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Immune checkpoint blockade has revolutionized opportunities for therapeutic intervention in cancer but demonstrates a low frequency of response in most patients and in some common types of tumors. An emerging paradigm supports the notion that trillions of normally beneficial microbes inhabiting the gastrointestinal tract, termed the microbiota, critically impact the success or failure of antitumor immunity induced by immune checkpoint blockade. Here, we briefly summarize the current knowledge on how interactions between the microbiota and immune system are contributing to the outcome of cancer immunotherapy. We propose that this immune–microbiota dialogue is particularly important in gastrointestinal cancers that exhibit striking resistance to immune checkpoint blockade and inherently develop in a unique environment that is rich in both immune-cell networks and direct exposure to the microbiota. Finally, we focus on how future studies should determine whether microbiota can be harnessed as a strategy to boost antitumor immunity in these contexts and beyond. See related article, p. 1291

PDF file - 56K, P values corresponding to different cutoff (minimum P value, first, second and third quartile) in CRC and RCC lung metastases. {section sign}P values were corrected by the formula proposed by Altman et al.

PDF file - 83K, Baseline characteristics of 52 patients with RCC lung metastasis. * determined by Heng et al.

PDF file - 90K, Baseline characteristics of 140 patients with CRC lung metastasis. The stage was determined by pathological examination at the time of diagnosis.

Purpose: By unlocking antitumor immunity, antibodies targeting programmed cell death 1 (PD-1) exhibit impressive clinical results in non–small cell lung cancer, underlining the strong interactions between tumor and immune cells. However, factors that can robustly predict long-lasting responses are still needed.Experimental Design: We performed in-depth immune profiling of lung adenocarcinoma using an integrative analysis based on immunohistochemistry, flow-cytometry, and transcriptomic data. Tumor mutational status was investigated using next-generation sequencing. The response to PD-1 blockers was analyzed from a prospective cohort according to tumor mutational profiles and PD-L1 expression, and a public clinical database was used to validate the results obtained.Results: We showed that distinct combinations of STK11, EGFR, and TP53 mutations were major determinants of the tumor immune profile (TIP) and of the expression of PD-L1 by malignant cells. Indeed, the presence of TP53 mutations without co-occurring STK11 or EGFR alterations (TP53-mut/STK11-EGFR-WT), independently of KRAS mutations, identified the group of tumors with the highest CD8 T-cell density and PD-L1 expression. In this tumor subtype, pathways related to T-cell chemotaxis, immune cell cytotoxicity, and antigen processing were upregulated. Finally, a prolonged progression-free survival (PFS: HR = 0.32; 95% CI, 0.16–0.63, P < 0.001) was observed in anti–PD-1-treated patients harboring TP53-mut/STK11-EGFR-WT tumors. This clinical benefit was even more remarkable in patients with associated strong PD-L1 expression.Conclusions: Our study reveals that different combinations of TP53, EGFR, and STK11 mutations, together with PD-L1 expression by tumor cells, represent robust parameters to identify best responders to PD-1 blockade. Clin Cancer Res; 24(22); 5710–23. ©2018 AACR.

Purpose: If immune cells are involved in tumor surveillance and have a prognostic impact in most primary tumors, little is known about their significance in metastases. Because patients' survival is heterogeneous, even at metastatic stages, we hypothesized that immune cells may be involved in the control of metastases. We therefore characterized the tumor immune microenvironment and its prognostic value in colorectal and renal cell carcinoma (RCC) metastases, and compared it to primary tumors.Experimental Design: We analyzed by immunohistochemistry (n = 192) and qPCR (n = 32) the immune environments of colorectal carcinoma and RCC lung metastases.Results: Metastases from colorectal carcinoma and RCC have different immune infiltrates. Higher densities of DC-LAMP+ mature dendritic cells (P < 0.0001) and lower densities of NKp46+ NK cells (P < 0.0001) were observed in colorectal carcinoma as compared to RCC metastases, whereas densities of T cells were similar. High densities of CD8+ and DC-LAMP+ cells correlated with longer overall survival (OS) in colorectal carcinoma (P = 0.008) and shorter OS in RCC (P < 0.0001). High NK-cell densities were associated with improved survival in RCC (P = 0.002) but not in colorectal carcinoma. Densities of immune cells correlated significantly from primary to relapsing metastases for the same patient. A TH1 orientation was found in colorectal carcinoma metastases, whereas a heterogeneous immune gene expression was found in RCC metastases.Conclusions: Our results show a major prognostic value of the immune pattern (CD8+/DC-LAMP+ cell densities) in colorectal carcinoma and RCC, reproducible from primary to metastatic tumors, although with opposite clinical impacts, and highlight the role of the tumor cell in shaping its immune environment. Clin Cancer Res; 19(15); 4079–91. ©2013 AACR.

PDF file - 101K, Influence of pre-operative patient treatments on the distribution of CD8+, DC-LAMP+ and NKp46+ immune cells in CRC (a, b and c) and RCC lung metastases (d, e and f). 63/140 patients with CRC lung metastases have been treated with neo-adjuvant chemotherapy and 9/52 patients with RCC lung metastases have been treated with IL2/IFN. Whiskers length represents 10-90 percentile. ns, not significant (Mann-Whitney test).

Figures S1-12 Tables S1-S4 Supplementary material Supplementary Table S1. Antibodies used in flow cytometry experiments. Supplementary Table S2. Baseline characteristics of the 221 patients with lung adenocarcinoma in each TIP. Supplementary Table S3. Univariate Cox proportional-hazard analyses for progression-free survival and overall survival according to the mutational status of tumors in nivolumab treated patients. Supplementary Table S4. Baseline characteristics of the 32 patients with advanced-stages lung adenocarcinoma treated by nivolumab according to the mutational status of their tumor. Supplementary Figure S1. Spearman-correlation matrix of immune cell densities. Supplementary Figure S2. Comparison of immune cell densities, overall survival, molecular alteration frequency, and PD-L1 expression by tumor cells, between patients from TIP-1a and 1b. Supplementary Figure S3. TP53, STK11 and EGFR mutations strongly impact lung adenocarcinoma immune profiles. Supplementary Figure S4. Patterns of TP53, EGFR and STK11 mutations in the three TIPs. Supplementary Figure S5. No impact of KRAS mutations on the immune composition of TP53-Mut/STK11-EGFR-WT tumors and of TP53-STK11-EGFR-WT tumors. Supplementary Figure S6. Flow cytometry gating strategy used to characterize CD8 T cells from freshly resected tumors of lung adenocarcinoma patients. Supplementary Figure S7. Effector functions of CD8 TILs in TP53-Mut/STK11-EGFR-WT tumors vs TP53-WT tumors. Supplementary Figure S8. Higher MHC-I expression by malignant cells in TP53-Mut/ STK11- EGFR-WT tumors and its impact on tumor immune profiles. Supplementary Figure S9. Impact on the survival of nivolumab treated patients of TP53, KRAS, STK11 and EGFR mutations. Supplementary Figure S10. Anti-PD-1 efficacy in advanced-stage NSCLC patients according to distinct combinations of TP53, EGFR and STK11 mutations. Supplementary Figure S11. Impact of KRAS mutations on anti-PD-1 efficacy in advanced-stage NSCLC patients with TP53-Mut/STK11-EGFR-WT, TP53-STK11-EGFR-WT and in STK11-Mut/TP53-EGFR-WT tumors. Supplementary Figure S12. Impact of PD-L1 expression on the PFS of patients treated by anti-PD-1 according to distinct combinations of TP53, EGFR and STK11 mutations.

PDF file - 88K, Gene expression in lung metastases from CRC and RCC. Expression of genes related to (a) immune cell populations, (b) Th1/Th2 orientations, (c) inflammation and angiogenesis, (d) immunosuppression, (e) cytotoxicity, (f) chemokines/chemokine receptors in lung metastases from CRC (grey bars) and RCC (white bars). Expression levels of genes were determined using threshold cycle (Ct) values normalized to actin B ACTB (ΔCt). Whiskers length represents 10-90 percentile. ns, not significant; *P

PDF file - 107K, Kaplan-Meier curves for the duration of OS according to the presence of CD8+ T cells in the center of the tumor (CT) (a), in the invasive margin (IM) (b) and in combined regions (CT+IM) (c) of CRC lung metastases. Statistical comparison was performed by the log-rank test.

PDF file - 148K, Kaplan-Meier curves for the duration of OS according to the presence of CD8+ T cells in the center of the tumor (CT) (a), in the invasive margin (IM) (b) and in combined regions (CT+IM) (c) of RCC lung metastases. Representation of the Kaplan-Meier curves for the duration of OS according to the presence of NKp46+ cells in the CT (d), in the IM (e) and in combined region (CT+IM) (f) of RCC lung metastases. Statistical comparison was performed by the log-rank test.

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Toll-like receptors (TLR) recognize pathogen molecules and danger-associated signals that stimulate inflammatory processes. TLRs have been studied mainly in antigen-presenting cells, where they exert important immune regulatory functions, but they are also expressed by epithelial tumor cells, where they have been implicated in tumor progression. In this study, we demonstrate that the injection of TLR7 agonist in NOD/SCID mice, in C57BL/6 wild-type, and TLR7-deficient mice grafted with lung adenocarcinoma tumor cells leads to increased tumor progression and chemotherapeutic resistance. In patients with non–small cell lung cancer, expression analyses revealed that high TLR7 expression was strongly associated with resistance to neoadjuvant chemotherapy and poor clinical outcomes. Our findings delineate a crucial role for TLR7 in lung cancer physiopathology. Cancer Res; 74(18); 5008–18. ©2014 AACR.

Supplemental Figures 1-10. Supplementary figure 1 shows that TLR7 antagonist IRS661 specifically blocks TLR7 expression by human A549 cells Supplementary figure 2 shows that TLR7 agonist induces chemoresistance in murine LL/2 cells Supplementary figure 3 shows the pro-tumoral effect of TLR7 stimulation with increasing doses of chemotherapy Supplementary figure 4 shows that Stimulation of TLR7 favors tumor growth and chemoresistance of murine LL/2 cells while TLR7 blockade with an antagonist inhibits tumor growth Supplementary figure 5 shows that TLR7 stimulation induces myeloid cell recruitment into LL/2 tumors in immunocompetent C57BL/6 mice Supplementary figure 6 shows Determining the optimal cutoff for the cohort of patients not treated with neo-adjuvant chemotherapy Supplementary figure 7 shows Determining the optimal cutoff for the cohort of patients treated with neo-adjuvant chemotherapy Supplementary figure 8 shows that non-responders patients to neo-adjuvant chemotherapy have a poor prognosis Supplementary figure 9 shows the correlation graph for TLR7 expression by tumor cells in metastatic lymph nodes and in primary lung tumor, after treatment with neo-adjuvant chemotherapy Supplementary figure 10 shows that High TLR7 expression on tumor cells is associated with low response to neo-adjuvant chemotherapy

Microbial colonization of the mammalian intestine elicits inflammatory or tolerogenic T cell responses, but the mechanisms controlling these distinct outcomes remain poorly understood and accumulating evidence indicates that aberrant immunity to intestinal microbiota is causally associated with infectious, inflammatory, and malignant diseases1–8. Here, we define a critical pathway controlling the fate of inflammatory versus tolerogenic T cells that are specific for the microbiota and express the transcription factor RORγt. We profiled all RORγt+ immune cells at single cell resolution from the intestine-draining lymph nodes of mice and reveal a dominant presence of Tregs and lymphoid tissue-induced (LTi)-like group 3 innate lymphoid cells (ILC3s), which co-localize at interfollicular regions. These ILC3s have interconverting potential with RORγt+ extrathymic Aire-expressing cells, abundantly express major histocompatibility complex class II, and are necessary and sufficient to promote microbiota-specific RORγt+ Tregs and prevent their expansion as inflammatory T helper (Th)17 cells. This occurs through ILC3-mediated antigen-presentation, interleukin-2 gradients, and αv integrin. Finally, single-cell analyses demonstrate that ILC3 and RORγt+ Treg interactions are impaired in inflammatory bowel disease. Our results define a novel paradigm whereby ILC3s positively select for antigen specific RORγt+ Tregs, and against Th17 cells, to establish immune tolerance to the microbiota and intestinal health.

BackgroundNatural killer (NK) cells play a crucial role in tumor immunosurveillance through their cytotoxic effector functions and their capacity to interact with other immune cells to build a coordinated antitumor immune response. Emerging data reveal NK cell dysfunction within the tumor microenvironment (TME) through checkpoint inhibitory molecules associated with a regulatory phenotype.ObjectiveWe aimed at analyzing the gene expression profile of intratumoral NK cells compared with non-tumorous NK cells, and to characterize their inhibitory function in the TME.MethodsNK cells were sorted from human lung tumor tissue and compared with non- tumoral distant lungs.ResultsIn the current study, we identify a unique gene signature of NK cell dysfunction in human non-small cell lung carcinoma (NSCLC). First, transcriptomic analysis reveals significant changes related to migratory pattern with a downregulation of sphingosine-1-phosphate receptor 1 (S1PR1) and CX3C chemokine receptor 1 (CX3CR1) and overexpression of C-X-C chemokine receptor type 5 (CXCR5) and C-X-C chemokine receptor type 6 (CXCR6). Second, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and killer cell lectin like receptor (KLRC1) inhibitory molecules were increased in intratumoral NK cells, and CTLA-4 blockade could partially restore MHC class II level on dendritic cell (DC) that was impaired during the DCs/NK cell cross talk. Finally, NK cell density impacts the positive prognostic value of CD8+ T cells in NSCLC.ConclusionsThese findings demonstrate novel molecular cues associated with NK cell inhibitory functions in NSCLC.

International audience; Interleukin (IL)-2 is a pleiotropic cytokine that is necessary to prevent chronic inflammation in the gastrointestinal tract 1-4. The protective effects of IL-2 involve the generation, maintenance and function of regulatory T (T reg) cells 4-8 , and the use of low doses of IL-2 has emerged as a potential therapeutic strategy for patients with inflammatory bowel disease 9. However, the cellular and molecular pathways that control the production of IL-2 in the context of intestinal health are undefined. Here we show, in a mouse model, that IL-2 is acutely required to maintain T reg cells and immunological homeostasis throughout the gastrointestinal tract. Notably, lineage-specific deletion of IL-2 in T cells did not reduce T reg cells in the small intestine. Unbiased analyses revealed that, in the small intestine, group-3 innate lymphoid cells (ILC3s) are the dominant cellular source of IL-2, which is induced selectively by IL-1β. Macrophages in the small intestine produce IL-1β, and activation of this pathway involves MYD88-and NOD2-dependent sensing of the microbiota. Our loss-of-function studies show that ILC3-derived IL-2 is essential for maintaining T reg cells, immunological homeostasis and oral tolerance to dietary antigens in the small intestine. Furthermore, production of IL-2 by ILC3s was significantly reduced in the small intestine of patients with Crohn's disease, and this correlated with lower frequencies of T reg cells. Our results reveal a previously unappreciated pathway in which a microbiota-and IL-1β-dependent axis promotes the production of IL-2 by ILC3s to orchestrate immune regulation in the intestine. To determine whether IL-2 is constitutively required for the maintenance of T reg cells and immunological homeostasis in the intestine, we administered isotype-control or anti-IL-2 neutralizing antibodies every other day to adult mice for two weeks. Within this short time period, the neutralization of IL-2 promoted an enlargement of the spleen and mesenteric lymph nodes, and caused significant reductions of T reg cells and significant increases in the proliferation of CD4 + T cells throughout the gastrointestinal tract and associated lymphoid tissues, including the mesenteric lymph nodes, large intestine and small intestine (Extended Data Fig. 1a-g). Blockade of IL-2 resulted in significantly enhanced IFNγ production by CD4 + T cells in both the small and large intestine, as well as increased IL-17A production in the large intestine (Extended Data Fig. 1h-k). Previous studies have suggested that CD4 + T cells are the dominant cellular source of IL-2 1,2. Therefore, we generated mice with a lineage-specific deletion of IL-2 in T cells by crossing IL-2-floxed mice 10 with Lck cre mice. Lck cre Il2 f/f mice exhibited a complete loss of IL-2 protein staining in T cells, and we observed a significant reduction in the number of T reg cells, and an increase in CD4 + T cell proliferation and effector function in the mesenteric lymph nodes and large intestine (Extended Data Fig. 2a-g). By contrast, deletion of

A high density of tumor-infiltrating CD8+ T cells and CD20+ B cells correlates with prolonged survival in patients with a wide variety of human cancers, including high-grade serous ovarian carcinoma (HGSC). However, the potential impact of mature dendritic cells (DCs) in shaping the immune contexture of HGSC, their role in the establishment of T cell-dependent antitumor immunity, and their potential prognostic value for HGSC patients remain unclear. We harnessed immunohistochemical tests and biomolecular analyses to demonstrate that a high density of tumor-infiltrating DC-LAMP+ DCs is robustly associated with an immune contexture characterized by TH1 polarization and cytotoxic activity. We showed that both mature DCs and CD20+ B cells play a critical role in the generation of a clinically favorable cytotoxic immune response in HGSC microenvironment. In line with this notion, robust tumor infiltration by both DC-LAMP+ DCs and CD20+ B cells was associated with most favorable overall survival in two independent cohorts of chemotherapy-naive HGSC patients. Our findings suggest that the presence of mature, DC-LAMP+ DCs in the tumor microenvironment may represent a novel, powerful prognostic biomarker for HGSC patients that reflects the activation of clinically relevant anticancer immunity. Citation Format: Iva Truxova, Lenka Kasikova, Michal Hensler, Petr Skapa, Jan Laco, Ladislav Pecen, Lucie Belicova, Ivan Praznovec, Michael J. Halaska, Tomas Brtnicky, Eva Salkova, Lukas Rob, Roman Kodet, Jeremy Goc, Catherine Sautes-Fridman, Wolf Herman Fridman, Ales Ryska, Lorenzo Galluzzi, Radek Spisek, Jitka Fucikova. Mature dendritic cells correlate with favorable immune infiltrate and improved prognosis in ovarian carcinoma patients [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B76.

A high density of tumor-infiltrating CD8+ T lymphocytes (CTLs), key mediator of anticancer immunity, and CD20+ B cells correlates with positive prognostic value and prolonged survival in patients with a various solid tumors including high-grade serous ovarian carcinoma (HGSC). The majority of HGSCs containing high frequencies of CD8+ CTLs are also robustly infiltrated by CD20+ B cells, and patients whose tumors exhibit abundant coinfiltration have higher survival rates than patients with tumors that only contain high amounts of CD8+ CTLs. However, the cellular mechanism, the potential impact of mature dendritic cells (DCs) in shaping the immune contexture of HGSC, their role in the establishment of T cell-dependent antitumor immunity, and their potential prognostic value for HGSC patients remain unclear. Three independent cohorts of patients with resectable HGSC who did not receive neoadjuvant chemotherapy were involved in the study. Lysosomal-associated membrane protein 3 (DC-LAMP) and its colocalization with CD20+B cells in samples was analyzed by immunohistochemistry (IHC) and cell quantification. Prognostic impact of DC-LAMP+ cells in patient cohort was stratified based on median density of DC-LAMP+ cells in the tumor stroma and nest, followed by retrospective RFS and OS analysis. Comparison of DC-LAMPHi and DC-LAMPLo patients and impact of mature DCs on the composition of the immune contexture characterized by TH1 polarization and cytotoxic activity was performed by biomolecular analyses (flow cytometry, NGS analysis). Patients with high density of DC-LAMP+ cells in the tumor stroma exhibited significantly longer RFS and OS than DC-LAMP- patients. Compared to DC-LAMPLo, DC-LAMPHi tumors exhibited an over-representation of gene sets specifically clustering to the following immunologic functions: T cells, CD8 cytotoxicity, TH1 polarization, T cell activation, NK cells, and plasma cells. Also, a significantly higher percentage of CD3+CD45+ and CD3+CD8+ T cells among live mononuclear cells and higher density of NK cells in the tumor stroma in DC-LAMPHi were observed. We found that both mature DCs and CD20+ B cells play a critical role in the generation of a clinically favorable cytotoxic immune response in HGSC microenvironment. Robust tumor infiltration by both DC-LAMP+ DCs and CD20+ B cells was associated with most favorable overall survival in independent cohorts of chemotherapy-naive HGSC patients. Our findings suggest that the presence of mature, DC-LAMP+ DCs in the tumor microenvironment may represent a novel, powerful prognostic biomarker for HGSC patients that reflects the activation of clinically relevant anticancer immunity. Citation Format: Iva Truxova, Lenka Kasikova, Michal Hensler, Petr Skapa, Jan Laco, Ladislav Pecen, Lucie Belicova, Sarka Vosahlikova, Ivan Praznovec, Michael Halaska, Tomas Brtnicky, Eva Salkova, Lukas Rob, Roman Kodet, Jeremy Goc, Catherine Sautes-Fridman, Wolf Herve Fridman, Ales Ryska, Lorenzo Galluzzi, Radek Spisek, Jitka Fucikova. Mature dendritic cells correlate with favorable immune infiltrate and improved prognosis in ovarian carcinoma patients [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A24.

Purpose: By unlocking antitumor immunity, antibodies targeting programmed cell death 1 (PD-1) exhibit impressive clinical results in non–small cell lung cancer, underlining the strong interactions between tumor and immune cells. However, factors that can robustly predict long-lasting responses are still needed. Experimental Design: We performed in-depth immune profiling of lung adenocarcinoma using an integrative analysis based on immunohistochemistry, flow-cytometry, and transcriptomic data. Tumor mutational status was investigated using next-generation sequencing. The response to PD-1 blockers was analyzed from a prospective cohort according to tumor mutational profiles and PD-L1 expression, and a public clinical database was used to validate the results obtained. Results: We showed that distinct combinations of STK11, EGFR, and TP53 mutations were major determinants of the tumor immune profile (TIP) and of the expression of PD-L1 by malignant cells. Indeed, the presence of TP53 mutations without co-occurring STK11 or EGFR alterations (TP53-mut/STK11-EGFR-WT), independently of KRAS mutations, identified the group of tumors with the highest CD8 T-cell density and PD-L1 expression. In this tumor subtype, pathways related to T-cell chemotaxis, immune cell cytotoxicity, and antigen processing were upregulated. Finally, a prolonged progression-free survival (PFS: HR = 0.32; 95% CI, 0.16–0.63, P < 0.001) was observed in anti–PD-1-treated patients harboring TP53-mut/STK11-EGFR-WT tumors. This clinical benefit was even more remarkable in patients with associated strong PD-L1 expression. Conclusions: Our study reveals that different combinations of TP53, EGFR, and STK11 mutations, together with PD-L1 expression by tumor cells, represent robust parameters to identify best responders to PD-1 blockade. Clin Cancer Res; 24(22); 5710–23. ©2018 AACR.

Interleukin (IL)-2 is a pleiotropic cytokine that is necessary to prevent chronic inflammation in the gastrointestinal tract

Patients with chronic obstructive pulmonary disease (COPD) have a higher prevalence of lung cancer. The chronic inflammation associated with COPD probably promotes the earliest stages of carcinogenesis. However, once tumors have progressed to malignancy, the impact of COPD on the tumor immune microenvironment remains poorly defined, and its effects on immune-checkpoint blockers' efficacy are still unknown.To study the impact of COPD on the immune contexture of non-small cell lung cancer.We performed in-depth immune profiling of lung tumors by immunohistochemistry and we determined its impact on patient survival (n = 435). Tumor-infiltrating T lymphocyte (TIL) exhaustion by flow cytometry (n = 50) was also investigated. The effectiveness of an anti-PD-1 (programmed cell death-1) treatment (nivolumab) was evaluated in 39 patients with advanced-stage non-small cell lung cancer. All data were analyzed according to patient COPD status.Remarkably, COPD severity is positively correlated with the coexpression of PD-1/TIM-3 (T-cell immunoglobulin and mucin domain-containing molecule-3) by CD8 T cells. In agreement, we observed a loss of CD8 T cell-associated favorable clinical outcome in COPDCOPD is associated with an increased sensitivity of CD8 tumor-infiltrating T lymphocytes to immune escape mechanisms developed by tumors, thus suggesting a higher sensitivity to PD-1 blockade in patients with COPD.

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.

Rationale:Itisnowwellestablishedthatimmuneresponsescantake 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 potentiallinkwithalocalhumoralimmuneresponseinpatientswith NSCLC. Methods: The cellular composition of TLS was investigated by immunohistochemistry. Characterization of B-cell subsets was performedby flowcytometry.Aretrospectivestudywasconductedin 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-termsurvival,bothinpatientswithearly-stageNSCLCandwith 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.

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. Cancer Res; 74(3); 705–15. ©2013 AACR.

Cancer is a major public health issue and figures among the leading causes of death in the world. Cancer development is a long process, involving the mutation, amplification or deletion of genes and chromosomal rearrangements. The transformed cells change morphologically, enlarge, become invasive and finally detach from the primary tumor to metastasize in other organs through the blood and/or lymph. During this process, the tumor cells interact with their microenvironment, which is complex and composed of stromal and immune cells that penetrate the tumor site via blood vessels and lymphoid capillaries. All subsets of immune cells can be found in tumors, but their respective density, functionality and organization vary from one type of tumor to another. Whereas inflammatory cells play a protumoral role, there is a large body of evidence of effector memory T cells controlling tumor invasion and metastasis. Thus, high densities of memory Th1/CD8 cytotoxic T cells in the primary tumors correlate with good prognosis in most tumor types. Tertiary lymphoid structures, which contain mature dendritic cells (DC) in a T cell zone, proliferating B cells and follicular DC, are found in the tumor stroma and they correlate with intratumoral Th1/CD8 T cell and B cell infiltration. Eventually, tumors undergo genetic and epigenetic modifications that allow them to escape being controlled by the immune system. This comprehensive review describes the immune contexture of human primary and metastatic tumors, how it impacts on patient outcomes and how it could be used as a predictive biomarker and guide immunotherapies.

Rationale: Tumor-infiltrating immune cells affect lung cancer outcome. However, the factors that influence the composition and function of the tumor immune environment remain poorly defined and need investigation, particularly in the era of immunotherapy.Objectives: To determine whether the tumoral immune environment is related to lung adenocarcinoma mutations.Methods: This retrospective cohort included 316 consecutive patients with lung adenocarcinoma (225 men; 258 smokers) studied from 2001 to 2005 in a single center. We investigated the association of densities of intratumoral mature dendritic cells (mDCs), CD8+ T cells, neutrophils, and macrophages with clinical and pathological variables and tumor cell mutation profiles obtained by next-generation sequencing.Measurements and Main Results: In 282 tumors, we found 460 mutations, mainly in TP53 (59%), KRAS (40%), STK11 (24%), and EGFR (14%). Intratumoral CD8+ T-cell density was high in smokers (P = 0.02) and TP53-mutated tumors (P = 0.02) and low in BRA...

De novo formation of tertiary lymphoid structures (TLS) has been described in lung cancers. Intratumoral TLS seem to be functional and are associated with a long-term survival for lung cancer patients, suggesting that they represent an activation site for tumor-specific T cells. Here, we characterized T-cell recruitment to TLS in human lung cancer to identify the adhesion molecules and chemoattractants orchestrating this migration. We found that most TLS T cells were CD62L+ and mainly of CD4+ memory phenotype, but naive T cells were highly enriched in these structures as compared with the rest of the tumor. A specific gene expression signature associated with T cell presence was identified in TLS, which included chemokines (CCL19, CCL21, CXCL13, CCL17, CCL22, and IL16), adhesion molecules (ICAM-2, ICAM-3, VCAM-1, and MAdCAM-1) and integrins (alphaL, alpha4, and alphaD). The presence of the corresponding receptors on TLS T cells was confirmed. Intratumoral PNAd+ high endothelial venules also were exclusively associated with TLS and colocalized with CD62L+ lymphocytes. Together, these data bring new insights into the T-cell recruitment to intratumoral TLS and suggest that blood T cell enter into TLS via high endothelial venules, which represent a new gateway for T cells to the tumor. Findings identify the molecules that mediate migration of tumor-specific T cells into TLS where T cell priming occurs, suggesting new strategies to enhance the efficacy of cancer immunotherapies. Cancer Res; 71(20); 6391–9. ©2011 AACR.

Chronic inflammation is recognized as a causative factor in the development of cancer and recent paradigms suggest that microbe-driven chronic inflammation is causally associated with the development and progression of cancer in the colon. Further, recent studies in mice have implicated innate lymphoid cells (ILC) as a key cell population that regulates intestinal inflammation. Group 3 innate lymphoid cells (ILC3) are essential regulators of immunity, inflammation and tissue homeostasis in the intestine, yet their role in cancer remains poorly defined. Here, we identify that ILC3 are associated with both human and mouse colon tumors. Tumor-associated ILC3 are selectively localized within lymphoid aggregates and exhibit a unique phenotypic profile as compared with nonmalignant tissue. Critically, mice with a selective deletion of ILC3-specific MHCII exhibit a striking increased susceptibility to intestinal tissue damage and develop highly invasive and flat colorectal tumors. These data demonstrate a protective role for antigen-presenting ILC3 in the context of cancer development and progression in the intestine, and suggest that further interrogation may lead to the development of novel immunotherapeutic strategies in colon cancer. Citation Format: Jeremy Goc, Nick Bessman, Sheena Sahota, Flamar Anne Laure, Gregory Putzel, David Withers, Janelle Arthur, Manish Shah, Gregory Sonnenberg. A protective role for group 3 innate lymphoid cells in colitis-associated colorectal cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A075.

There is now growing evidence that the immune contexture influences cancer progression and clinical outcome of patients with non-small cell lung cancer (NSCLC). If chemotherapy is widely used to treat patients with advanced-stage NSCLC, it remains unclear how it could modify the immune contexture and impact its prognostic value. Here, we analyzed two retrospective cohorts, respectively composed of 122 stage III-N2 NSCLC patients treated with chemotherapy before surgery and 39 stage-matched patients treated by surgery only. In patients treated with neoadjuvant chemotherapy, the histological characteristics, the expression of PD-L1 protein, and the tumor immune microenvironment (CD8+ T cells, DC-LAMP+ mature dendritic cells, and CD68+ macrophages) were evaluated and their prognostic value assessed together with standard clinical parameters. By analyzing pre- and post-treatment specimens, we did not find any changes in the PD-L1 expression. We also found that the tumor immune contexture in patients treated with neoadjuvant chemotherapy exhibited a similar pattern that the one found in chemotherapy-naive patients, with comparable densities of tumor-infiltrating CD8+ and DC-LAMP+ cells and a similar spatial organization. The percentage of residual viable tumor cells and the immune pattern (CD8+ and DC-LAMP+ cell densities) were significantly associated with the clinical outcome and allowed the identification of short- and long-term survivors, respectively. In multivariate analysis, the immune pattern was found to be the strongest independent prognostic factor. In conclusion, this study decrypts the complex interplay between cancer and immune cells in patients undergoing chemotherapy and supports potential beneficial synergistic effect of immunotherapy and chemotherapy.

Physical separation between the mammalian immune system and commensal bacteria is necessary to limit chronic inflammation. However, selective species of commensal bacteria can reside within intestinal-associated lymphoid tissues of healthy mammals. Here, we demonstrate that lymphoid tissue-resident commensal bacteria (LRC) colonized murine dendritic cells and modulate their cytokine production. In germ-free and antibiotic-treated mice, LRCs colonized intestinal-associated lymphoid tissues and induced multiple members of the IL-10 cytokine family, including dendritic cell-derived IL-10 and group 3 innate lymphoid cell (ILC3)-derived IL-22. Notably, IL-10 limited the development of pro-inflammatory Th17 cell responses, and IL-22 production enhanced LRC colonization in the steady state. Furthermore, LRC colonization protected mice from lethal intestinal damage in an IL-10-IL-10R-dependent manner. Collectively, our data reveal a unique host-commensal bacteria dialogue whereby selective subsets of commensal bacteria interact with dendritic cells to facilitate tissue-specific responses that are mutually beneficial for both the host and the microbe.

A delicate balance exists between the mammalian immune system and normally beneficial commensal bacteria that colonize the gastrointestinal tract, which is necessary to maintain tissue homeostasis. Dysregulation of these interactions between the host and commensal bacteria is causally associated with chronic inflammation and the development of cancer. In contrast, recent reports have highlighted that commensal bacteria also play an essential role in promoting anti-tumor immune responses in several contexts, highlighting a paradox whereby interactions between the host and commensal bacteria can influence both pro- and anti-tumor immunity. Given the critical roles for group 3 innate lymphoid cells (ILC3s) in regulating inflammation, tissue repair and host–microbe interactions in the intestine, here we discuss new evidence that ILC3s may profoundly influence the development, progression and control of tumors. In this review, we provide an overview of recent advances in understanding the impact of commensal bacteria on tumorigenesis, discuss recent findings identifying ILC3s as critical regulators of host–microbe interactions and highlight the emerging role of this immune cell population in cancer and their potential implication as a therapeutic target.

A high density of tumor-infiltrating mature dendritic cells (DC) and CD8+ T cells correlates with a positive prognosis in a majority of human cancers. The recruitment of activated lymphocytes to the tumor microenvironment, primed to recognize tumor-associated antigens, can occur in response to immunogenic cell death (ICD) of tumor cells. ICD is characterized by the preapoptotic translocation of calreticulin (CRT) from the endoplasmic reticulum (ER) to the cell surface as a result of an ER stress response accompanied by the phosphorylation of eukaryotic initiation factor 2α (eIF2α). We conducted a retrospective study on two independent cohorts of patients with non–small cell lung cancer (NSCLC) to investigate the prognostic potential of CRT. We report that the level of CRT expression on tumor cells, which correlated with eIF2α phosphorylation, positively influenced the clinical outcome of NSCLC. High CRT expression on tumor cells was associated with a higher density of infiltrating mature DC and effector memory T-cell subsets, suggesting that CRT triggers the activation of adaptive immune responses in the tumor microenvironment. Accordingly, patients with elevated CRT expression and dense intratumoral infiltration by DC or CD8+ T lymphocytes had the best prognosis. We conclude that CRT expression constitutes a new powerful prognostic biomarker that reflects enhanced local antitumor immune responses in the lung. Cancer Res; 76(7); 1746–56. ©2016 AACR.

Toll-like receptors (TLR) recognize pathogen molecules and danger-associated signals that stimulate inflammatory processes. TLRs have been studied mainly in antigen-presenting cells, where they exert important immune regulatory functions, but they are also expressed by epithelial tumor cells, where they have been implicated in tumor progression. In this study, we demonstrate that the injection of TLR7 agonist in NOD/SCID mice, in C57BL/6 wild-type, and TLR7-deficient mice grafted with lung adenocarcinoma tumor cells leads to increased tumor progression and chemotherapeutic resistance. In patients with non–small cell lung cancer, expression analyses revealed that high TLR7 expression was strongly associated with resistance to neoadjuvant chemotherapy and poor clinical outcomes. Our findings delineate a crucial role for TLR7 in lung cancer physiopathology. Cancer Res; 74(18); 5008–18. ©2014 AACR.

A high density of intratumoral effector memory CD8+/Th1 T cells is associated with favorable prognosis in most cancers and may be induced or increased by immunotherapy. Efficient adaptive immune reactions are shaped in tumor adjacent tertiary lymphoid structures, which exhibit all characteristics of immunity generating lymphoid formations in reactive lymph nodes. Malignant tumor cells impact favorably or deleteriously their immune microenvironment if they bear genetic mutations that result in neo-antigens or by producing chemokines and cytokines that recruit lymphocytes and myeloid cells or increase inflammation and neo-angiogenesis. This intricate network of interactions results in control or escape of tumors, and its understanding will help define goals to monitor efficiency of immunotherapies.

T-cell infiltrates are robust prognostic biomarkers in the majority of human cancers. However, the mechanisms that shape a protective T-cell response remain unclear. Our recent study implicates tertiary lymphoid structures in the shaping of an efficient and beneficial immune response in lung cancer patients.

International audience; BackgroundHypothesizing 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 FindingsClinical, 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 p285 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.ConclusionsOur data show that nutrition, systemic inflammation and tumoral immune contexture are prognostic determinants that, taken together, may predict outcome.

Purpose: If immune cells are involved in tumor surveillance and have a prognostic impact in most primary tumors, little is known about their significance in metastases. Because patients' survival is heterogeneous, even at metastatic stages, we hypothesized that immune cells may be involved in the control of metastases. We therefore characterized the tumor immune microenvironment and its prognostic value in colorectal and renal cell carcinoma (RCC) metastases, and compared it to primary tumors. Experimental Design: We analyzed by immunohistochemistry (n = 192) and qPCR (n = 32) the immune environments of colorectal carcinoma and RCC lung metastases. Results: Metastases from colorectal carcinoma and RCC have different immune infiltrates. Higher densities of DC-LAMP+ mature dendritic cells (P < 0.0001) and lower densities of NKp46+ NK cells (P < 0.0001) were observed in colorectal carcinoma as compared to RCC metastases, whereas densities of T cells were similar. High densities of CD8+ and DC-LAMP+ cells correlated with longer overall survival (OS) in colorectal carcinoma (P = 0.008) and shorter OS in RCC (P < 0.0001). High NK-cell densities were associated with improved survival in RCC (P = 0.002) but not in colorectal carcinoma. Densities of immune cells correlated significantly from primary to relapsing metastases for the same patient. A TH1 orientation was found in colorectal carcinoma metastases, whereas a heterogeneous immune gene expression was found in RCC metastases. Conclusions: Our results show a major prognostic value of the immune pattern (CD8+/DC-LAMP+ cell densities) in colorectal carcinoma and RCC, reproducible from primary to metastatic tumors, although with opposite clinical impacts, and highlight the role of the tumor cell in shaping its immune environment. Clin Cancer Res; 19(15); 4079–91. ©2013 AACR.

Inflammatory CD4+ T cell responses to self or commensal bacteria underlie the pathogenesis of autoimmunity and inflammatory bowel disease (IBD), respectively. While selection of self-specific T cells in the thymus limits responses to mammalian tissue antigens, the mechanisms that control selection of commensal bacteria-specific T cells remain poorly understood. Here we demonstrate that group 3 innate lymphoid cell (ILC3)-intrinsic expression of major histocompatibility complex class II (MHCII) is regulated similarly to thymic epithelial cells, and that MHCII+ ILC3s directly induce cell death of activated commensal bacteria-specific T cells. Further, MHCII on colonic ILC3s was reduced in pediatric IBD patients. Collectively, these results define a selection pathway for commensal bacteria-specific CD4+ T cells in the intestine and suggest that this process is dysregulated in human IBD. On going studies are interrogating the role and regulation of MHCII+ ILC3s in the context of allergy, autoimmunity and tumorigenesis.

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 based on the expression of FoxP3 marker in cancer patients. Here, we decipher the phenotype of Tregs infiltrating human lung cancer to determine their role in shaping the immune response against tumor cells. Our aim was to study the differentiation, activation, and immunosuppression status of Ti-Tregs in different areas of human lung tumors. Immunohistochemistry was performed on paraffin-embedded tissue sections for CD3 and FoxP3 stainings, and phenotypic analysis was carried out on fresh human lung cancer specimens (n=50) by flow cytometry. In lung cancer patients, Ti-Tregs localize in the different part of the tumor i.e. tumor nests, stroma, and tertiary lymphoid structures (TLS). Ti-Tregs are CD4+ and show central-memory and effector-memory phenotype. Interestingly, Ti-Tregs exhibit different phenotypes based on the differential expression of activation and immunosuppression molecules. In conclusion, the presence of several Ti-Treg subsets in lung tumor suggests that they may play different roles in distinct tumor areas. Thus, the next step will be to decipher the mechanisms and functions used by these Treg populations in order to manipulate them for therapeutic intervention.

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

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.

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

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 data's were compared to clinical data's and patient's 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 patient's 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