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ObjectiveT cells are major effectors of the antitumoural immune response. Their activation by tumour-associated antigens can unleash their proliferation and cytotoxic functions, leading to tumour cell elimination. However, tumour-related immunosuppressive mechanisms including the overexpression of immune checkpoints like programmed cell death protein-1 (PD-1), are also engaged, promoting immune escape. Current immunotherapies targeting these pathways have demonstrated weak efficacy in colorectal cancer (CRC). It is thus crucial to find new targets for immunotherapy in this cancer type.DesignIn a prospective cohort of patients with CRC, we investigated the phenotype of tumour-related and non-tumour related intestinal T cells (n=44), particularly the adenosinergic pathway, correlating with clinical phenotype. An autologous coculture model was developed between patient-derived primary tumour spheroids and their autologous tumour-associated lymphocytes. We used this relevant model to assess the effects of CD39 blockade on the antitumour T cell response.ResultsWe show the increased expression of CD39, and its co-expression with PD-1, on tumour infiltrating T cells compared with mucosal lymphocytes. CD39 expression was higher in the right colon and early-stage tumours, thus defining a subset of patients potentially responsive to CD39 blockade. Finally, we demonstrate in autologous conditions that CD39 blockade triggers T cell infiltration and tumour spheroid destruction in cocultures.ConclusionIn CRC, CD39 is strongly expressed on tumour infiltrating lymphocytes and its inhibition represents a promising therapeutic strategy for treating patients.

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: 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).

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 - 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 - 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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PDF file - 95K, Comparison of different methods used for the stratification of patients according to the density of mature DC, stromal CD8+ T cells or tumor nest CD8+ T cells.

PDF file - 308K, Characterization of immune cells in and out TLS.

PDF file - 131K, Clinical characteristic of NSCLC patients with DC-Lamp High versus DC-Lamp Low tumors.

PDF file - 123K, CD62L- T cells represent the main T cell population in human lung tumors.

PDF file - 380K, Gene expression levels related to immune populations, TLS, Th-orientation, cytotoxicity, T-cell activation, immuno-suppression, inflammation and angiogenesis according to the high and low density of mature DC.

PDF file - 200K, Twelve-color analysis of the immune cell populations in freshly resected tumors.

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.

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PDF file - 151K, Expression on tumor-infiltrating T cells of molecules involved in cytotoxicity, activation, and Th1 orientation.

PDF file - 168K, Gene expression on tumors stratified according to the high/low density of DC-Lamp+ mature DC.

PDF file - 271K, Coordination of gene expression levels related to immune populations, TLS, Th-orientation, cytotoxicity, T-cell activation, immuno-suppression, inflammation and angiogenesis according to the high density of mature DC.

PDF file - 99K, Baseline characteristics of the NSCLC fresh tumors enrolled in the prospective study.

PDF file - 98K, Baseline characteristics of the NSCLC frozen tumors enrolled in the prospective study.

PDF file - 78K, Multivariate Cox proportional hazards analysis for overall survival in NSCLC patients.

PDF file - 191K, Antibodies used in the study.

Background: MICA and MICB are tightly regulated stress-induced proteins that trigger the immune system by binding to the activating receptor NKG2D on cytotoxic lymphocytes. MICA and MICB are highly polymorphic molecules with prevalent expression on several types of solid tumors and limited expression in normal/healthy tissues, making them attractive targets for therapeutic intervention. Methods: We have generated a series of anti-MICA and MICB cross-reactive antibodies with the unique feature of binding to the most prevalent isoforms of both these molecules. Results: The anti-MICA and MICB antibody MICAB1, a human IgG1 Fc-engineered monoclonal antibody (mAb), displayed potent antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) of MICA/B-expressing tumor cells in vitro. However, it showed insufficient efficiency against solid tumors in vivo, which prompted the development of antibody-drug conjugates (ADC). Indeed, optimal tumor control was achieved with MICAB1-ADC format in several solid tumor models, including patient-derived xenografts (PDX) and carcinogen-induced tumors in immunocompetent MICAgen transgenic mice. Conclusions: These data indicate that MICA and MICB are promising targets for cytotoxic immunotherapy.

International audience; Harnessing innate immunity is emerging as a promising therapeutic approach in cancer. We report here the design of tetraspecific molecules engaging natural killer (NK) cell-activating receptors NKp46 and CD16a, the β-chain of the interleukin-2 receptor (IL-2R), and a tumor-associated antigen (TAA). In vitro, these tetraspecific antibody-based natural killer cell engager therapeutics (ANKETs) induce a preferential activation and proliferation of NK cells, and the binding to the targeted TAA triggers NK cell cytotoxicity and cytokine and chemokine production. In vivo, tetraspecific ANKETs induce NK cell proliferation and their accumulation at the tumor bed, as well as the control of local and disseminated tumors. Treatment of non-human primates with CD20-directed tetraspecific ANKET leads to CD20+ circulating B cell depletion, with minimal systemic cytokine release and no sign of toxicity. Tetraspecific ANKETs, thus, constitute a technological platform for harnessing NK cells as next-generation cancer immunotherapies.

International audience

Background & AimsAnti–GM-CSF autoantibodies (aGMAb) are detected in ileal Crohn’s Disease (CD) patients. Their induction and mode of action impacting homeostasis during, or prior to disease are not well understood. We aimed to investigate the underlying mechanisms leading to the induction of aGMAb, from functional orientation to recognized epitopes, for their impact on intestinal immune homeostasis and use as predictive biomarker for complicated CD.MethodsUsing longitudinally collected sera from active component US personnel, we characterize naturally occurring aGMAb in a subset of CD patients years before disease onset. We employed biochemical, cellular, and transcriptional analysis to uncover a mechanism that governs the impaired immune balance in CD years prior to diagnosis.ResultsNeutralizing aGMAb are specific to posttranslational glycosylations on GM-CSF, detectable years prior to diagnosis, and associated with complicated CD at presentation. Glycosylation and production of GM-CSF change in CD patients, altering myeloid homeostasis and destabilizing group 3 innate lymphoid cells. Perturbations in immune homeostasis precede the inflammation and are detectable in the non-inflamed CD mucosa of patients presenting with anti-GM-CSF autoantibodies.ConclusionsAnti-GM-CSF autoantibodies predict the diagnosis of complicated CD, have unique epitopes, and impair myeloid cell homeostasis across the ILC3-GM-CSF-myeloid cell axis, altering intestinal immune homeostasis long before the diagnosis of disease.

Anti-granulocyte macrophage-colony stimulating factor autoantibodies (aGMAbs) are detected in patients with ileal Crohn's disease (CD). Their induction and mode of action during or before disease are not well understood. We aimed to investigate the underlying mechanisms associated with aGMAb induction, from functional orientation to recognized epitopes, for their impact on intestinal immune homeostasis and use as a predictive biomarker for complicated CD.We characterized using enzyme-linked immunosorbent assay naturally occurring aGMAbs in longitudinal serum samples from patients archived before the diagnosis of CD (n = 220) as well as from 400 healthy individuals (matched controls) as part of the US Defense Medical Surveillance System. We used biochemical, cellular, and transcriptional analysis to uncover a mechanism that governs the impaired immune balance in CD mucosa after diagnosis.Neutralizing aGMAbs were found to be specific for post-translational glycosylation on granulocyte macrophage-colony stimulating factor (GM-CSF), detectable years before diagnosis, and associated with complicated CD at presentation. Glycosylation of GM-CSF was altered in patients with CD, and aGMAb affected myeloid homeostasis and promoted group 1 innate lymphoid cells. Perturbations in immune homeostasis preceded the diagnosis in the serum of patients with CD presenting with aGMAb and were detectable in the noninflamed CD mucosa.Anti-GMAbs predict the diagnosis of complicated CD long before the diagnosis of disease, recognize uniquely glycosylated epitopes, and impair myeloid cell and innate lymphoid cell balance associated with altered intestinal immune homeostasis.

Background Coronavirus disease 2019 (COVID-19) is a new pandemic disease caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The C5a anaphylatoxin and its receptor C5aR1 (CD88) play a key role in the initiation and maintenance of several inflammatory responses, by recruiting and activating neutrophils and monocytes in the lungs. Methods We provide a longitudinal analysis of immune responses, including immune cell phenotyping and assessments of the soluble factors present in the blood and broncho-alveolar lavage fluid (BALF) of patients at various stages of COVID-19 severity: paucisymptomatic, pneumonia and acute respiratory distress syndrome (ARDS) Results We report an increase in soluble C5a levels proportional to COVID-19 severity and high levels of C5aR1 expression in blood and pulmonary myeloid cells, supporting a role for the C5a-C5aR1 axis in the pathophysiology of ARDS. Avdoralimab, an anti-C5aR1 therapeutic monoclonal antibodies (mAbs) prevented C5a-mediated human myeloid cell recruitment and activation, and inhibited acute lung injury (ALI) in human C5aR1 knockin mice. Conclusions These results support the evaluation of avdoralimab to block C5a-C5aR1 axis as a mean of limiting myeloid cell infiltration in damaged organs and preventing the excessive lung inflammation and endothelialitis associated with ARDS in COVID-19 patients Acknowledgements The Explore COVID-19 IPH group, the Explore COVID-19 Marseille Immunopole group. Ethics Approval Human study protocol was approved by the Committee for the Protection of Persons Ile-de-France III – France (#2020-A00757-32). Animal experiments were approved by the ministere de l’enseignement superieur, de la recherche et de l’innovation – France (APAFIS#25418-2020051512242806 v2).

Disease states and cellular compartments can display a remarkable amount of heterogeneity, and truly appreciating this heterogeneity requires the ability to detect and probe each subpopulation present. A myriad of recent single-cell assays has allowed for in-depth analysis of these diverse cellular populations; however, fully understanding the interplay between each cell type requires knowledge not only of their mere presence but also of their spatial organization and their relation one to the other. Immunohistochemistry allows for the visualization of cells and tissue; however, standard techniques only allow for the use of very few probes on a single specimen, not allowing for in-depth analysis of complex cellular heterogeneity. A number of multiplex imaging techniques, such as immunofluorescence and multiplex immunohistochemistry, have been proposed to allow probing more cellular markers at once; however, many of these techniques still have their limitations. The use of fluorescent markers has an inherent limitation to the number of probes that can be simultaneously used due to spectral overlap. Moreover, other proposed multiplex IHC methods are time-consuming and require expensive reagents. Still, many of the methods rely on frozen tissue, which deviates from standards in human pathological evaluation. Here, we describe a multiplex IHC technique, staining for consecutive markers on a single slide, which utilizes similar steps and similar reagents as standard IHC, thus making it possible for any lab with standard IHC capabilities to perform this useful procedure. This method has been validated and confirmed that consecutive markers can be stained without the risk of cross-reactivity between staining cycles. Furthermore, we have validated that this technique does not lead to decreased antigenicity of subsequent epitopes probed, nor does it lead to steric hindrance.

Hogstad et al. show that the somatic BRAFV600E mutation in myeloid dendritic cell precursors in Langerhans cell histiocytosis promotes lesion formation through impaired dendritic cell migration and resistance to apoptosis, which can be rescued with targeted MAPK pathway inhibition., Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia characterized by granulomatous lesions containing pathological CD207+ dendritic cells (DCs) with constitutively activated mitogen-activated protein kinase (MAPK) pathway signaling. Approximately 60% of LCH patients harbor somatic BRAFV600E mutations localizing to CD207+ DCs within lesions. However, the mechanisms driving BRAFV600E+ LCH cell accumulation in lesions remain unknown. Here we show that sustained extracellular signal–related kinase activity induced by BRAFV600E inhibits C-C motif chemokine receptor 7 (CCR7)–mediated DC migration, trapping DCs in tissue lesions. Additionally, BRAFV600E increases expression of BCL2-like protein 1 (BCL2L1) in DCs, resulting in resistance to apoptosis. Pharmacological MAPK inhibition restores migration and apoptosis potential in a mouse LCH model, as well as in primary human LCH cells. We also demonstrate that MEK inhibitor-loaded nanoparticles have the capacity to concentrate drug delivery to phagocytic cells, significantly reducing off-target toxicity. Collectively, our results indicate that MAPK tightly suppresses DC migration and augments DC survival, rendering DCs in LCH lesions trapped and resistant to cell death.

Background Crohn’s disease (CD) is a heterogenous, chronic inflammatory disorder driven by a combination of genetic, environmental, and microbiota-dependent risk factors. Mononuclear phagocytes (MNP) are crucial cells that maintain intestinal homeostasis. An important cytokine for MNP survival and function is granulocyte-macrophage colony stimulating factor (GM-CSF). Interestingly, several studies reported CD-associated genetic risk variants within the GM-CSF receptor and its downstream signaling components. Furthermore, high titers of autoantibodies specific to GM-CSF can be detected in CD patients. Taken together, this data suggests an important role for GM-CSF in abrogation of CD development in a subgroup of patients. Aims This study sought to investigate the function of GM-CSF autoantibodies in CD. Methods We retrospectively quantified and characterized GM-CSF autoantibodies in sera of 220 CD, 200 ulcerative colitis (UC) patients, and 220 healthy controls (HC) sampled at 3 time points prior to disease diagnosis and one time point after diagnosis. ELISA was used to determine GM-CSF autoantibody titers and isotypes followed by in vitro multiplexed mass cytometry (CyTOF) neutralization assays on peripheral blood mononuclear cells. Flow cytometry and CyTOF were used to map the profile of immune cells isolated from inflamed and non-inflamed CD mucosa. Results Our data demonstrates that GM-CSF autoantibodies are specific to CD, significantly elevated up to 7 years prior to diagnosis of disease, and correlate with disease location, severity, and complications at the time of diagnosis. Moreover, in contrast to GM-CSF autoantibodies in pulmonary alveolar proteinosis patients, CD-associated autoantibodies neutralize GM-CSF via specific recognition of post-translational modifications (PTM), affecting MNP function. Removal of PTM enabled GM-CSF to escape autoantibody binding and restored MNP response to GM-CSF in the presence of neutralizing antibodies, indicating a potential therapeutic avenue. Furthermore, we identified group 3 innate lymphoid cells (ILC3) as a major source of GM-CSF in the healthy intestinal tract, suggesting intriguing crosstalk of MNP and ILC3 across the GM-CSF-GM-CSFR axis. Conclusions Our results identify GM-CSF autoantibodies as predictive serological biomarker for CD in a subgroup of patients presenting with severe and complicated form of disease at the time of diagnosis. The presence of GM-CSF autoantibodies precedes the onset of CD by several years and likely abrogates homeostatic immune cell crosstalk involving ILC3 and MNP, suggesting the development of a pre-diseased state in CD patients. Funding Agencies CIHRDr. Edward Ketchum Graduate Scholarship

Background: MICA and MICB are tightly regulated stress-induced proteins that trigger the immune system by binding to the activating receptor NKG2D on cytotoxic lymphocytes. MICA and MICB are highly polymorphic molecules with prevalent expression on several types of solid tumors and limited expression in normal/healthy tissues, making them attractive targets for therapeutic intervention. Methods: We have generated a series of anti-MICA and MICB cross-reactive antibodies with the unique feature of binding to the most prevalent isoforms of both these molecules. Results: The anti-MICA and MICB antibody MICAB1, a human IgG1 Fc-engineered monoclonal antibody (mAb), displayed potent antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) of MICA/B-expressing tumor cells in vitro. However, it showed insufficient efficiency against solid tumors in vivo, which prompted the development of antibody-drug conjugates (ADC). Indeed, optimal tumor control was achieved with MICAB1-ADC format in several solid tumor models, including patient-derived xenografts (PDX) and carcinogen-induced tumors in immunocompetent MICAgen transgenic mice. Conclusions: These data indicate that MICA and MICB are promising targets for cytotoxic immunotherapy.

SUMMARYAtherosclerosis is driven by multifaceted contributions of the immune system within the circulation and at vascular focal sites. Yet the specific immune dysregulations within the atherosclerotic lesions that lead to clinical cerebro- and cardiovascular complications (i.e. ischemic stroke and myocardial infarction) are poorly understood. Here, using single-cell mass cytometry with Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) we found that atherosclerotic plaques were enriched in activated, differentiated, and exhausted subsets of T cells vs. blood. Next, using single-cell proteomic, transcriptomic, and cell-to-cell interaction analyses we found unique functional dysregulations of both T cells and macrophages in plaques of patients with clinically symptomatic (SYM; recent stroke of TIA) or asymptomatic (ASYM, no recent stroke) carotid artery disease. SYM plaques were enriched with a distinct CD4+T cell subset, and T cells were activated, differentiated and presented subset specific exhaustion. SYM macrophages presented alternatively activated phenotypes including subsets associated with plaque vulnerability. In ASYM plaques, T cells and macrophages were activated and displayed a strong IL-1β signaling across cell types, that was absent in SYM plaques. The identification of plaque-specific innate and adaptive immune dysregulations associated with cerebrovascular events provides the basis for the design of precisely tailored cardiovascular immunotherapies.

Although common evolutionary principles drive the growth of cancer cells regardless of the tissue of origin, the microenvironment in which tumours arise substantially differs across various organ sites. Recent studies have established that, in addition to cell-intrinsic effects, tumour growth regulation also depends on local cues driven by tissue environmental factors. In this Review, we discuss how tissue-specific determinants might influence tumour development and argue that unravelling the tissue-specific contribution to tumour immunity should help the development of precise immunotherapeutic strategies for patients with cancer.

Background In uveal melanomas, immune infiltration is a marker of poor prognosis. This work intended to decipher the biological characteristics of intra‐tumor immune population, compare it to other established biomarkers and to patients' outcome. Methods Primary, untreated, and mainly large uveal melanomas with retinal detachment were analyzed using: transcriptomic profiling (n = 15), RT‐qPCR (n = 36), immunohistochemistry (n = 89), Multiplex Ligation‐dependent Probe Amplification (MLPA) for copy number alterations (CNA) analysis (n = 89), array‐CGH (n = 17), and survival statistics (n = 86). Results Gene expression analysis divided uveal melanomas into two groups, according to the IFNγ/STAT1‐IRF1 pathway activation. Tumors with IFNγ‐signature had poorer prognosis and showed increased infiltration of CD8+ T lymphocytes and macrophages. Cox multivariate analyses of immune cell infiltration with MLPA data delineated better prognostic value for three prognostic groups (three‐tier stratification) than two (two‐tier stratification). CNA‐based model comprising monosomy 3, 8q amplification, and LZTS1and NBL1 deletions emerged as the best predictor for disease‐free survival. It outperformed immune cell infiltration in receiver operating characteristic curves. The model that combined CNA and immune infiltration defined risk‐groups according to the number of DNA alterations. Immune cell infiltration was increased in the high‐risk group (73.7%), where it did not correlate with patient survival, while it was associated with poorer outcome in the intermediate risk‐group. Conclusions High degree of immune cell infiltration occurs in a subset of uveal melanomas, is interferon‐gamma‐related, and associated with poor survival. It allows for two‐tier stratification, which is prognostically less efficient than a three‐tier one. The best prognostic stratification is by CNA model with three risk‐groups where immune cell infiltration impacts only some subgroups.

Background T lymphocytes and HLA expression on tumor cell both influence prognostic of localized colorectal cancer, but their role following chemotherapy in patients with liver metastatic colorectal cancer (mCRC) was not addressed. Methods One hundred fourteen patients treated in curative intend of liver mCRC were included in this retrospective study. Patients were either untreated or treated with neoadjuvant therapy containing an anti-EGFR, bevacizumab or oxaliplatin. Immune densities were quantified in the tumor core and in invasive margin of metastases, using Qupath software or a pathologist’s quantification. CD8, NKp46, Foxp3, CD163, HLA, PD-L1 were analyzed and were correlated with progression free survival (PFS) and overall survival (OS) using multivariable Cox proportional hazards models. Results In the whole cohort only a high CD8+ cells infiltrate, a high HLA-I expression and wild-type RAS/RAF status were associated with a better overall survival in both univariate and multivariate model. Moreover, CD8+ cells immune infiltrate at invasive margin combined to HLA expression in cancer cell could increase patient’s outcome prediction. RAS status but not immune cell infiltrate was associated with HLA expression on tumor cells. In comparison to untreated patients, neoadjuvant chemotherapy induced CD8+ cells recruitment and increased PD-L1 staining in immune infiltrates only for WT RAS patients. In this context, anti-EGFR and oxaliplatin based chemotherapy are the most powerful to induce CD8+ cells mobilization within the metastatic site. Conclusions While CD8 infiltrate and HLA expression appear to be prognostic for mCRC, CD8 and PD-L1 infiltrate are enhanced by neoadjuvant chemotherapy in mCRC under RAS status dependence. Electronic supplementary material The online version of this article (10.1186/s40425-018-0438-3) contains supplementary material, which is available to authorized users.

Large numbers of melanoma lesions develop resistance to targeted inhibition of mutant BRAF or fail to respond to checkpoint blockade. We explored whether modulation of intratumoral antigen-presenting cells (APCs) could increase responses to these therapies. Using mouse melanoma models, we found that CD103(+) dendritic cells (DCs) were the only APCs transporting intact antigens to the lymph nodes and priming tumor-specific CD8(+) T cells. CD103(+) DCs were required to promote anti-tumoral effects upon blockade of the checkpoint ligand PD-L1; however, PD-L1 inhibition only led to partial responses. Systemic administration of the growth factor FLT3L followed by intratumoral poly I:C injections expanded and activated CD103(+) DC progenitors in the tumor, enhancing responses to BRAF and PD-L1 blockade and protecting mice from tumor rechallenge. Thus, the paucity of activated CD103(+) DCs in tumors limits checkpoint-blockade efficacy and combined FLT3L and poly I:C therapy can enhance tumor responses to checkpoint and BRAF blockade.

6551 Background: Immune cell (IC) infiltrates in primary tumors (PT) have been identified as prognostic markers in head and neck squamous cell carcinoma (HNSCC). IC densities may differ among PT, lymph node metastasis (LNM) and recurrent disease (RD) and by primary disease site (oral cavity- OC, oropharynx- OP, hypopharynx- HP, larynx- L). Here, we compare CD3 and CD20 IC densities in PT, LNM and RD in paired samples from different disease sites and determine the prognostic impact of IC infiltrates. Methods: Tissue microarrays with 425 PT, 198 LNM and 46 RD samples--each in triplicate--were stained immunohistochemically for CD3 and CD20 in the same slide. Immune cell densities per mm2 were determined using a digital image analysis software (QuPath). Individual means were calculated from triplicates of each sample. IC infiltrates from different sample types (PT, LNM, RD) and primary tumor sites were compared using Kruskal-Wallis and Mann-Whitney-U tests. Paired samples were compared using Wilcoxon signed rank test. IC densities were classified as CD3 high/low and CD20 high/low for each primary tumor site using the individual median as a cut-off. Overall survival (OS) was calculated using the Kaplan-Meier method. P-values for each hypothesis were corrected using a false discovery rate of 5%. Results: CD3 and CD20 IC densities differed significantly by sample type (both p0.05). CD3 densities were significantly higher than CD20 densities in all sample types. CD3high patients had the best prognosis in all sites except for OC (qlow/CD20high patients had the worst OS compared to CD3low/CD20low and CD3high/CD20high or even CD3high/CD20low patients (p=0.018) who had the best prognosis. Conclusions: IC densities of CD3 and CD20 vary by sample type and primary site. Except for OC, in all sites the prognostic impact is determined by CD3high, whereas in OSCC only the combination of CD3 and CD20 IC densities achieves a good prognostic value. Interestingly, CD3low/CD20high patients have the worst overall survival in OC patients. Further work is needed to understand the interaction of B- and T cell infiltrates in the tumor, especially in OC.

Atherosclerosis is driven by multifaceted contributions of the immune system within the circulation and at vascular focal sites. However, specific characteristics of dysregulated immune cells within atherosclerotic lesions that lead to clinical events such as ischemic stroke or myocardial infarction are poorly understood. Here, using single-cell proteomic and transcriptomic analyses, we uncovered distinct features of both T cells and macrophages in carotid artery plaques of patients with clinically symptomatic disease (recent stroke or transient ischemic attack) compared to asymptomatic disease (no recent stroke). Plaques from symptomatic patients were characterized by a distinct subset of CD4+ T cells and by T cells that were activated and differentiated. Moreover, some T cell subsets in these plaques presented markers of T cell exhaustion. Additionally, macrophages from these plaques contained alternatively activated phenotypes, including subsets associated with plaque vulnerability. In plaques from asymptomatic patients, T cells and macrophages were activated and displayed evidence of interleukin-1β signaling. The identification of specific features of innate and adaptive immune cells in plaques that are associated with cerebrovascular events may enable the design of more precisely tailored cardiovascular immunotherapies.

Ulcerated melanomas may have a unique biology and microenvironment. We test whether markers of immune infiltration correlate with clinical outcome in ulcerated compared to non-ulcerated primary melanoma tumors.Sixty-two stage II-III cutaneous melanomas, 32 ulcerated and 30 non-ulcerated, were analyzed for tumor-infiltrating lymphocytes (TILs). Immunohistochemistry (IHC) was performed for CD2, a marker previously shown to correlate with overall survival (OS) and recurrence-free survival (RFS) in this patient population. IHC using antibody, VE1, to BRAF V600E was also performed on a subset of 41 tumors to assess the relationship of BRAF mutation to immune markers.We found, using Cox regression models, that the presence of TILs was associated with improved OS (p = 0.034) and RFS (p = 0.002) in ulcerated melanoma tumors, but not in non-ulcerated melanoma (p = 0.632, 0.416). CD2 expression also was correlated with improved OS (p = 0.021) and RFS (p = 0.001) in ulcerated melanoma, but no relationship was seen in non-ulcerated melanoma (p = 0.427, 0.682). In this small population, BRAF status did not correlate with TILs or CD2+ count.Our data show that immune markers including TILs and CD2 count correlate more closely with survival in ulcerated melanomas than that in non-ulcerated melanomas. We propose that immune biomarkers may be particularly relevant to ulcerated, as compared to non-ulcerated, melanomas and that this merits study in larger populations.