Tertiary lymphoid structures and B cells: An intratumoral immunity cycle.

The generation of anti-tumor immunity in the draining lymph nodes is known as the cancer immunity cycle. Accumulating evidence supports the occurrence of such a cycle at tumor sites in the context of chronic inflammation. Here, we review the role of tertiary lymphoid structures (TLS) in the generation of T and B cell immunities, focusing on the impact of B cells that undergo full maturation, resulting in the generation of plasma cells (PCs) producing high-affinity IgG and IgA antibodies. In this context, we propose that antibodies binding to tumor cells induce macrophage or natural killer (NK)-cell-dependent apoptosis. Subsequently, released antigen-antibody complexes are internalized and processed by dendritic cells (DCs), amplifying antigen presentation to T cells. Immune complexes may also be fixed by follicular DCs (FDCs) in TLS, thereby increasing memory B cell responses. This amplification loop creates an intra-tumoral immunity cycle, capable of increasing sensitivity of tumors to immunotherapy even in cancers with low mutational burden.

Tertiary lymphoid structures generate and propagate anti-tumor antibody-producing plasma cells in renal cell cancer.

The presence of intratumoral tertiary lymphoid structures (TLS) is associated with positive clinical outcomes and responses to immunotherapy in cancer. Here, we used spatial transcriptomics to examine the nature of B cell responses within TLS in renal cell carcinoma (RCC). B cells were enriched in TLS, and therein, we could identify all B cell maturation stages toward plasma cell (PC) formation. B cell repertoire analysis revealed clonal diversification, selection, expansion in TLS, and the presence of fully mature clonotypes at distance. In TLS+ tumors, IgG- and IgA-producing PCs disseminated into the tumor beds along fibroblastic tracks. TLS+ tumors exhibited high frequencies of IgG-producing PCs and IgG-stained and apoptotic malignant cells, suggestive of anti-tumor effector activity. Therapeutic responses and progression-free survival correlated with IgG-stained tumor cells in RCC patients treated with immune checkpoint inhibitors. Thus, intratumoral TLS sustains B cell maturation and antibody production that is associated with response to immunotherapy, potentially via direct anti-tumor effects.

Activation of B cells in Tertiary Lymphoid Structures in cancer: Anti-tumor or anti-self?

Whereas T cells in the tumor microenvironment have been the main focus as cancer controlling cells and targets of immunotherapies, B cells have recently gained strong attention. Being associated to Tertiary Lymphoid Structures (TLS) located at the vicinity of tumor nests, the fate of B cell depends on TLS maturity. In immature TLS they may evolve as regulatory B cells producing immunosuppressive cytokines and promote tumor growth. In mature TLS with a germinal center, B cells are selected, amplified, undergo affinity maturation and isotypic switching, resulting in plasma cell generation and production of anti-tumor antibodies. In that case, they are associated with longer patient's survival and therapeutic response to immunotherapy. Identification of tumor specific, or tumor overexpressed, antigens recognized by "in situ" produced antibodies and their discrimination from self-antigens induced by ICI treatments is a major challenge to develop novel antibody-based immunotherapies.

B cells are associated with survival and immunotherapy response in sarcoma.

Soft-tissue sarcomas represent a heterogeneous group of cancer, with more than 50 histological subtypes1,2. The clinical presentation of patients with different subtypes is often atypical, and responses to therapies such as immune checkpoint blockade vary widely3,4. To explain this clinical variability, here we study gene expression profiles in 608 tumours across subtypes of soft-tissue sarcoma. We establish an immune-based classification on the basis of the composition of the tumour microenvironment and identify five distinct phenotypes: immune-low (A and B), immune-high (D and E), and highly vascularized (C) groups. In situ analysis of an independent validation cohort shows that class E was characterized by the presence of tertiary lymphoid structures that contain T cells and follicular dendritic cells and are particularly rich in B cells. B cells are the strongest prognostic factor even in the context of high or low CD8+ T cells and cytotoxic contents. The class-E group demonstrated improved survival and a high response rate to PD1 blockade with pembrolizumab in a phase 2 clinical trial. Together, this work confirms the immune subtypes in patients with soft-tissue sarcoma, and unravels the potential of B-cell-rich tertiary lymphoid structures to guide clinical decision-making and treatments, which could have broader applications in other diseases.

Impact of metronomic trabectedin combined with low-dose cyclophosphamide on sarcoma microenvironment and correlation with clinical outcome: results from the TARMIC study.

Soft tissue sarcomas (STS) are diverse mesenchymal tumors with few therapeutic options in advanced stages. Trabectedin has global approval for treating STS patients resistant to anthracycline-based regimens. Recent pre-clinical data suggest that trabectedin's antitumor activity extends beyond tumor cells to influencing the tumor microenvironment (TME), especially affecting tumor-associated macrophages and their pro-tumoral functions. We present the phase I/II results evaluating a combination of metronomic trabectedin and low-dose cyclophosphamide on the TME in patients with advanced sarcomas. 50 patients participated: 20 in phase I and 30 in phase II. Changes in the TME were assessed in 28 patients using sequential tumor samples at baseline and day two of the cycle. Treatment notably decreased CD68 + CD163 + macrophages in biopsies from tumor lesions compared to pre-treatment samples in 9 of the 28 patients after 4 weeks. Baseline CD8 + T cell presence increased in 11 of these patients. In summary, up to 57% of patients exhibited a positive immunological response marked by reduced M2 macrophages or increased CD8 + T cells post-treatment. This positive shift in the TME correlated with improved clinical benefit and progression-free survival. This study offers the first prospective evidence of trabectedin's immunological effect in advanced STS patients, highlighting a relationship between TME modulation and patient outcomes.This study was registered with ClinicalTrial.gov, number NCT02406781.

Dendritic cells in the tumor microenvironment: prognostic and theranostic impact.

Among all immune cells, dendritic cells (DC) are the most potent APCs in the immune system and are central players of the adaptive immune response. There are phenotypically and functionally distinct DC populations derived from blood and lymphoid organ including plasmacytoid DC (pDC), conventional DC (cDC1 and cDC2) and monocyte-derived DC (moDC). The interaction between these different DCs and tumors is a dynamic process where DC-mediated cross-priming of tumor specific T cells is critical in initiating and sustaining anti-tumor immunity. Their presence within the tumor tends to induce T cell responses and to reduce cancer progression and is associated with improved patient survival. This review will focus on the distinct tumor-associated DCs (TADC) subsets in the tumor microenvironment (TME), their roles in tumor immunology and their prognostic and/or predictive impact in human cancers. The development of therapeutic immunity strategies targeting TADC is promising to enhance their immune-stimulatory capacity in cancers and improve the efficacy of current immunotherapies including immune checkpoint inhibitor (ICI) blockade and DC-based therapies.

Tertiary Lymphoid Structures and B cells: Clinical impact and therapeutic modulation in cancer.

Tumors progression is under the control of a heterogeneous microenvironment composed of immune cells, fibroblasts, blood and lymphatic vessels, in which T cells have been demonstrated to be major actors, through their cytotoxic and cytokine producing effector functions and their long term memory that protects against metastasis. In this scenario, lessons from mouse models taught that B cells exert a protumoral role, via macrophage-dependent activation of inflammation. However, it became progressively evident from studies in patients with human cancers that the anti-tumor responses can be generated and controlled in tertiary lymphoid structures (TLS) that concentrate most of the intratumoral B cells and where B cells can differentiate into plasma cells and memory cells. Furthermore, recent studies demonstrated that the presence in tumors of B cells and TLS are associated with favorable outcome in patients treated by immunotherapy, unraveling TLS as a new predictive marker of anti-tumor response human cancers. This review encompasses the characteristics and functions of TLS and of B cells in human tumors, their prognostic and theranostic impact and summarizes the mouse models used to induce TLS neogenesis in tumors.

Nivolumab, nivolumab-ipilimumab, and VEGFR-tyrosine kinase inhibitors as first-line treatment for metastatic clear-cell renal cell carcinoma (BIONIKK): a biomarker-driven, open-label, non-comparative, randomised, phase 2 trial.

BACKGROUND: We previously reported a 35-gene expression classifier identifying four clear-cell renal cell carcinoma groups (ccrcc1 to ccrcc4) with different tumour microenvironments and sensitivities to sunitinib in metastatic clear-cell renal cell carcinoma. Efficacy profiles might differ with nivolumab and nivolumab-ipilimumab. We therefore aimed to evaluate treatment efficacy and tolerability of nivolumab, nivolumab-ipilimumab, and VEGFR-tyrosine kinase inhibitors (VEGFR-TKIs) in patients according to tumour molecular groups. METHODS: This biomarker-driven, open-label, non-comparative, randomised, phase 2 trial included patients from 15 university hospitals or expert cancer centres in France. Eligible patients were aged 18 years or older, had an Eastern Cooperative Oncology Group performance status of 0-2, and had previously untreated metastatic clear-cell renal cell carcinoma. Patients were randomly assigned (1:1) using permuted blocks of varying sizes to receive either nivolumab or nivolumab-ipilimumab (ccrcc1 and ccrcc4 groups), or either a VEGFR-TKI or nivolumab-ipilimumab (ccrcc2 and ccrcc3 groups). Patients assigned to nivolumab-ipilimumab received intravenous nivolumab 3 mg/kg plus ipilimumab 1 mg/kg every 3 weeks for four doses followed by intravenous nivolumab 240 mg every 2 weeks. Patients assigned to nivolumab received intravenous nivolumab 240 mg every 2 weeks. Patients assigned to VEGFR-TKIs received oral sunitinib (50 mg/day for 4 weeks every 6 weeks) or oral pazopanib (800 mg daily continuously). The primary endpoint was the objective response rate by investigator assessment per Response Evaluation Criteria in Solid Tumors version 1.1. The primary endpoint and safety were assessed in the population who received at least one dose of study drug. This trial is registered with ClinicalTrials.gov, NCT02960906, and with the EU Clinical Trials Register, EudraCT 2016-003099-28, and is closed to enrolment. FINDINGS: Between June 28, 2017, and July 18, 2019, 303 patients were screened for eligibility, 202 of whom were randomly assigned to treatment (61 to nivolumab, 101 to nivolumab-ipilimumab, 40 to a VEGFR-TKI). In the nivolumab group, two patients were excluded due to a serious adverse event before the first study dose and one patient was excluded from analyses due to incorrect diagnosis. Median follow-up was 18·0 months (IQR 17·6-18·4). In the ccrcc1 group, objective responses were seen in 12 (29%; 95% CI 16-45) of 42 patients with nivolumab and 16 (39%; 24-55) of 41 patients with nivolumab-ipilimumab (odds ratio [OR] 0·63 [95% CI 0·25-1·56]). In the ccrcc4 group, objective responses were seen in seven (44%; 95% CI 20-70) of 16 patients with nivolumab and nine (50% 26-74) of 18 patients with nivolumab-ipilimumab (OR 0·78 [95% CI 0·20-3·01]). In the ccrcc2 group, objective responses were seen in 18 (50%; 95% CI 33-67) of 36 patients with a VEGFR-TKI and 19 (51%; 34-68) of 37 patients with nivolumab-ipilimumab (OR 0·95 [95% CI 0·38-2·37]). In the ccrcc3 group, no objective responses were seen in the four patients who received a VEGFR-TKI, and in one (20%; 95% CI 1-72) of five patients who received nivolumab-ipilimumab. The most common treatment-related grade 3-4 adverse events were hepatic failure and lipase increase (two [3%] of 58 for both) with nivolumab, lipase increase and hepatobiliary disorders (six [6%] of 101 for both) with nivolumab-ipilimumab, and hypertension (six [15%] of 40) with a VEGFR-TKI. Serious treatment-related adverse events occurred in two (3%) patients in the nivolumab group, 38 (38%) in the nivolumab-ipilimumab group, and ten (25%) patients in the VEGFR-TKI group. Three deaths were treatment-related: one due to fulminant hepatitis with nivolumab-ipilimumab, one death from heart failure with sunitinib, and one due to thrombotic microangiopathy with sunitinib. INTERPRETATION: We demonstrate the feasibility and positive effect of a prospective patient selection based on tumour molecular phenotype to choose the most efficacious treatment between nivolumab with or without ipilimumab and a VEGFR-TKI in the first-line treatment of metastatic clear-cell renal cell carcinoma. FUNDING: Bristol Myers Squibb, ARTIC.

Immune-Desert Tumor Microenvironment in Thoracic SMARCA4-Deficient Undifferentiated Tumors with Limited Efficacy of Immune Checkpoint Inhibitors.

BACKGROUND: Thoracic SMARCA4-deficient undifferentiated tumors (SMARCA4-UT) are aggressive neoplasms. Data linking BAF alterations with tumor microenvironment (TME) and efficacy of immune checkpoint inhibitors (ICI) are contradictory. The TME of SMARCA4-UT and their response to ICI are unknown. MATERIALS AND METHODS: Patients diagnosed with SMARCA4-UT in our institution were included. Immunostainings for tertiary lymphoid structures (TLS), immune cell markers, and checkpoints were assessed. Validation was performed using an independent transcriptome dataset including SMARCA4-UT, non-small cell lung cancers (NSCLC) with/without SMARCA4 mutations, and unclassified thoracic sarcomas (UTS). CXCL9 and PD-L1 expressions were assessed in NSCLC and thoracic fibroblast cell lines, with/without SMARCA4 knockdown, treated with/without interferon gamma. RESULTS: Nine patients were identified. All samples but one showed no TLS, consistent with an immune desert TME phenotype. Four patients received ICI as part of their treatment, but the only one who responded, had a tumor with a TLS and immune-rich TME. Unsupervised clustering of the validation cohort using immune cell scores identified 2 clusters associated with cell ontogeny and immunity (cluster 1 enriched for NSCLC independently of SMARCA4 status (n = 9/10; P = .001); cluster 2 enriched for SMARCA4-UT (n = 11/12; P = .005) and UTS (n = 5/5; P = .0005). SMARCA4 loss-of-function experiments revealed interferon-induced upregulation of CXCL9 and PD-L1 expression in the NSCLC cell line with no effect on the thoracic fibroblast cell line. CONCLUSION: SMARCA4-UT mainly have an immune desert TME with limited efficacy to ICI. TME of SMARCA4-driven tumors varies according to the cell of origin questioning the interplay between BAF alterations, cell ontogeny and immunity.

Therapeutic Targeting of the Colorectal Tumor Stroma.

Colorectal tumors have been classified based on histologic factors, genetic factors, and consensus molecular subtypes, all of which affect the tumor microenvironment. Elements of the tumor microenvironment serve as therapeutic targets and might be used as prognostic factors. For example, immune checkpoint inhibitors are used to treat tumors with microsatellite instability, and anti-angiogenic agents may be used in combination with other drugs to slow or inhibit tumor growth. We review the features of the colorectal tumor stroma that are associated with patient outcomes and discuss potential therapeutic agents that target these features.

Tertiary lymphoid structures, drivers of the anti-tumor responses in human cancers.

The characterization of the microenvironment of human tumors led to the description of tertiary lymphoid structures (TLS) characterized by mature dendritic cells in a T-cell zone adjacent to B-cell follicle including a germinal center. TLS represent sites of lymphoid neogenesis that develop in most solid cancers. Analysis of the current literature shows that the TLS presence is associated with a favorable clinical outcome for cancer patients, regardless of the approach used to quantify TLS and the stage of the disease. Using several approaches that combine immunohistochemistry, gene expression assays, and flow cytometry on large series of lung tumors, our work demonstrated that TLS are important sites for the initiation and/or maintenance of the local and systemic T- and B-cell responses against tumors. Surrounded by high endothelial venules, they represent a privileged area for the recruitment of lymphocytes into tumors and generation of central-memory T and B cells that circulate and limit cancer progression. TLS can be considered as a novel biomarker to stratify the overall survival risk of untreated cancer patients and as a marker of efficient immunotherapies. The induction and manipulation of cancer-associated TLS using drug agonists and/or biotherapies should open new avenues to treat cancer patients.

The clinical role of the TME in solid cancer.

The highly complex and heterogenous ecosystem of a tumour not only contains malignant cells, but also interacting cells from the host such as endothelial cells, stromal fibroblasts, and a variety of immune cells that control tumour growth and invasion. It is well established that anti-tumour immunity is a critical hurdle that must be overcome for tumours to initiate, grow and spread and that anti-tumour immunity can be modulated using current immunotherapies to achieve meaningful anti-tumour clinical responses. Pioneering studies in melanoma, ovarian and colorectal cancer have demonstrated that certain features of the tumour immune microenvironment (TME)-in particular, the degree of tumour infiltration by cytotoxic T cells-can predict a patient's clinical outcome. More recently, studies in renal cell cancer have highlighted the importance of assessing the phenotype of the infiltrating T cells to predict early relapse. Furthermore, intricate interactions with non-immune cellular players such as endothelial cells and fibroblasts modulate the clinical impact of immune cells in the TME. Here, we review the critical components of the TME in solid tumours and how they shape the immune cell contexture, and we summarise numerous studies evaluating its clinical significance from a prognostic and theranostic perspective.

Revisiting immune escape in colorectal cancer in the era of immunotherapy.

In colorectal cancer (CRC), T-cell checkpoint blockade is only effective in patients diagnosed with mismatch repair-deficient (MMR-d) cancers. However, defects in Human Leukocyte Antigen (HLA) class I expression were reported to occur in most MMR-d CRCs, which would preclude antigen presentation in these tumours, considered essential for the clinical activity of this immunotherapeutic modality. We revisited this paradox by characterising HLA class I expression in two independent cohorts of CRC. We determined that loss of HLA class I expression occurred in the majority (73-78%) of MMR-d cases. This phenotype was rare in CRC liver metastases, irrespective of MMR status, whereas weak, inducible expression of HLA class I molecules was frequent in liver lesions. We propose that HLA class I is an important determinant of metastatic homing in CRCs. This observation is paramount to understand CRC carcinogenesis and for the application of immunotherapies in the metastatic setting.

Intra-tumoral tertiary lymphoid structures are associated with a low risk of early recurrence of hepatocellular carcinoma.

BACKGROUND & AIMS: Tertiary lymphoid structures (TLSs) provide a local and critical microenvironment for generating anti-tumor cellular and humoral immune responses. TLSs are associated with improved clinical outcomes in most solid tumors investigated to date. However, their role in hepatocellular carcinoma (HCC) is debated, as they have recently been shown to promote the growth of malignant hepatocyte progenitors in the non-tumoral liver. METHODS: We aimed to determine, by pathological review, the prognostic significance of both intra-tumoral and non-tumoral TLSs in a series of 273 patients with HCC treated by surgical resection in Henri Mondor University Hospital. Findings were further validated by gene expression profiling using a public data set (LCI cohort). RESULTS: TLSs were identified in 47% of the tumors, by pathological review, with lymphoid aggregates, primary and secondary follicles in 26%, 16% and 5% of the cases, respectively. Univariate and multivariate analyses showed that intra-tumoral TLSs significantly correlated with a lower risk of early relapse (<2 years after surgery, hazard ratio 0.46, p = 0.005). Interestingly, the risk of recurrence was also related to the degree of TLS maturation (primary or secondary follicles vs. lymphoid aggregates, p = 0.01). A gene expression signature associated with the presence of intra-tumoral TLS was also independently associated with a lower risk of early relapse in the LCI cohort. No association between the density of TLSs located in the adjacent non-tumoral liver and early or late recurrence was observed. CONCLUSIONS: We have shown that intra-tumoral TLSs are associated with a lower risk of early relapse in 2 independent cohorts of patients with HCC treated by surgical resection. Thus, intra-tumoral TLSs may reflect the existence of ongoing, effective anti-tumor immunity. LAY SUMMARY: Tertiary lymphoid structures provide a critical microenvironment for generating anti-tumor immune responses, and are associated with improved clinical outcome in most cancers investigated. Their role in hepatocellular carcinoma is however debated. We show in the present study that intra-tumoral tertiary lymphoid structures are associated with a low risk of early relapse after surgical resection, suggesting that they reflect the existence of in situ, effective anti-tumor immunity.

Association of IL-36γ with tertiary lymphoid structures and inflammatory immune infiltrates in human colorectal cancer.

IL-1 family cytokines play a dual role in the gut, with different family members contributing either protective or pathogenic effects. IL-36γ is an IL-1 family cytokine involved in polarizing type-1 immune responses. However, its function in the gut, including in colorectal cancer pathogenesis, is not well appreciated. In a murine model of colon carcinoma, IL-36γ controls tertiary lymphoid structure formation and promotes a type-1 immune response concurrently with a decrease in expression of immune checkpoint molecules in the tumor microenvironment. Here, we demonstrate that IL-36γ plays a similar role in driving a pro-inflammatory phenotype in human colorectal cancer. We analyzed a cohort of 33 primary colorectal carcinoma tumors using imaging, flow cytometry, and transcriptomics to determine the pattern and role of IL-36γ expression in this disease. In the colorectal tumor microenvironment, we observed IL-36γ to be predominantly expressed by M1 macrophages and cells of the vasculature, including smooth muscle cells and high endothelial venules. This pattern of IL-36γ expression is associated with a CD4+ central memory T cell infiltrate and an increased density of B cells in tertiary lymphoid structures, as well as with markers of fibrosis. Conversely, expression of the antagonist to IL-36 signaling, IL-1F5, was associated with intratumoral expression of checkpoint molecules, including PD-1, PD-L1, and CTLA4, which can suppress the immune response. These data support a role for IL-36γ in the physiologic immune response to colorectal cancer by sustaining inflammation within the tumor microenvironment.

TLS in Tumors: What Lies Within.

The role of tertiary lymphoid structures (TLS) in cancer has recently been illustrated and revisited using mouse models. Joshi et al. describe regulatory T (Treg) cell infiltration and functional heterogeneity of TLS in lung tumors. Finkin et al. report that inflammation-associated TLS serve as niche for tumor progenitor cells, which may lead to recurrence in hepatocellular-carcinoma.

Transcriptomic analysis of the tumor microenvironment to guide prognosis and immunotherapies.

Tumors are highly heterogeneous tissues where malignant cells are surrounded by and interact with a complex tumor microenvironment (TME), notably composed of a wide variety of immune cells, as well as vessels and fibroblasts. As the dialectical influence between tumor cells and their TME is known to be clinically crucial, we need tools that allow us to study the cellular composition of the microenvironment. In this focused research review, we report MCP-counter, a methodology based on transcriptomic markers that assesses the proportion of several immune and stromal cell populations in the TME from transcriptomic data, and we highlight how it can provide a way to decipher the complex mechanisms at play in tumors. In several malignancies, MCP-counter scores have been used to show various prognostic impacts of the TME, which we also show to be linked with the mutational burden of tumors. We also compared established molecular classifications of colorectal cancer and clear-cell renal cell carcinoma with the output of MCP-counter, and show that molecular subgroups have different TME profiles, and that these profiles are consistent within a given subgroup. Finally, we provide insights as to how knowing the TME composition may shape patient care in the near future.

Tertiary lymphoid structures in cancer and beyond.

Tertiary lymphoid structures (TLS) are ectopic lymphoid formations found in inflamed, infected, or tumoral tissues. They exhibit all the characteristics of structures in the lymph nodes (LN) associated with the generation of an adaptive immune response, including a T cell zone with mature dendritic cells (DC), a germinal center with follicular dendritic cells (FDC) and proliferating B cells, and high endothelial venules (HEV). In this review, we discuss evidence for the roles of TLS in chronic infection, autoimmunity, and cancer, and address the question of whether TLS present beneficial or deleterious effects in these contexts. We examine the relationship between TLS in tumors and patient prognosis, and discuss the potential role of TLS in building and/or maintaining local immune responses and how this understanding may guide therapeutic interventions.