Tertiary lymphoid structures in anticancer immunity.

Tertiary lymphoid structures (TLS) are transient ectopic lymphoid aggregates where adaptive antitumour cellular and humoral responses can be elaborated. Initially described in non-small cell lung cancer as functional immune lymphoid structures associated with better clinical outcome, TLS have also been found in many other carcinomas, as well as melanomas and sarcomas, and associated with improved response to immunotherapy. The manipulation of TLS as a therapeutic strategy is now coming of age owing to the likely role of TLS in the improved survival of patients with cancer receiving immune checkpoint inhibitor treatment. TLS have also garnered considerable interest as a predictive biomarker of the response to antitumour therapies, including immune checkpoint blockade and, possibly, chemotherapy. However, several important questions still remain regarding the definition of TLS in terms of both their cellular composition and functions. Here, we summarize the current views on the composition of TLS at different stages of their development. We also discuss the role of B cells and T cells associated with TLS and their dialogue in mounting antibody and cellular antitumour responses, as well as some of the various mechanisms that negatively regulate antitumour activity of TLS. The prognostic value of TLS to the clinical outcome of patients with cancer and the relationship between TLS and the response to therapy are then addressed. Finally, we present some preclinical evidence that favours the idea that manipulating the formation and function of TLS could lead to a potent next-generation cancer immunotherapy.

HEV-associated dendritic cells are observed in metastatic tumor-draining lymph nodes of cutaneous melanoma patients with longer distant metastasis-free survival after adjuvant immunotherapy.

Introduction: Tissue biomarkers that aid in identifying cutaneous melanoma (CM) patients who will benefit from adjuvant immunotherapy are of crucial interest. Metastatic tumor-draining lymph nodes (mTDLN) are the first encounter site between the metastatic CM cells and an organized immune structure. Therefore, their study may reveal mechanisms that could influence patients´ outcomes. Methods: Twenty-nine stage-III CM patients enrolled in clinical trials to study the vaccine VACCIMEL were included in this retrospective study. After radical mTDLN dissection, patients were treated with VACCIMEL (n=22) or IFNα-2b (n=6), unless rapid progression (n=1). Distant Metastasis-Free Survival (DMFS) was selected as an end-point. Two cohorts of patients were selected: one with a good outcome (GO) (n=17; median DMFS 130.0 months), and another with a bad outcome (BO) (n=12; median DMFS 8.5 months). We analyzed by immunohistochemistry and immunofluorescence the expression of relevant biomarkers to tumor-cell biology and immune cells and structures in mTDLN, both in the tumor and peritumoral areas. Results: In BO patients, highly replicating Ki-67+ tumor cells, low tumor HLA-I expression and abundant FoxP3+ lymphocytes were found (p=0.037; p=0.056 and p=0.021). In GO patients, the most favorable biomarkers for prolonged DMFS were the abundance of peri- and intra-tumoral CD11c+ cells (p=0.0002 and p=0.001), peri-tumoral DC-LAMP+ dendritic cells (DCs) (p=0.001), and PNAd+ High Endothelial Venules (HEVs) (p=0.004). Most strikingly, we describe in GO patients a peculiar, heterogeneous structure that we named FAPS (Favoring Antigen-Presenting Structure), a triad composed of DC, HEV and CD62L+ naïve lymphocytes, whose postulated role would be to favor tumor antigen (Ag) priming of incoming naïve lymphocytes. We also found in GO patients a preferential tumor infiltration of CD8+ and CD20+ lymphocytes (p=0.004 and p=0.027), as well as peritumoral CD20+ aggregates, with no CD21+ follicular dendritic cells detected (p=0.023). Heterogeneous infiltration with CD64+CD68-CD163-, CD64+CD68+CD163- and CD64+CD68+CD163+ macrophages were observed in both cohorts. Discussion: The analysis of mTDLN in GO and BO patients revealed marked differences. This work highlights the importance of analyzing resected mTDLN from CM patients and suggests a correlation between tumor and immune characteristics that may be associated with a spontaneous or vaccine-induced long DMFS. These results should be confirmed in prospective studies.

Regulatory T cells infiltrate the tumor-induced tertiary lymphoïd structures and are associated with poor clinical outcome in NSCLC.

On one hand, regulatory T cells (Tregs) play an immunosuppressive activity in most solid tumors but not all. On the other hand, the organization of tumor-infiltrating immune cells into tertiary lymphoid structures (TLS) is associated with long-term survival in most cancers. Here, we investigated the role of Tregs in the context of Non-Small Cell Lung Cancer (NSCLC)-associated TLS. We observed that Tregs show a similar immune profile in TLS and non-TLS areas. Autologous tumor-infiltrating Tregs inhibit the proliferation and cytokine secretion of CD4+ conventional T cells, a capacity which is recovered by antibodies against Cytotoxic T-Lymphocyte-Associated protein-4 (CTLA-4) and Glucocorticoid-Induced TNFR-Related protein (GITR) but not against other immune checkpoint (ICP) molecules. Tregs in the whole tumor, including in TLS, are associated with a poor outcome of NSCLC patients, and combination with TLS-dendritic cells (DCs) and CD8+ T cells allows higher overall survival discrimination. Thus, Targeting Tregs especially in TLS may represent a major challenge in order to boost anti-tumor immune responses initiated in TLS.

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.