Jeremy Goc

Dendritic Cells in Tumor-Associated Tertiary Lymphoid Structures Signal a Th1 Cytotoxic Immune Contexture and License the Positive Prognostic Value of Infiltrating CD8+ T Cells

Tumor-infiltrating T cells, particularly CD45RO(+)CD8(+) memory T cells, confer a positive prognostic value in human cancers. However, the mechanisms that promote a protective T-cell response in the tumor microenvironment remain unclear. In chronic inflammatory settings such as the tumor microenvironment, lymphoid neogenesis can occur to create local lymph node-like structures known as tertiary lymphoid structures (TLS). These structures can exacerbate a local immune response, such that TLS formation in tumors may help promote an efficacious immune contexture. However, the role of TLS in tumors has yet to be investigated carefully. In lung tumors, mature dendritic cells (DC) present in tumor-associated TLS can provide a specific marker of these structures. In this study, we evaluated the influence of TLS on the characteristics of the immune infiltrate in cohorts of prospective and retrospective human primary lung tumors (n = 458). We found that a high density of mature DC correlated closely to a strong infiltration of T cells that are predominantly of the effector-memory phenotype. Moreover, mature DC density correlated with expression of genes related to T-cell activation, T-helper 1 (Th1) phenotype, and cytotoxic orientation. Lastly, a high density of TLS-associated DC correlated with long-term survival, which also allowed a distinction of patients with high CD8(+) T-cell infiltration but a high risk of death. Taken together, our results show how tumors infiltrated by TLS-associated mature DC generate a specific immune contexture characterized by a strong Th1 and cytotoxic orientation that confers the lowest risk of death. Furthermore, our findings highlight the pivotal function of TLS in shaping the immune character of the tumor microenvironment, in promoting a protective immune response mediated by T cells against cancer.

Presence of B Cells in Tertiary Lymphoid Structures Is Associated with a Protective Immunity in Patients with Lung Cancer

RATIONALE It is now well established that immune responses can take place outside of primary and secondary lymphoid organs. We previously described the presence of tertiary lymphoid structures (TLS) in patients with non-small cell lung cancer (NSCLC) characterized by clusters of mature dendritic cells (DCs) and T cells surrounded by B-cell follicles. We demonstrated that the density of these mature DCs was associated with favorable clinical outcome. OBJECTIVES To study the role of follicular B cells in TLS and the potential link with a local humoral immune response in patients with NSCLC. METHODS The cellular composition of TLS was investigated by immunohistochemistry. Characterization of B-cell subsets was performed by flow cytometry. A retrospective study was conducted in two independent cohorts of patients. Antibody specificity was analyzed by ELISA. MEASUREMENTS AND MAIN RESULTS Consistent with TLS organization, all stages of B-cell differentiation were detectable in most tumors. Germinal center somatic hypermutation and class switch recombination machineries were activated, associated with the generation of plasma cells. Approximately half of the patients showed antibody reactivity against up to 7 out of the 33 tumor antigens tested. A high density of follicular B cells correlated with long-term survival, both in patients with early-stage NSCLC and with advanced-stage NSCLC treated with chemotherapy. The combination of follicular B cell and mature DC densities allowed the identification of patients with the best clinical outcome. CONCLUSIONS B-cell density represents a new prognostic biomarker for NSCLC patient survival, and makes the link between TLS and a protective B cell-mediated immunity.

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.

Characteristics of tertiary lymphoid structures in primary cancers

Tumors are sustained by complex networks of interactions between malignant cells, stromal cells and tumor-infiltrating immune cells. These networks differ from patient to patient in terms of nature, composition and organization as well as with regard to the precise localization of tumor-infiltrating cells. Of note, the heterogeneity of the immunological component of the tumor microenvironment, as opposed to its mere abundance, has been shown to influence disease outcome. However, a key question remains: where does the activation of tumor-specific T cells take place? The recently described, tumor-associated lymph node-like entities termed “tertiary lymphoid structures” exhibit a structural organization that is reminiscent of secondary lymphoid organs, and thus may imprint the local immune contexture. Here, we discuss how cancer-associated tertiary lymphoid structures impact on the tumor micro-architecture, immune microenvironment, and ultimately, patient survival.

Systemic Inflammation, Nutritional Status and Tumor Immune Microenvironment Determine Outcome of Resected Non-Small Cell Lung Cancer

Background Hypothesizing that nutritional status, systemic inflammation and tumoral immune microenvironment play a role as determinants of lung cancer evolution, the purpose of this study was to assess their respective impact on long-term survival in resected non-small cell lung cancers (NSCLC). Methods and Findings Clinical, pathological and laboratory data of 303 patients surgically treated for NSCLC were retrospectively analyzed. C-reactive protein (CRP) and prealbumin levels were recorded, and tumoral infiltration by CD8+ lymphocytes and mature dendritic cells was assessed. We observed that factors related to nutritional status, systemic inflammation and tumoral immune microenvironment were correlated; significant correlations were also found between these factors and other relevant clinical-pathological parameters. With respect to outcome, at univariate analysis we found statistically significant associations between survival and the following variables: Karnofsky index, American Society of Anesthesiologists (ASA) class, CRP levels, prealbumin concentrations, extent of resection, pathologic stage, pT and pN parameters, presence of vascular emboli, and tumoral infiltration by either CD8+ lymphocytes or mature dendritic cells and, among adenocarcinoma type, tumor grade (all p<0.05). In multivariate analysis, prealbumin levels (Relative Risk (RR): 0.34 [0.16–0.73], p = 0.0056), CD8+ cell count in tumor tissue (RR = 0.37 [0.16–0.83], p = 0.0162), and disease stage (RR 1.73 [1.03–2.89]; 2.99[1.07–8.37], p = 0.0374- stage I vs II vs III-IV) were independent prognostic markers. When taken together, parameters related to systemic inflammation, nutrition and tumoral immune microenvironment allowed robust prognostic discrimination; indeed patients with undetectable CRP, high (>285 mg/L) prealbumin levels and high (>96/mm2) CD8+ cell count had a 5-year survival rate of 80% [60.9–91.1] as compared to 18% [7.9–35.6] in patients with an opposite pattern of values. When stages I-II were considered alone, the prognostic significance of these factors was even more pronounced. Conclusions Our data show that nutrition, systemic inflammation and tumoral immune contexture are prognostic determinants that, taken together, may predict outcome.

Development of Methods for Selective Gene Expression Profiling in Tertiary Lymphoid Structure Using Laser Capture Microdissection

Tertiary lymphoid structures (TLS) are de novo lymphoid formations that are induced within tissues during inflammatory episodes. TLS have been reported at various anatomic sites and in many different contexts like cancer, infections, autoimmunity, graft rejection, and idiopathic diseases. These inducible, ectopic, and transient lymphoid structures exhibit the prototypical architecture found within secondary lymphoid organs (SLO) and have been recently appreciated as a major driver of the local adaptive immune reaction. As TLS emerge within tissues, the isolation in situ and the molecular characterization of these structures are challenging to operate. Laser capture microdissection (LCM) is a powerful tool to isolate selective structural components and cells from frozen or paraffin-embedded tissues. We and other groups previously applied LCM to decipher the molecular network within TLS and uncover their intrinsic connection with the local microenvironment. In this chapter, we describe a detailed LCM method for selecting and isolating TLS in situ to perform comprehensive downstream molecular analyses.

Abstract LB-273: Identity card of tumor-infiltrating regulatory T cells in the context of tertiary lymphoid structures in lung cancer patients

Regulatory T cells (Tregs) exert various suppressive mechanisms to dampen the host immune response which can help tumor cells to escape immune surveillance. However, the prognostic value of tumor-infiltrating Tregs (Ti-Tregs) is controversial. We described, tumor-associated tertiary lymphoid structures (TLS) in lung cancers, shown that they are critical for the local coordination and polarization of protective immunity and are able to imprint the behavior of intra-tumoral CD8+ T cells. The aim of the study was to determine the role of Ti-Tregs in the shaping of the local immune response in the context of TLS. We studied the differentiation, activation, immunosuppression, and immune checkpoint (ICP) status of Ti-Tregs in different areas of human lung tumors on a prospective cohort of 50 lung cancer specimens. In lung cancer patients, CD3+FoxP3+ cells were detected in different zones of the tumor i.e. the tumor stroma (TLS and non-TLS areas) and to a lesser extend in tumor nests. They express CD4 and all markers of human Tregs. As observed for conventional CD4+ T cells, Ti-Tregs show central-memory and effector-memory phenotype. Based on the expression of activation, immunosuppression and ICP (gene and protein level), Tregs exhibit a fully activated phenotype in tumor compared to distant sites of NSCLC patients. Interestingly, Ti-Tregs display a common phenotype in TLS versus out TLS but with some key specificities regarding immune checkpoint expression. In conclusion, the presence of several Ti-Treg subsets in lung tumor suggests they may use distinct mechanisms to exert their immunosuppressive functions in the different tumor areas. Citation Format: Priyanka DEVI, Sylvain Leveugle, Jeremy Goc, Helene Kaplon, Claire Germain, Samantha Knockaert, Pierre Validire, Diane Damotte, Sandrine Katsahian, Wolf Herve Fridman, Catherine Sautes-Fridman, Myriam Lawand, Marie-Caroline Dieu-Nosjean. Identity card of tumor-infiltrating regulatory T cells in the context of tertiary lymphoid structures in lung cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-273.

Abstract 1650: Prognostic importance of both stage of the disease and immune infiltrate in the outcome of NSCLC patients

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The majority of prognostic studies has highlighted a positive impact of intratumoral T cells on the survival of patients affected by solid tumors. The high reproducibility of these data strongly support the idea that one should take into account immune criteria for the prognostic staging of neoplastic lesions. Several studies have indeed reported a favorable clinical outcome for lung cancers patients whose neoplastic lesions contain a high density of CD8+ T cells. Moreover, we recently showed that mature dendritic cells (DC) were selectively detected in tertiary lymphoid structures (TLS) in lung cancer. We showed that TLS exhibit strong structural analogies with canonical secondary lymphoid organs with clusters of mature DC and T cells surrounded by B cell follicles. Lung cancer-associated TLS also present features of an ongoing immune reaction site. Interestingly, we reported that a high density of mature DC is correlated with the long-term survival of patients affected by early-stage, advanced and metastatic lung cancer. The combination of mature DC and CD8+ T cells allows the identification of patients with the highest risk of death. Thus, the survival of lung cancer patients appears to be positively influenced by the abundance of both cell types. This raises the question of evaluating the clinical impact of these tumor-infiltrating immune cells in combination with pathological parameters in lung cancer patients. A total of 372 patients operated for a lung cancer were enrolled in a retrospective study. Using immunohistochemistry, we demonstrated that combination of the density of mature DC and CD8+ T cells, with the pT stage better predicts the overall survival of lung cancer patients than the gold standard pT stage alone. The DC/CD8 score with the N stage allow a better discrimination of patients with high- versus low-risk of death, especially in the group of patients with lymph node invasion. We also demonstrated that the combination of DC/CD8 and emboli parameters allow a better stratification of lung cancer patients for survival. Finally, the DC/CD8 score enhances the prognostic value of the TNM stage. Combining DC/CD8 score with the pTNM stage yields a more refined view of the prognosis of lung cancer patients. In conclusion, DC/CD8 score becomes prognosticator of survival for patients with first signs of metastasis (lymph node involvement, vascular and/or lymphatic tumor invasion). This study contributes to a better understanding of beneficial elements of immune responses to tumors. Combining DC/CD8 score with the stage of the tumor could guide better treatment choices. Citation Format: Marie-Caroline Dieu-Nosjean, Jeremy GOC, Claire GERMAIN, Samantha KNOCKAERT, Marco Alifano, Audrey LUPO, Diane Damotte, Pierre Validire, Scott SA HAMMOND, Wolf-Herman Fridman, Catherine Sautes-Fridman. Prognostic importance of both stage of the disease and immune infiltrate in the outcome of NSCLC patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1650. doi:10.1158/1538-7445.AM2014-1650

Abstract LB-497: Primary tumor localization determines the metastatic immune profile

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Primary tumor localization determines the metastatic immune profile Background Most cancer patients die from their metastatic disease and not from their primary tumor. However, little is known on the immune microenvironment of metastatic lesions and on its impact on clinical outcome. Indeed, the immune pattern of the tumor microenvironment has been recently identified as being a major prognostic factor in primary tumors. Thus, a high density of memory T cells with a Th1 and CD8 cytotoxic orientation is beneficial in many cancers. However, there are a few exceptions since CD8+ T cells have been reported to be associated with bad prognosis in renal cell carcinoma. The question of the respective roles of the tumor cell and organ microenvironment to explain these differences has not been addressed. Methods We studied the density and organization of tumor-infiltrating immune cells (CD3+, CD8+, Foxp3+, granzyme B+, NK cells and mature DCs) in retrospective cohorts of primary and lung metastases of colorectal cancer (CRC, n=124) and renal cell carcinoma (RCC, n=55) by immunohistochemistry. We investigated by qPCR the gene expression levels related to immune populations and their functions in the lung metastases from 19 CRC and 12 RCC. Biological datas were compared to clinical datas and patients outcome. Results The cell composition (CD3+, CD4+ and Foxp3+ T cells, mature DCs and NK cells) and organization (tertiary lymphoid structures) of the immune infiltrate in metastases depend on the origin of the primary tumor. In both cases (colorectal cancer and renal cell carcinoma lung metastases), the density of CD8+ T cells and mature DCs appeared to be strongest prognosticator of patients survival as reported for many primary tumors, but with opposite impact. High densities of mature DCs and CD8+ cells correlate with good overall survival in lung metastases of colorectal cancer and with poor survival in lung metastases of renal cell cancer. In the latter, a strong Th2 and inflammatory context was present associated with a strong Th1 polarization. Conclusions Altogether, our data demonstrate that the immune pattern depends on the tumor origin and has a major prognostic role in advanced cancer stages with an impact on patient therapeutic management. We demonstrated also the complexity of the immune response polarization and its impact upon tumor immune surveillance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-497. doi:1538-7445.AM2012-LB-497

Abstract LB-498: Density of tertiary lymphoid structures is associated with activated and effector-memory T lymphocyte infiltration in human lung tumor

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: An increasing number of studies have demonstrated a strong correlation between T cell infiltrate and clinical outcome in several types of human solid cancers. Nevertheless, the mechanisms governing T-cell infiltration and activation into tumors remain poorly characterized. In lung cancer, our team has observed the presence of tertiary lymphoid structures called Ti-BALT (Tumor-induced Bronchus-Associated Lymphoid Tissues). These structures present features of an ongoing immune response and their density is associated with long-term survival for lung cancer patients, suggesting their implication in local T cell recruitment and activation. We recently published a specific gene expression signature associated with T cell presence in Ti-BALT, which includes lymphoid chemokines, adhesion molecules, and integrins (De Chaisemartin L, et al. Cancer Research 2011). This chemoattractant gene expression signature strongly suggests an active recruitment of immune cells in these structures. Our aim was to study the influence of these structures on the composition, density and functionality of the immune infiltrate in lung tumors. Methods: The expression of relevant molecules was assessed on fresh tumor-infiltrating lymphocytes by multicolor flow cytometry, on tissue sections by immunohistochemistry and on frozen tumors by Low Density Array analysis. Results: Large-scale flow cytometry analysis revealed an increased infiltration of T and B but not NK cells in tumors with a high density of Ti-BALT as compared to tumors with a low density. A significant increase proportion of activated and effector-memory T cell subsets was observed in Ti-BALT high versus low tumors. Whereas most T cells located in Ti-BALT had a naive and early memory T cell phenotype, as observed in canonical secondary lymphoid organs, T cells located outside Ti-BALT were significantly enriched in effector-memory T cell phenotype. Finally, Ki-67+ proliferating T cells were found in close contact with mature dendritic cells in Ti-BALT, suggesting a local priming of T cells in these structures. Conclusion: Together, these data suggest that Ti-BALT represent a privileged area for T cell recruitment and activation in the primary site of the tumor. This mechanism could lead to the establishment and maintenance of a protective anti-tumor immune response directly in the tumor. These findings give news insights about mechanisms of T cell infiltration and activation in tumor microenvironment, suggesting new strategies to enhance the efficacy of cancer immunotherapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-498. doi:1538-7445.AM2012-LB-498