Germinal center-dependent and -independent immune responses of tumor-infiltrating B cells in human cancers.

B cells play essential roles in immunity, mainly through the production of high affinity plasma cells (PCs) and memory B (Bmem) cells. The affinity maturation and differentiation of B cells rely on the integration of B-cell receptor (BCR) intrinsic and extrinsic signals provided by antigen binding and the microenvironment, respectively. In recent years, tumor infiltrating B (TIL-B) cells and PCs (TIL-PCs) have been revealed as important players in antitumor responses in human cancers, but their interplay and dynamics remain largely unknown. In lymphoid organs, B-cell responses involve both germinal center (GC)-dependent and GC-independent pathways for Bmem cell and PC production. Affinity maturation of BCR repertoires occurs in GC reactions with specific spatiotemporal dynamics of signal integration by B cells. In general, the reactivation of high-affinity Bmem cells by antigens triggers GC-independent production of large numbers of PC without BCR rediversification. Understanding B-cell dynamics in immune responses requires the integration of multiple tools and readouts such as single-cell phenotyping and RNA-seq, in situ analyses, BCR repertoire analysis, BCR specificity and affinity assays, and functional tests. Here, we review how those tools have recently been applied to study TIL-B cells and TIL-PC in different types of solid tumors. We assessed the published evidence for different models of TIL-B-cell dynamics involving GC-dependent or GC-independent local responses and the resulting production of antigen-specific PCs. Altogether, we highlight the need for more integrative B-cell immunology studies to rationally investigate TIL-B cells as a leverage for antitumor therapies.

Autologous T cell responses to primary human colorectal cancer spheroids are enhanced by ectonucleotidase inhibition.

OBJECTIVE: T 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. DESIGN: In 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. RESULTS: We 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. CONCLUSION: In CRC, CD39 is strongly expressed on tumour infiltrating lymphocytes and its inhibition represents a promising therapeutic strategy for treating patients.

Antitumor immunity induced by antibody-based natural killer cell engager therapeutics armed with not-alpha IL-2 variant.

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 ß-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.

Neutralizing Anti-Granulocyte Macrophage-Colony Stimulating Factor Autoantibodies Recognize Post-Translational Glycosylations on Granulocyte Macrophage-Colony Stimulating Factor Years Before Diagnosis and Predict Complicated Crohn's Disease.

BACKGROUND & AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Targeting MICA/B with cytotoxic therapeutic antibodies leads to tumor control.

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.

Association of COVID-19 inflammation with activation of the C5a-C5aR1 axis.

Coronavirus disease 2019 (COVID-19) is a disease caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has resulted in a pandemic1. The C5a complement factor and its receptor C5aR1 (also known as CD88) have a key role in the initiation and maintenance of several inflammatory responses by recruiting and activating neutrophils and monocytes1. Here we provide a longitudinal analysis of immune responses, including phenotypic analyses of immune cells and assessments of the soluble factors that are present in the blood and bronchoalveolar lavage fluid of patients at various stages of COVID-19 severity, including those who were paucisymptomatic or had pneumonia or acute respiratory distress syndrome. The levels of soluble C5a were increased in proportion to the severity of COVID-19 and high expression levels of C5aR1 receptors were found in blood and pulmonary myeloid cells, which supports a role for the C5a-C5aR1 axis in the pathophysiology of acute respiratory distress syndrome. Anti-C5aR1 therapeutic monoclonal antibodies prevented the C5a-mediated recruitment and activation of human myeloid cells, and inhibited acute lung injury in human C5aR1 knock-in mice. These results suggest that blockade of the C5a-C5aR1 axis could be used to limit the infiltration of myeloid cells in damaged organs and prevent the excessive lung inflammation and endothelialitis that are associated with acute respiratory distress syndrome in patients with COVID-19.

Multiplexed Immunohistochemical Consecutive Staining on Single Slide (MICSSS): Multiplexed Chromogenic IHC Assay for High-Dimensional Tissue Analysis.

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.

Single-cell immune landscape of human atherosclerotic plaques.

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-1ß 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.

Multifunctional Natural Killer Cell Engagers Targeting NKp46 Trigger Protective Tumor Immunity.

Over the last decade, various new therapies have been developed to promote anti-tumor immunity. Despite interesting clinical results in hematological malignancies, the development of bispecific killer-cell-engager antibody formats directed against tumor cells and stimulating anti-tumor T cell immunity has proved challenging, mostly due to toxicity problems. We report here the generation of trifunctional natural killer (NK) cell engagers (NKCEs), targeting two activating receptors, NKp46 and CD16, on NK cells and a tumor antigen on cancer cells. Trifunctional NKCEs were more potent in vitro than clinical therapeutic antibodies targeting the same tumor antigen. They had similar in vivo pharmacokinetics to full IgG antibodies and no off-target effects and efficiently controlled tumor growth in mouse models of solid and invasive tumors. Trifunctional NKCEs thus constitute a new generation of molecules for fighting cancer. VIDEO ABSTRACT.

DNA alteration-based classification of uveal melanoma gives better prognostic stratification than immune infiltration, which has a neutral effect in high-risk group.

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.

Cocultures of human colorectal tumor spheroids with immune cells reveal the therapeutic potential of MICA/B and NKG2A targeting for cancer treatment.

BACKGROUND: Immunotherapies still fail to benefit colorectal cancer (CRC) patients. Relevant functional assays aimed at studying these failures and the efficacy of cancer immunotherapy in human are scarce. 3D tumor cultures, called tumor organoids or spheroids, represent interesting models to study cancer treatments and could help to challenge these issues. METHODS: We analyzed heterotypic cocultures of human colon tumor-derived spheroids with immune cells to assess the infiltration, activation and function of T and NK cells toward human colorectal tumors in vitro. RESULTS: We showed that allogeneic T and NK cells rapidly infiltrated cell line-derived spheroids, inducing immune-mediated tumor cell apoptosis and spheroid destruction. NKG2D, a key activator of cytotoxic responses, was engaged on infiltrating cells. We thus assessed the therapeutic potential of an antibody targeting the specific ligands of NKG2D, MICA and MICB, in this system. Anti-MICA/B enhanced immune-dependent destruction of tumor spheroid by driving an increased NK cells infiltration and activation. Interestingly, tumor cells reacted to immune infiltration by upregulating HLA-E, ligand of the inhibitory receptor NKG2A expressed by CD8 and NK cells. NKG2A was increased after anti-MICA/B treatment and, accordingly, combination of anti-MICA/B and anti-NKG2A was synergistic. These observations were ultimately confirmed in a clinical relevant model of coculture between CRC patients-derived spheroids and autologous tumor-infiltrating lymphocytes. CONCLUSIONS: Altogether, we show that tumor spheroids represent a relevant tool to study tumor-lymphocyte interactions on human tissues and revealed the antitumor potential of immunomodulatory antibodies targeting MICA/B and NKG2A.

Host tissue determinants of tumour immunity.

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.

Anti-NKG2A mAb Is a Checkpoint Inhibitor that Promotes Anti-tumor Immunity by Unleashing Both T and NK Cells.

Checkpoint inhibitors have revolutionized cancer treatment. However, only a minority of patients respond to these immunotherapies. Here, we report that blocking the inhibitory NKG2A receptor enhances tumor immunity by promoting both natural killer (NK) and CD8+ T cell effector functions in mice and humans. Monalizumab, a humanized anti-NKG2A antibody, enhanced NK cell activity against various tumor cells and rescued CD8+ T cell function in combination with PD-x axis blockade. Monalizumab also stimulated NK cell activity against antibody-coated target cells. Interim results of a phase II trial of monalizumab plus cetuximab in previously treated squamous cell carcinoma of the head and neck showed a 31% objective response rate. Most common adverse events were fatigue (17%), pyrexia (13%), and headache (10%). NKG2A targeting with monalizumab is thus a novel checkpoint inhibitory mechanism promoting anti-tumor immunity by enhancing the activity of both T and NK cells, which may complement first-generation immunotherapies against cancer.

RAS status and neoadjuvant chemotherapy impact CD8+ cells and tumor HLA class I expression in liver metastatic colorectal cancer.

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.

RAF/MEK/extracellular signal-related kinase pathway suppresses dendritic cell migration and traps dendritic cells in Langerhans cell histiocytosis lesions.

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.

Identification of unique neoantigen qualities in long-term survivors of pancreatic cancer.

Pancreatic ductal adenocarcinoma is a lethal cancer with fewer than 7% of patients surviving past 5 years. T-cell immunity has been linked to the exceptional outcome of the few long-term survivors, yet the relevant antigens remain unknown. Here we use genetic, immunohistochemical and transcriptional immunoprofiling, computational biophysics, and functional assays to identify T-cell antigens in long-term survivors of pancreatic cancer. Using whole-exome sequencing and in silico neoantigen prediction, we found that tumours with both the highest neoantigen number and the most abundant CD8+ T-cell infiltrates, but neither alone, stratified patients with the longest survival. Investigating the specific neoantigen qualities promoting T-cell activation in long-term survivors, we discovered that these individuals were enriched in neoantigen qualities defined by a fitness model, and neoantigens in the tumour antigen MUC16 (also known as CA125). A neoantigen quality fitness model conferring greater immunogenicity to neoantigens with differential presentation and homology to infectious disease-derived peptides identified long-term survivors in two independent datasets, whereas a neoantigen quantity model ascribing greater immunogenicity to increasing neoantigen number alone did not. We detected intratumoural and lasting circulating T-cell reactivity to both high-quality and MUC16 neoantigens in long-term survivors of pancreatic cancer, including clones with specificity to both high-quality neoantigens and predicted cross-reactive microbial epitopes, consistent with neoantigen molecular mimicry. Notably, we observed selective loss of high-quality and MUC16 neoantigenic clones on metastatic progression, suggesting neoantigen immunoediting. Our results identify neoantigens with unique qualities as T-cell targets in pancreatic ductal adenocarcinoma. More broadly, we identify neoantigen quality as a biomarker for immunogenic tumours that may guide the application of immunotherapies.

Innate Immune Landscape in Early Lung Adenocarcinoma by Paired Single-Cell Analyses.

To guide the design of immunotherapy strategies for patients with early stage lung tumors, we developed a multiscale immune profiling strategy to map the immune landscape of early lung adenocarcinoma lesions to search for tumor-driven immune changes. Utilizing a barcoding method that allows a simultaneous single-cell analysis of the tumor, non-involved lung, and blood cells, we provide a detailed immune cell atlas of early lung tumors. We show that stage I lung adenocarcinoma lesions already harbor significantly altered T cell and NK cell compartments. Moreover, we identified changes in tumor-infiltrating myeloid cell (TIM) subsets that likely compromise anti-tumor T cell immunity. Paired single-cell analyses thus offer valuable knowledge of tumor-driven immune changes, providing a powerful tool for the rational design of immune therapies. VIDEO ABSTRACT.

Quantification of hepatocellular carcinoma heterogeneity with multiparametric magnetic resonance imaging.

Tumour heterogeneity poses a significant challenge for treatment stratification. The goals of this study were to quantify heterogeneity in hepatocellular carcinoma (HCC) using multiparametric magnetic resonance imaging (mpMRI), and to report preliminary data correlating quantitative MRI parameters with advanced histopathology and gene expression in a patient subset. Thirty-two HCC patients with 39 HCC lesions underwent mpMRI including diffusion-weighted imaging (DWI), blood-oxygenation-level-dependent (BOLD), tissue-oxygenation-level-dependent (TOLD) and dynamic contrast-enhanced (DCE)-MRI. Histogram characteristics [central tendency (mean, median) and heterogeneity (standard deviation, kurtosis, skewness) MRI parameters] in HCC and liver parenchyma were compared using Wilcoxon signed-rank tests. Histogram data was correlated between MRI methods in all patients and with histopathology and gene expression in 14 patients. HCCs exhibited significantly higher intra-tissue heterogeneity vs. liver with all MRI methods (P < 0.030). Although central tendency parameters showed significant correlations between MRI methods and with each of histopathology and gene expression, heterogeneity parameters exhibited additional complementary correlations between BOLD and DCE-MRI and with histopathologic hypoxia marker HIF1α and gene expression of Wnt target GLUL, pharmacological target FGFR4, stemness markers EPCAM and KRT19 and immune checkpoint PDCD1. Histogram analysis combining central tendency and heterogeneity mpMRI features is promising for non-invasive HCC characterization on the imaging, histologic and genomics levels.

MAGE expression in head and neck squamous cell carcinoma primary tumors, lymph node metastases and respective recurrences-implications for immunotherapy.

Melanoma associated antigens (MAGE) are potential targets for immunotherapy and have been associated with poor overall survival (OS) in head and neck squamous cell carcinoma (HNSCC). However, little is known about MAGE in lymph node metastases (LNM) and recurrent disease (RD) of HNSCC.To assess whether MAGE expression increases with metastasis or recurrence, a tissue microarray (TMA) of 552 primary tumors (PT), 219 LNM and 75 RD was evaluated by immunohistochemistry for MAGE antigens using three monoclonal antibodies to multiple MAGE family members. Mean expression intensity (MEI) was obtained from triplicates of each tumor specimen.The median MEI compared between PT, LNM and RD was significantly higher in LNM and RD. In paired samples, MEI was comparable in PT to respective LNM, but significantly different from RD. Up to 25% of patients were negative for pan-MAGE or MAGE-A3/A4 in PT, but positive in RD. The prognostic impact of MAGE expression was validated in the TMA cohort and also in TCGA data (mRNA). OS was significantly lower for patients expressing pan-MAGE or MAGE-A3/A4 in both independent cohorts.MAGE expression was confirmed as a prognostic marker in HNSCC and may be important for immunotherapeutic strategies as a shared antigen.

Host-Protozoan Interactions Protect from Mucosal Infections through Activation of the Inflammasome.

While conventional pathogenic protists have been extensively studied, there is an underappreciated constitutive protist microbiota that is an integral part of the vertebrate microbiome. The impact of these species on the host and their potential contributions to mucosal immune homeostasis remain poorly studied. Here, we show that the protozoan Tritrichomonas musculis activates the host epithelial inflammasome to induce IL-18 release. Epithelial-derived IL-18 promotes dendritic cell-driven Th1 and Th17 immunity and confers dramatic protection from mucosal bacterial infections. Along with its role as a "protistic" antibiotic, colonization with T. musculis exacerbates the development of T-cell-driven colitis and sporadic colorectal tumors. Our findings demonstrate a novel mutualistic host-protozoan interaction that increases mucosal host defenses at the cost of an increased risk of inflammatory disease.