Tumor-associated Tertiary Lymphoid Structures Research Articles

Tertiary lymphoid structures in ovarian cancer

Ovarian cancer (OC) is a significant cause of cancer-related mortality in women worldwide. Despite advances in treatment modalities, including surgery and chemotherapy, the overall prognosis for OC patients remains poor, particularly for patients with advanced or recurrent disease. Immunotherapy, particularly immune checkpoint blockade (ICB), has revolutionized cancer treatment in various malignancies but has shown limited efficacy in treating OC, which is primarily attributed to the immunologically. Tertiary lymphoid structures (TLSs), which are ectopic aggregates of immune cells, have emerged as potential mediators of antitumor immunity. This review explores the composition, formation, and induction of tumor associated TLS (TA-TLS) in OC, along with their role and therapeutic implications in disease development and treatment. By elucidating the roles TA-TLSs and their cellular compositions played in OC microenvironment, novel therapeutic targets may be identified to overcome immune suppression and enhance immunotherapy efficacy in ovarian cancer.

Abstract 4220: Combined light sheet and multispectral imaging facilitates the detection of tumor-associated tertiary lymphoid structures in the KP NSCLC model

Abstract Tertiary lymphoid structures (TLS) have recently emerged as a predictive biomarker in cancer immunotherapy. Their consideration in preclinical drug development is impeded by rare and transient appearance and random distribution, making them difficult to analyze with conventional sampling methods. We applied whole-organ tissue clearing and 3D staining of B and T cell clusters to comprehensively identify TLS in a KP GEMM of NSCLC using 3D Light Sheet Fluorescence Microscopy (3D LSFM). To further investigate cellular composition of TLS we performed 3D LSFM-educated sectioning of relevant ROIs and cyclic immunofluorescence of the same samples. 3D LSFM facilitated correlation of tumor features and location with T cell infiltration as well as the analysis of number, size and location of TLS. The majority of TLS were located in proximity to bronchi instead of intratumorally. LSFM-guided multiplex immunofluorescence revealed further details on cellular composition and maturation status of TLS. This combination of 3D and 2D imaging modalities offers a holistic approach for the identification of TLS in whole murine tumors or organs. The technology could help studying effects of immunotherapy on de novo formation or maturation of TLS. Citation Format: Nils O'Brien, Birte Appelt, Marie-Lena Moerbitz, Joerg P. Mueller, Frank Herting, Pablo Umaña, Claudio Murgia, Sara Colombetti, Thomas Pöschinger. Combined light sheet and multispectral imaging facilitates the detection of tumor-associated tertiary lymphoid structures in the KP NSCLC model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4220.

Exploiting Tertiary Lymphoid Structures to Stimulate Antitumor Immunity and Improve Immunotherapy Efficacy.

Tumor-associated tertiary lymphoid structures (TLS) have been associated with favorable clinical outcomes and response to immune checkpoint inhibitors in many cancer types, including non-small cell lung cancer. Although the detailed cellular and molecular mechanisms underlying these clinical associations have not been fully elucidated, growing preclinical and clinical studies are helping to elucidate the mechanisms at the basis of TLS formation, composition, and regulation of immune responses. However, a major challenge remains how to exploit TLS to enhance naïve and treatment-mediated antitumor immune responses. Here, we discuss the current understanding of tumor-associated TLS, preclinical models that can be used to study them, and potential therapeutic interventions to boost TLS formation, with a particular focus on lung cancer research.

Detection and quantitative analysis of tumor-associated tertiary lymphoid structures.

Tumor-associated tertiary lymphoid structures (TLSs) are ectopic lymphoid formations within tumor tissue, with mainly B and T cell populations forming the organic aggregates. The presence of TLSs in tumors has been strongly associated with patient responsiveness to immunotherapy regimens and improving tumor prognosis. Researchers have been motivated to actively explore TLSs due to their bright clinical application prospects. Various studies have attempted to decipher TLSs regarding their formation mechanism, structural composition, induction generation, predictive markers, and clinical utilization. Meanwhile, the scientific approaches to qualitative and quantitative descriptions are crucial for TLS studies. In terms of detection, hematoxylin and eosin (H&E), multiplex immunohistochemistry (mIHC), multiplex immunofluorescence (mIF), and 12-chemokine gene signature have been the top approved methods. However, no standard methods exist for the quantitative analysis of TLSs, such as absolute TLS count, analysis of TLS constituent cells, structural features, TLS spatial location, density, and maturity. This study reviews the latest research progress on TLS detection and quantification, proposes new directions for TLS assessment, and addresses issues for the quantitative application of TLSs in the clinic.

Turning Tertiary Lymphoid Structures (TLS) into Hot Spots: Values of TLS in Gastrointestinal Tumors.

Tertiary lymphoid structures (TLSs) are ectopic lymphocyte aggregation structures found in the tumor microenvironment (TME). Emerging evidence shows that TLSs are significantly correlated with the progression of gastrointestinal tumors, patients' prognosis, and the efficacy of adjuvant therapy. Besides, there are still some immunosuppressive factors in the TLSs that may affect the anti-tumor responses of TLSs, including negative regulators of anti-tumor immune responses, the immune checkpoint molecules, and inappropriate tumor metabolism. Therefore, a more comprehensive understanding of TLSs' responses in gastrointestinal tumors is essential to fully understand how TLSs can fully exert their anti-tumor responses. In addition, targeting TLSs with immune checkpoint inhibitors and vaccines to establish mature TLSs is currently being developed to reprogram the TME, further benefiting cancer immunotherapies. This review summarizes recent findings on the formation of TLSs, the mechanisms of their anti-tumor immune responses, and the association between therapeutic strategies and TLSs, providing a novel perspective on tumor-associated TLSs in gastrointestinal tumors.

S1842 Tertiary Lymphoid Structure Mimicking Pancreatic Mass

Introduction: Tertiary lymphoid structures (TLS) are defined as non-congenital ectopic lymphoid tissues that can be found in non-lymphoid organs and form in response to chronic inflammation. TLS’s etiology as well as it’s physiological and pathological roles remain a highly debated topic. Case Description/Methods: An asymptomatic 50-year-old obese diabetic female with a history of biopsy proven cirrhosis secondary to non-alcoholic fatty liver disease (NAFLD) presented to the clinic after a routine hepatocellular carcinoma (HCC) surveillance MRI. A 1.3 cm enhancing nodule at the neck of pancreas was noted. Due to concern for pancreatic cancer an endoscopic ultrasound with fine needle biopsy (EUS-FNB) was planned. During EUS an 11.4 mm by 8.5mm oval mass with irregular margins was identified in the pancreatic body. Fine needle biopsy with 22-gauge needle was performed using trans gastric approach. An enlarged lymph node was identified in peripancreatic region measuring 24.2 mm by 10.9 mm, an additional fine needle biopsy was obtained. Pathology of pancreatic mass and lymph node later revealed fragments of lymphoid tissue consistent with reactive lymph node with single cluster of cytologically mildly atypical cells however no evidence of metastatic malignancy was appreciated. Immunohistochemical staining was positive for CD3 and CD20. CD56, chromogranin, synaptophysin, CD10, e-cadherin and AMACR stains were negative. Flow cytometry analysis was ordered. It demonstrated no evidence of a lymphoproliferative disorder. A three-month post procedure MRI was obtained which demonstrated a 1.6 cm nodule at neck of the pancreas increased in size when compared to prior image. (Figure) Discussion: In immunology, TLS are a highly debated topic as to whether they serve as mediators of protective or pathologic immune responses in certain chronic inflammatory diseases and in the regulation of immune responses. When associated with both primary and or metastatic tumors they’re known as tumor-associated TLS (TA-TLS). In cancer their presence is associated with prolonged increased rates of disease-free survival. When identified flow cytometry should always be ordered in order to rule out a lymphoproliferative disorder. TLS and its implications are still an area of active study, clear guidelines in terms of management are still being developed. Furthermore as to whether they represent premalignant lesion is still unknown. Our patient remains asymptomatic and continues to be monitored with serial imaging.Figure 1.: A. Magnification 200x. Small fragment of benign pancreatic acinar cells near lymphoid cluster. B. Magnification 100x. Aggregates of lymphoid tissue with intervening capillaries representing lymph node like structure within pancreas. C. MRI Abdomen with contrast showing 1.3 cm enhancing nodule at neck of pancreas. D. EUS. Fine needle aspiration of 11.4 mm by 8.5 mm intrapancreatic mass.

Tumor necrosis factor receptor regulation of peripheral node addressin biosynthetic components in tumor endothelial cells.

Tumor-associated tertiary lymphoid structures are ectopic lymphoid aggregates that have considerable morphological, cellular, and molecular similarity to secondary lymphoid organs, particularly lymph nodes. Tumor vessels expressing peripheral node addressin (PNAd) are hallmark features of these structures. Previous work from our laboratory demonstrated that PNAd is displayed on intratumoral vasculature of murine tumors, and its expression is controlled by the engagement of lymphotoxin-α3, secreted by effector CD8 T cells, with tumor necrosis factor receptors (TNFR) on tumor endothelial cells (TEC). The goals of the present work were: 1) to identify differences in expression of genes encoding the scaffolding proteins and glycosyl transferases associated with PNAd biosynthesis in TEC and lymph node blood endothelial cells (LN BEC); and 2) to determine which of these PNAd associated components are regulated by TNFR signaling. We found that the same genes encoding scaffolding proteins and glycosyl transferases were upregulated in PNAd+ LN BEC and PNAd+ TEC relative to their PNAdneg counterparts. The lower level of PNAd expression on TEC vs LN BEC was associated with relatively lower expression of these genes, particularly the carbohydrate sulfotransferase Chst4. Loss of PNAd on TEC in the absence of TNFR signaling was associated with lack of upregulation of these same genes. A small subset of PNAd+ TEC remaining in the absence of TNFR signaling showed normal upregulation of a subset of these genes, but reduced upregulation of genes encoding the scaffolding proteins podocalyxin and nepmucin, and carbohydrate sulfotransferase Chst2. Lastly, we found that checkpoint immunotherapy augmented both the fraction of TEC expressing PNAd and their surface level of this ligand. This work points to strong similarities in the regulation of PNAd expression on TEC by TNFR signaling and on LN BEC by lymphotoxin-β receptor signaling, and provides a platform for the development of novel strategies that manipulate PNAd expression on tumor vasculature as an element of cancer immunotherapy.

Stromal and Immune Cell Dynamics in Tumor Associated Tertiary Lymphoid Structures and Anti-Tumor Immune Responses.

Tertiary lymphoid structures (TLS) are ectopic lymphoid organs that have been observed in chronic inflammatory conditions including cancer, where they are thought to exert a positive effect on prognosis. Both immune and non-immune cells participate in the genesis of TLS by establishing complex cross-talks requiring both soluble factors and cell-to-cell contact. Several immune cell types, including T follicular helper cells (Tfh), regulatory T cells (Tregs), and myeloid cells, may accumulate in TLS, possibly promoting or inhibiting their development. In this manuscript, we propose to review the available evidence regarding specific aspects of the TLS formation in solid cancers, including 1) the role of stromal cell composition and architecture in the recruitment of specific immune subpopulations and the formation of immune cell aggregates; 2) the contribution of the myeloid compartment (macrophages and neutrophils) to the development of antibody responses and the TLS formation; 3) the immunological and metabolic mechanisms dictating recruitment, expansion and plasticity of Tregs into T follicular regulatory cells, which are potentially sensitive to immunotherapeutic strategies directed to costimulatory receptors or checkpoint molecules.

Functional Th1-oriented T follicular helper cells that infiltrate human breast cancer promote effective adaptive immunity.

We previously demonstrated that tumor-infiltrating lymphocytes (TIL) in human breast cancer sometimes form organized tertiary lymphoid structures (TLS) characterized by CXCL13-producing T follicular helper (Tfh) cells. The present study found that CD4+ Tfh TIL, CD8+ TIL, and TIL-B, colocalizing in TLS, all express the CXCL13 receptor CXCR5. An ex vivo functional assay determined that only activated, functional Th1-oriented Tfh TIL (PD-1hiICOSint phenotype) provide help for immunoglobulin and IFN-γ production. A functional Tfh TIL presence signals an active TLS, characterized by humoral (immunoglobulins, Ki-67+ TIL-B in active germinal centers) and cytotoxic (GZMB+CD8+ and GZMB+CD68+ TIL plus Th1 gene expression) immune responses. Analysis of active versus inactive TLS in untreated patients revealed that the former are associated with positive clinical outcomes. TLS also contain functional T follicular regulatory (Tfr) TIL, which are characterized by a CD25+CXCR5+GARP+FOXP3+ phenotype and a demethylated FOXP3 gene. Functional Tfr inhibited functional Tfh activities via a glycoprotein A repetitions predominant (GARP)-associated TGF-β-dependent mechanism. The activity of tumor-associated TLS was dictated by the relative balance between functional Tfh TIL and functional Tfr TIL. These data provide mechanistic insight into TLS processes orchestrated by functional Th1-oriented Tfh TIL, including TIL-B and CD8+ TIL activation and immunological memory generation. Tfh TIL, regulated by functional Tfr TIL, are an expected key target of PD-1/PD-L1 blockade.

A Sampling of Highlights from the Literature: Article Recommendations from Our Deputy and Senior Editors.

Modulating RNA splicing can augment antitumor T-cell responses (by Srosenbe via Wikimedia Commons)Altered RNA splicing in cancer cells can give rise to neoantigens, but it is unclear whether this is clinically relevant and therapeutically targetable. Using B16-F10 and MC38 tumor models, Lu et al. find several compounds that modulate RNA splicing can inhibit tumor growth and improve responses to anti-PD1 checkpoint blockade. These effects are dependent on T cells and MHC class I peptide presentation. Neoepitopes induced by pharmacologic modulation of RNA splicing in B16-F10 cells can trigger antitumor T-cell responses in mice. The data suggest potential new approaches to enhancing cancer immunotherapy.Lu SX, …, Bradley RK. Cell 2021 Jul 22;184:4032–47.e31.Arming CAR T cells with BATF enhances efficacy (from Spezadams via Wikimedia Commons)The transcriptional networks underlying T-cell exhaustion, including exhaustion in CAR T cells, are not fully known. Seo et al. show that basic leucine zipper ATF-like transcription factor (BATF) and interferon regulatory factor 4 (IRF4) act in a cooperative manner to offset T-cell exhaustion. Overexpression of BATF in CAR T cells increases cell persistence and cytokine production and decreases expression of inhibitory receptors and exhaustion-related markers. These effects are dependent on interactions between BATF and IRF4. The data highlight potential therapeutic targets to improve CAR T-cell therapy.Seo H, …, Hogan PG. Nat Immunol 2021 Aug 1;22:983–95.TILs with differing specificities have different functional states (from Brandon Dilbeck via Wikimedia Commons)Understanding of the relationship between the functional state of tumor-infiltrating lymphocytes (TIL) and the specificity of their TCRs is limited, but advances in single-cell technology are changing this. Using such technologies, Caushi et al. show that non–small cell lung cancer TILs with TCRs specific for mutation-associated neoantigens (MANA) have a unique transcriptional profile that is largely characteristic of tissue-resident memory cells and that MANA-specific T cells from tumors nonresponsive to anti-PD1 express significantly reduced levels of genes associated with T-cell dysfunction. Oliveira et al. show that melanoma-reactive TILs are predominantly in an exhausted state and only rarely acquire memory properties, and that disease persistence correlates with persistence of exhausted melanoma-reactive cells in peripheral blood. These data will inform further development of cancer immunotherapy.Caushi JX, …, Smith KN. Nature 2021 Aug 5;596:126–32.Oliveira G, …, Wu CJ. Nature 2021 Aug 5;596:119–25.A targeted radionuclide and checkpoint inhibition combination enhances antitumor immunity (by MichaelFrey via Wikimedia Commons)External beam radiotherapy enhances responses to immune checkpoint blockade (ICB) in preclinical models. Patel et al. show that a targeted radionuclide therapy comprising the alkylphosphocholine analogue NM600 chelated to yittrium-90 (90Y) enhances responses to ICB in mouse models of melanoma, neuroblastoma, and breast cancer without causing toxicity to normal tissues. The 90Y-NM600 plus ICB combination increases immune-cell infiltration into the tumor, and production of proinflammatory cytokines and clonal expansion of CD8+ T cells in the tumor, highlighting the immunologic mechanisms behind this potential new therapeutic combination.Patel RB, …, Morris ZS. Sci Transl Med 2021 Jul 14;13:eabb3631.TFR cells can suppress antitumor responses induced by ICB (from Fig. 1C of Rodriguez and Engelhard, Cancer Immunol Res 2020)The mechanisms behind the effects of immune checkpoint blockade (ICB) are not completely understood. Eschweiler et al. show that follicular regulatory T (TFR) cells are present in tumor-associated tertiary lymphoid structures (TLS) and that they are more suppressive than regulatory T cells. Anti-PD1 can increase the number of TFR cells in tumors, and administration of anti-CTLA4 to deplete regulatory cells prior to anti-PD1 improves treatment efficacy in mice and humans.Eschweiler S, …, Vijayanand P. Nat Immunol 2021 Aug 1;22:1052–63.β2M can accumulate in the tumor microenvironment (β2M staining, from Fig. 1A of Roemer et al., Cancer Immunol Res 2016)β2-microglobulin (β2M) concentrations increase during progression of multiple myeloma (MM), but β2M's role in disease is not well-known. Hofbauer et al. find that MM-associated macrophages can phagocytose β2M, which then leads to NLRP3-mediated inflammasome activation. The resulting cytokine secretion and inflammation contribute to MM severity. Inhibition of the inflammasome or related cytokines ameliorates disease in a mouse model of MM, thus highlighting potential therapeutic targets for the treatment of MM.Hofbauer D, …, Bruns H. Immunity 2021 Jul 20. DOI: 10.1016/j.immuni.2021.07.002.

Tertiary Lymphoid Structures in the Central Nervous System: Implications for Glioblastoma.

Glioblastoma is the most common and aggressive brain tumor, which is uniformly lethal due to its extreme invasiveness and the absence of curative therapies. Immune checkpoint inhibitors have not yet proven efficacious for glioblastoma patients, due in part to the low prevalence of tumor-reactive T cells within the tumor microenvironment. The priming of tumor antigen-directed T cells in the cervical lymph nodes is complicated by the shortage of dendritic cells and lack of appropriate lymphatic vessels within the brain parenchyma. However, recent data suggest that naive T cells may also be primed within brain tumor-associated tertiary lymphoid structures. Here, we review the current understanding of the formation of these structures within the central nervous system, and hypothesize that promotion of tertiary lymphoid structures could enhance priming of tumor antigen-targeted T cells and sensitize glioblastomas to cancer immunotherapy.

Tertiary Lymphoid Structures in Cancer: The Double-Edged Sword Role in Antitumor Immunity and Potential Therapeutic Induction Strategies.

The complex tumor microenvironment (TME) plays a vital role in cancer development and dramatically determines the efficacy of immunotherapy. Tertiary lymphoid structures (TLSs) within the TME are well recognized and consist of T cell-rich areas containing dendritic cells (DCs) and B cell-rich areas containing germinal centers (GCs). Accumulating research has indicated that there is a close association between tumor-associated TLSs and favorable clinical outcomes in most types of cancers, though a minority of studies have reported an association between TLSs and a poor prognosis. Overall, the double-edged sword role of TLSs in the TME and potential mechanisms need to be further investigated, which will provide novel therapeutic perspectives for antitumor immunoregulation. In this review, we focus on discussing the main functions of TLSs in the TME and recent advances in the therapeutic manipulation of TLSs through multiple strategies to enhance local antitumor immunity.

Immune mechanisms orchestrate tertiary lymphoid structures in tumors via cancer-associated fibroblasts.

SUMMARYTumor-associated tertiary lymphoid structures (TA-TLS) are associated with enhanced patient survival and responsiveness to cancer therapies, but the mechanisms underlying their development are unknown. We show here that TA-TLS development in murine melanoma is orchestrated by cancer-associated fibroblasts (CAF) with characteristics of lymphoid tissue organizer cells that are induced by tumor necrosis factor receptor signaling. CAF organization into reticular networks is mediated by CD8 T cells, while CAF accumulation and TA-TLS expansion depend on CXCL13-mediated recruitment of B cells expressing lymphotoxin-α1β2. Some of these elements are also overrepresented in human TA-TLS. Additionally, we demonstrate that immunotherapy induces more and larger TA-TLS that are more often organized with discrete T and B cell zones, and that TA-TLS presence, number, and size are correlated with reduced tumor size and overall response to checkpoint immunotherapy. This work provides a platform for manipulating TA-TLS development as a cancer immunotherapy strategy.

PD-1+CXCR5−CD4+ Th-CXCL13 cell subset drives B cells into tertiary lymphoid structures of nasopharyngeal carcinoma

BackgroundA major current challenge is to exploit tertiary lymphoid structures (TLSs) to promote the lymphocyte infiltration, activation and differentiation by tumor antigens to increase antitumor immune responses. The mechanisms that...

Tumor-Associated Tertiary Lymphoid Structures: From Basic and Clinical Knowledge to Therapeutic Manipulation.

The tumor microenvironment is a complex ecosystem almost unique to each patient. Most of available therapies target tumor cells according to their molecular characteristics, angiogenesis or immune cells involved in tumor immune-surveillance. Unfortunately, only a limited number of patients benefit in the long-term of these treatments that are often associated with relapses, in spite of the remarkable progress obtained with the advent of immune checkpoint inhibitors (ICP). The presence of “hot” tumors is a determining parameter for selecting therapies targeting the patient immunity, even though some of them still do not respond to treatment. In human studies, an in-depth analysis of the organization and interactions of tumor-infiltrating immune cells has revealed the presence of an ectopic lymphoid organization termed tertiary lymphoid structures (TLS) in a large number of tumors. Their marked similarity to secondary lymphoid organs has suggested that TLS are an “anti-tumor school” and an “antibody factory” to fight malignant cells. They are effectively associated with long-term survival in most solid tumors, and their presence has been recently shown to predict response to ICP inhibitors. This review discusses the relationship between TLS and the molecular characteristics of tumors and the presence of oncogenic viruses, as well as their role when targeted therapies are used. Also, we present some aspects of TLS biology in non-tumor inflammatory diseases and discuss the putative common characteristics that they share with tumor-associated TLS. A detailed overview of the different pre-clinical models available to investigate TLS function and neogenesis is also presented. Finally, new approaches aimed at a better understanding of the role and function of TLS such as the use of spheroids and organoids and of artificial intelligence algorithms, are also discussed. In conclusion, increasing our knowledge on TLS will undoubtedly improve prognostic prediction and treatment selection in cancer patients with key consequences for the next generation immunotherapy.

B Cells in Patients With Melanoma: Implications for Treatment With Checkpoint Inhibitor Antibodies.

The contributions of the humoral immune response to melanoma are now widely recognized, with reports of positive prognostic value ascribed to tumor-infiltrating B cells (TIL-B) and increasing evidence of B cells as key predictors of patient response to treatment. There are disparate views as to the pro- and anti-tumor roles of B cells. B cells appear to play an integral role in forming tumor-associated tertiary lymphoid structures (TLSs) which can further modulate T cell activation. Expressed antibodies may distinctly influence tumor regulation in the tumor microenvironment, with some isotypes associated with strong anti-tumor immune response and others with progressive disease. Recently, B cells have been evaluated in the context of cancer immunotherapy. Checkpoint inhibitors (CPIs), targeting T cell effector functions, have revolutionized the management of melanoma for many patients; however, there remains a need to accurately predict treatment responders. Increasing evidence suggests that B cells may not be simple bystanders to CPI immunotherapy. Mature and differentiated B cell phenotypes are key positive correlates of CPI response. Recent evidence also points to an enrichment in activatory B cell phenotypes, and the contribution of B cells to TLS formation may facilitate induction of T cell phenotypes required for response to CPI. Contrastingly, specific B cell subsets often correlate with immune-related adverse events (irAEs) in CPI. With increased appreciation of the multifaceted role of B cell immunity, novel therapeutic strategies and biomarkers can be explored and translated into the clinic to optimize CPI immunotherapy in melanoma.

Tumor-Associated Tertiary Lymphoid Structures: A Cancer Biomarker and a Target for Next-generation Immunotherapy.

The different forms of lymphoid organization that coexist in our bodies appeared at distinct time points during the evolution of the animal kingdom. Some of these forms are constitutive, either in fully dedicated organs, such as lymph nodes, or in tissue interfacing with the external environment, such as mucosal-associated lymphoid tissues. Others, known as tertiary lymphoid structures (TLS), are selectively induced in response to inflammation in any peripheral tissues and organs. In this chapter, we discuss the functional interest of each of these lymphoid organizations under different physiopathological conditions. In the context of cancer, recent findings have identified TLS formation as a hallmark of active T- and B-cell immune responses against tumors. TLS are thus a powerful prognostic factor in nearly all solid cancers, which must be taken into account along with the tumor microenvironment. The presence of TLS also predicts the response to immunotherapy including immune checkpoint blockade. With tumor-associated TLS now a key target for the next generation of immunotherapy, this chapter discusses their potential therapeutic manipulations in oncology.

Insights into Tumor-Associated Tertiary Lymphoid Structures: Novel Targets for Antitumor Immunity and Cancer Immunotherapy.

Tertiary lymphoid structures (TLS) are ectopic lymphoid aggregates that phenotypically resemble conventional secondary lymphoid organs and are commonly found at sites of chronic inflammation. They are also found in a wide variety of primary and metastatic human tumors. The presence of tumor-associated TLS (TA-TLS) is associated with prolonged patient survival, higher rates of disease-free survival, and a favorable response to current cancer therapies. However, the immune responses that occur in these structures, and how they contribute to improved clinical outcomes, remain incompletely understood. In addition, it is unknown how heterogeneity in TA-TLS cellular composition, structural organization, and anatomic location influences their functionality and prognostic significance. Understanding more about TA-TLS development, formation, and function may offer new therapeutic options to modulate antitumor immunity.

Human NKp44+ Group 3 Innate Lymphoid Cells Associate with Tumor-Associated Tertiary Lymphoid Structures in Colorectal Cancer.

Innate lymphoid cells (ILC) are responsible for mucosal tissue homeostasis and are involved in the progression and suppression of several types of cancer. However, the effects of ILCs on colorectal cancer are poorly understood. We characterized human ILCs in normal colon and colorectal cancer tissue, investigating their role in the tumor immune microenvironment. Normal mucosa and tumor tissues were obtained from patients with colorectal cancer, and the cells were isolated by enzymatic digestion. NKp44+ ILC3s with high expression of tertiary lymphoid structure (TLS) formation-related genes, including LTA, LTB, and TNF, accumulated in the normal colonic mucosa and T1/T2 tumors. However, the number of NKp44+ ILC3s was significantly reduced in T3/T4 tumors compared with normal colonic mucosa and T1/T2 tumors. NKp44+ ILC3s present in T3/T4 tumors had decreased expression of TLS formation-related genes, whereas stromal cells had decreased expression of CXCL13, CCL19, and CCL21 The decreasing number of NKp44+ ILC3s during tumor progression correlated with the TLS density in tumors. Thus, our results indicate that NKp44+ ILC3s infiltrate colorectal cancer tissue, but the number of cells decreases in T3/T4 tumors with associated decreases in TLS induction.

Therapeutic vascular normalization to promote tumor-associated tertiary lymphoid structures

Abstract Tertiary lymphoid structures (TLS) are non-encapsulated immune cell aggregates that form at sites of chronic inflammation. Recent studies have shown that the presence of TLS in human tumors indicates positive clinical outcome. However, many tumors have poorly organized and leaky vasculature that impedes entry of immune effector cells into tumors and consequently TLS formation. Recently, studies have shown that low doses of antiangiogenic agents normalize tumor vasculature, leading us to hypothesize that treating tumors with low doses of vascular normalizing (VN) therapies will improve immune cell infiltration and TLS formation within the tumor microenvironment (TME). To test this hypothesis, tumor-bearing mice were treated intratumorally with VN agents. RNA was isolated from digested tumors and transcript levels of TLS-promoting factors and markers of inflammation/immune cell infiltration were measured and compared to PBS treated controls. Changes in tumor vasculature were evaluated using immunofluorescence microscopy. Additionally, primary cultures of murine T cells and DCs and the BPR melanoma cell line were treated in vitro with VN agents. We observed that the VN agents dasatinib, STING agonist, bevacizumab, and agonist anti-TNFR1 antibody each induced global changes in the TME that are potentially supportive of immune cell infiltration and TLS formation. In vitro, dasatinib induced DCs and BPR melanoma cells to express higher levels of co-stimulatory receptors, MHC class I and II, and TLS-promoting chemokines. Treatment with a STING agonist was able to enhance T cell activation, while treatment with dasatinib inhibited T cell activation.