (95% CI 0·00-0·92) after axillary lymph node dissection versus 1·19% (0·31-2·08) after axillary radiotherapy. The planned non-inferiority test was underpowered because of the low number of events. The one-sided 95% CI for the underpowered non-inferiority test on the hazard ratio was 0·00-5·27, with a non-inferiority margin of 2. Lymphoedema in the ipsilateral arm was noted significantly more often after axillary lymph node dissection than after axillary radiotherapy at 1 year, 3 years, and 5 years.Axillary lymph node dissection and axillary radiotherapy after a positive sentinel node provide excellent and comparable axillary control for patients with T1-2 primary breast cancer and no palpable lymphadenopathy. Axillary radiotherapy results in significantly less morbidity.EORTC Charitable Trust.Copyright © 2014 Elsevier Ltd. All rights reserved.