Defining biomarkers in oral cancer according to smoking and drinking status.

Introduction: Oral Squamous Cell Carcinomas (OSCC) are mostly related to tobacco consumption eventually associated to alcohol (Smoker/Drinker patients: SD), but 25-30% of the patients have no identified risk factors (Non-Smoker/Non-Drinker patients: NSND). We hypothesized that these patients have distinguishable immune profiles that could be useful for prognosis. Materials and Methods: Cells present in immune tumor microenvironment (TME) and blood from 87 OSCC HPV-negative patients were analyzed using a multiparameter flow cytometry assay, in a prospective case-control study. Cytokine levels in tumor supernatants and blood were determined by a cytometric bead array (CBA) assay. Results: Normal gingiva and blood from healthy donors (HD) were used as controls. A significant increase of granulocytes (p<0.05 for blood), of monocytes-macrophages (p<0.01 for blood) and of CD4+ T cells expressing CD45RO and CCR6 (p<0.001 for blood; p<0.0001 for TME) as well as higher levels of IL-6 (p<0.01 for sera, p<0.05 for tumor supernatant) were observed in SD patients as compared to NSND OSCC patients and HD. High percentages of CD4+ T cells expressing CD45RO and CCR6 cells in tumor tissue (p=0.05) and blood (p=0.05) of SD OSCC patients were also associated with a poorer prognosis while a high percentage of regulatory T cells (Treg) in tumor tissue was associated with a more favorable prognostic factor (p=0.05). Also, a higher percentage of blood CD8+ T lymphocytes among CD45+ cells in NSND patients was associated with a better disease-free survival (p=0.004). Conclusion: Granulocytes, monocytes-macrophages, and CD4+ T cells expressing CD45RO and CCR6 in blood and TME as well as serum IL-6 can therefore distinguish OSCC SD and NSND patients. Quantifying the proportion of CD4+ T cells expressing CD45RO and CCR6 and of Treg in SD patients and CD8+ T cells in NSND patients could help defining the prognostic of OSCC patients.

Tumor-Associated Tertiary Lymphoid Structures: From Basic and Clinical Knowledge to Therapeutic Manipulation.

The tumor microenvironment is a complex ecosystem almost unique to each patient. Most of available therapies target tumor cells according to their molecular characteristics, angiogenesis or immune cells involved in tumor immune-surveillance. Unfortunately, only a limited number of patients benefit in the long-term of these treatments that are often associated with relapses, in spite of the remarkable progress obtained with the advent of immune checkpoint inhibitors (ICP). The presence of "hot" tumors is a determining parameter for selecting therapies targeting the patient immunity, even though some of them still do not respond to treatment. In human studies, an in-depth analysis of the organization and interactions of tumor-infiltrating immune cells has revealed the presence of an ectopic lymphoid organization termed tertiary lymphoid structures (TLS) in a large number of tumors. Their marked similarity to secondary lymphoid organs has suggested that TLS are an "anti-tumor school" and an "antibody factory" to fight malignant cells. They are effectively associated with long-term survival in most solid tumors, and their presence has been recently shown to predict response to ICP inhibitors. This review discusses the relationship between TLS and the molecular characteristics of tumors and the presence of oncogenic viruses, as well as their role when targeted therapies are used. Also, we present some aspects of TLS biology in non-tumor inflammatory diseases and discuss the putative common characteristics that they share with tumor-associated TLS. A detailed overview of the different pre-clinical models available to investigate TLS function and neogenesis is also presented. Finally, new approaches aimed at a better understanding of the role and function of TLS such as the use of spheroids and organoids and of artificial intelligence algorithms, are also discussed. In conclusion, increasing our knowledge on TLS will undoubtedly improve prognostic prediction and treatment selection in cancer patients with key consequences for the next generation immunotherapy.

Slug, a Cancer-Related Transcription Factor, is Involved in Vascular Smooth Muscle Cell Transdifferentiation Induced by Platelet-Derived Growth Factor-BB During Atherosclerosis.

Background Heart attacks and stroke often result from occlusive thrombi following the rupture of vulnerable atherosclerotic plaques. Vascular smooth muscle cells (VSMCs) play a pivotal role in plaque vulnerability because of their switch towards a proinflammatory/macrophage-like phenotype when in the context of atherosclerosis. The prometastatic transcription factor Slug/Snail2 is a critical regulator of cell phenotypic transition. Here, we aimed to investigate the role of Slug in the transdifferentiation process of VSMCs occurring during atherogenesis. Methods and Results In rat and human primary aortic smooth muscle cells, Slug protein expression is strongly and rapidly increased by platelet-derived growth factor-BB (PDGF-BB). PDGF-BB increases Slug protein without affecting mRNA levels indicating that this growth factor stabilizes Slug protein. Immunocytochemistry and subcellular fractionation experiments reveal that PDGF-BB triggers a rapid accumulation of Slug in VSMC nuclei. Using pharmacological tools, we show that the PDGF-BB-dependent mechanism of Slug stabilization in VSMCs involves the extracellular signal-regulated kinase 1/2 pathway. Immunohistochemistry experiments on type V and type VI atherosclerotic lesions of human carotids show smooth muscle-specific myosin heavy chain-/Slug-positive cells surrounding the prothrombotic lipid core. In VSMCs, Slug siRNAs inhibit prostaglandin E2 secretion and prevent the inhibition of cholesterol efflux gene expression mediated by PDGF-BB, known to be involved in plaque vulnerability and/or thrombogenicity. Conclusions Our results highlight, for the first time, a role of Slug in aortic smooth muscle cell transdifferentiation and enable us to consider Slug as an actor playing a role in the atherosclerotic plaque progression towards a life-threatening phenotype. This also argues for common features between acute cardiovascular events and cancer.

A thioredoxin-mimetic peptide exerts potent anti-inflammatory, antioxidant, and atheroprotective effects in ApoE2.Ki mice fed high fat diet.

Aims: Oxidative stress and inflammation play a pathogenic role in atherosclerosis. Thioredoxin-1 (Trx-1) is an anti-oxidative, anti-inflammatory protein with atheroprotective effects. However, in vivo cleavage of Trx-1 generates a truncated pro-inflammatory protein, Trx-80, which compromises the therapeutic use of Trx-1. Here we analysed whether the thioredoxin-mimetic peptide (TxMP), CB3 might exert anti-oxidative, anti-inflammatory, and atheroprotective effects in ApoE2.Ki mice. Methods and results: We synthesized a small TxMP, Ac-Cys-Pro-Cys-amide, CB3 and characterized its antioxidant and anti-inflammatory effects on cultured peritoneal murine macrophages. CB3 significantly and dose-dependently reduced the level of reactive oxygen species in lipopolysaccharides (LPS)-activated macrophages. In addition, it efficiently lowered LPS-induced inflammatory process through NF-κB inhibition, as evidenced by the reduced secretion of monocyte chemoattractant protein-1, interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α by macrophages. Nevertheless, CB3 did not affect cholesterol accumulation in macrophages. A daily-administered dose of 10 µg/g body weight CB3 to ApoE2.Ki mice on high fat diet did not affect plasma of total cholesterol and triglycerides levels but significantly reduced the plasma levels of pro-inflammatory cytokines (IL-33 and TNF-α) and oxidative markers. In contrast, it significantly induced the plasma levels of anti-inflammatory proteins (adiponectin, IL-10). In addition, CB3 reduced the number of pro-inflammatory M1 macrophages in spleen and decreased the ratio of M1/M2 macrophages in atherosclerotic lesion areas. Finally, CB3 significantly reduced the surface area of aortic lesions. Conclusions: Our results clearly showed that similar to the full length Trx-1, CB3 exerts protective effects, by reducing inflammation and oxidative stress in macrophages and in ApoE2.Ki mice. The atheroprotective effect of CB3 opens promising therapeutic approaches for treatment of atherosclerosis.

Intra-cheek immunization as a novel vaccination route for therapeutic vaccines of head and neck squamous cell carcinomas using plasmo virus-like particles.

Despite current therapy, head and neck squamous cell carcinomas (HNSCCs) arising from various mucosal sites of the upper aero-digestive tract frequently relapse in a loco-regional manner and have a poor prognosis. Our objective was to validate an innovative mucosal route of vaccination using plasmo virus-like particles (pVLPs) in a pre-clinical orthotopic model of HNSCCs. For this purpose, we used pVLP-E7, that are plasmid DNA encoding retroviral virus-like particles carrying a truncated E7 oncoprotein from HPV-16 as antigen model, to vaccinate mice bearing pre-established TC-1 tumors implanted into the buccal mucosa. pVLP-E7 were combined with clinical grade TLR agonists (Imiquimod and CpG-ODN). In this pre-clinical orthotopic model, whose tumor microenvironment resembles to those of human HNSCCs, different mucosal vaccination routes were tested for their ability to elicit efficient immune and antitumoral responses. Results showed that mucosal intra-cheek (IC) vaccinations using pVLP-E7, comparatively to intradermic vaccinations (ID), gave rise to higher mobilization of mucosal (CD49a(+)) CD8(+) specific effector T cells in both tumor draining lymph nodes (TdLNs) and tumor microenvironment resulting in better antitumor effects and in a long-term protection against tumor rechallenge. In vivo CD8(+) depletion demonstrated that antitumoral effects were fully dependent upon the presence of CD8(+) T cells. Validation of IC mucosal vaccinations with pVLPs combined with adjuvants using a pre-clinical orthotopic model of HNSCC provides valuable pre-clinical data to rapidly envision the use of such therapeutic vaccines in patients with HNSCCs, inasmuch as vaccinal components and adjuvants can be easily obtained as clinical grade reagents.

Anti-inflammatory and antiatherogenic effects of the NLRP3 inflammasome inhibitor arglabin in ApoE2.Ki mice fed a high-fat diet.

BACKGROUND: This study was designed to evaluate the effect of arglabin on the NLRP3 inflammasome inhibition and atherosclerotic lesion in ApoE2Ki mice fed a high-fat Western-type diet. METHODS AND RESULTS: Arglabin was purified, and its chemical identity was confirmed by mass spectrometry. It inhibited, in a concentration-dependent manner, interleukin (IL)-1ß and IL-18, but not IL-6 and IL-12, production in lipopolysaccharide and cholesterol crystal-activated cultured mouse peritoneal macrophages, with a maximum effect at ≈50 nmol/L and EC50 values for both cytokines of ≈ 10 nmol/L. Lipopolysaccharide and cholesterol crystals did not induce IL-1ß and IL-18 production in Nlrp3(-/-) macrophages. In addition, arglabin activated autophagy as evidenced by the increase in LC3-II protein. Intraperitoneal injection of arglabin (2.5 ng/g body weight twice daily for 13 weeks) into female ApoE2.Ki mice fed a high-fat diet resulted in a decreased IL-1ß plasma level compared with vehicle-treated mice (5.2±1.0 versus 11.7±1.1 pg/mL). Surprisingly, arglabin also reduced plasma levels of total cholesterol and triglycerides to 41% and 42%, respectively. Moreover, arglabin oriented the proinflammatory M1 macrophages into the anti-inflammatory M2 phenotype in spleen and arterial lesions. Finally, arglabin treatment markedly reduced the median lesion areas in the sinus and whole aorta to 54% (P=0.02) and 41% (P=0.02), respectively. CONCLUSIONS: Arglabin reduces inflammation and plasma lipids, increases autophagy, and orients tissue macrophages into an anti-inflammatory phenotype in ApoE2.Ki mice fed a high-fat diet. Consequently, a marked reduction in atherosclerotic lesions was observed. Thus, arglabin may represent a promising new drug to treat inflammation and atherosclerosis.

Human TCR alpha/beta+ CD4-CD8- double-negative T cells in patients with autoimmune lymphoproliferative syndrome express restricted Vbeta TCR diversity and are clonally related to CD8+ T cells.

The peripheral expansion of alpha/beta+-CD4-CD8- double negative (DN) T cells in patients with autoimmune lymphoproliferative syndrome (ALPS) is a consistent feature of this disease, and part of the diagnostic criteria of ALPS. The origin of these cells remains undetermined. They could derive from mature T cells that have lost coreceptor expression, or represent a special minor cell lineage. To investigate relationship of DN and single positive (SP) T cells in ALPS, we used Immunoscope technology to analyze the TCRVbeta repertoire diversity of sorted DN and SP T cells, and we performed CDR3 sequence analyses of matching clonotypes. We show that DN T cells express all the Vbeta gene families that are used by their SP counterparts, though they dominantly use some Vbeta genes. Analysis of CDR3 length distribution revealed a diverse polyclonal TCR repertoire for sorted CD4+ T cells, whereas both DN and CD8+ T cells showed a skewed TCR repertoire with oligoclonal expansions throughout most of the Vbeta families. CDR3 sequencing of matching clonotypes revealed a significant sharing of CDR3 sequences from selected Vbeta-Jbeta transcripts between DN and CD8+ T cells. Altogether, these data strongly argue for a CD8 origin of DN T cells in ALPS.

Perforin-dependent apoptosis functionally compensates Fas deficiency in activation-induced cell death of human T lymphocytes.

Activation-induced cell death (AICD) is involved in peripheral tolerance by controlling the expansion of repeatedly stimulated T cells via an apoptotic Fas (CD95; APO-1)-dependent pathway. The TNFRSF-6 gene encoding Fas is mutated in children suffering from autoimmune lymphoproliferative syndrome (ALPS), which is characterized by lymphoproliferation and autoimmunity. We examined AICD in Fas-deficient T cells from ALPS patients. We showed that primary activated Fas-deficient T cells die by apoptosis after repeated T cell antigen receptor (TCR) stimulation despite resistance to Fas-mediated cell death. This Fas-independent AICD was found to be mediated through a cytotoxic granules-dependent pathway. Cytotoxic granules-mediated AICD was also detected in normal T lymphocytes though to a lesser extent. As expected, the cytotoxic granules-dependent AICD was abolished in T cells from Rab27a- or perforin-deficient patients who exhibited defective granules-dependent cytotoxicity. Supporting an in vivo relevance of the cytotoxic granules-dependent AICD in ALPS patients, we detected an increased number of circulating T lymphocytes expressing granzymes A and B. Altogether, these data indicated that the cytotoxic granules-dependent cell death in ALPS may compensate for Fas deficiency in T lymphocytes. Furthermore, they identified a novel AICD pathway as a unique alternative to Fas apoptosis in human peripheral T lymphocytes.

XIAP deficiency in humans causes an X-linked lymphoproliferative syndrome.

The homeostasis of the immune response requires tight regulation of the proliferation and apoptosis of activated lymphocytes. In humans, defects in immune homeostasis result in lymphoproliferation disorders including autoimmunity, haemophagocytic lymphohystiocytosis and lymphomas. The X-linked lymphoproliferative syndrome (XLP) is a rare, inherited immunodeficiency that is characterized by lymphohystiocytosis, hypogammaglobulinaemia and lymphomas, and that usually develops in response to infection with Epstein-Barr virus (EBV). Mutations in the signalling lymphocyte activation molecule (SLAM)-associated protein SAP, a signalling adaptor molecule, underlie 60% of cases of familial XLP. Here, we identify mutations in the gene that encodes the X-linked inhibitor-of-apoptosis XIAP (also termed BIRC4) in patients with XLP from three families without mutations in SAP. These mutations lead to defective expression of XIAP. We show that apoptosis of lymphocytes from XIAP-deficient patients is enhanced in response to various stimuli including the T-cell antigen receptor (TCR)-CD3 complex, the death receptor CD95 (also termed Fas or Apo-1) and the TNF-associated apoptosis-inducing ligand receptor (TRAIL-R). We also found that XIAP-deficient patients, like SAP-deficient patients, have low numbers of natural killer T-lymphocytes (NKT cells), indicating that XIAP is required for the survival and/or differentiation of NKT cells. The observation that XIAP-deficiency and SAP-deficiency are both associated with a defect in NKT cells strengthens the hypothesis that NKT cells have a key role in the immune response to EBV. Furthermore, by identifying an XLP immunodeficiency that is caused by mutations in XIAP, we show that XIAP is a potent regulator of lymphocyte homeostasis in vivo.

Sildenafil and vardenafil, types 5 and 6 phosphodiesterase inhibitors, induce caspase-dependent apoptosis of B-chronic lymphocytic leukemia cells.

Type 4 phosphodiesterase (PDE4) inhibitors reportedly induce apoptosis in chronic lymphocytic leukemia (CLL) cells. Following clinical improvement of one previously untreated CLL patient with sildenafil therapy, we evaluated the in vitro induction of apoptosis in CLL cells by 4 PDE5/6 inhibitors, including sildenafil, vardenafil, zaprinast, and methoxyquinazoline (MQZ). After 24 hours of culture, the various PDE inhibitors differed in their ability to induce apoptosis, with zaprinast displaying no killing effect. Normal B cells isolated from control donors were totally resistant to PDE-induced apoptosis. Vardenafil was 3 and 30 times more potent an inducer of apoptosis than sildenafil and MQZ, respectively. Both vardenafil and sildenafil failed to elevate adenosine 3'5' cyclic monophosphate (cAMP) levels, largely excluding an inhibitory effect on cAMP-PDE3, -PDE4, and -PDE7. Vardenafil- or sildenafil-treated B-CLL cells displayed up to 30% intracellular active caspase 3. Drug-induced apoptosis was inhibited by the caspase inhibitor z-VAD.fmk, prevented by interleukin-4 (IL-4), and significantly reduced by stromal-derived factor1-alpha (SDF-1alpha). We conclude that vardenafil and sildenafil induce caspase-dependent apoptosis of B-CLL cells in vitro and thus might be considered in the treatment of CLL patients. However, further in vivo investigations should be warranted.

Monoglycerides induce apoptosis in human leukemic cells while sparing normal peripheral blood mononuclear cells.

A major drawback of the current antineoplastic treatments is their lack of specificity toward cancer cells, because they are most often cytotoxic to normal cells, thus creating related side effects. Hence, the identification of new apoptosis-inducing agents, specifically targeting malignant cells while sparing their normal counterparts, is of crucial interest. We show here that monoglycerides, a family of lipids consisting of a single fatty acid attached to a glycerol backbone, induce cell death in several human leukemic cell lines. Importantly, treatment of primary leukemic cells, obtained from B-cell chronic lymphocytic leukemia patients, resulted in rapid apoptosis. In striking contrast, resting or activated human peripheral blood mononuclear cells from healthy individuals were resistant to the same treatment. Therefore, these compounds could represent potential antileukemic drugs or could allow for the design of novel therapeutic agents applied to leukemia.

Mechanisms of CD47-induced caspase-independent cell death in normal and leukemic cells: link between phosphatidylserine exposure and cytoskeleton organization.

Dying cells, apoptotic or necrotic, are swiftly eliminated by professional phagocytes. We previously reported that CD47 engagement by CD47 mAb or thrombospondin induced caspase-independent cell death of chronic lymphocytic leukemic B cells (B-CLL). Here we show that human immature dendritic cells (iDCs) phagocytosed the CD47 mAb-killed leukemic cells in the absence of caspases 3, 7, 8, and 9 activation in the malignant lymphocytes. Yet the dead cells displayed the cytoplasmic features of apoptosis, including cell shrinkage, phosphatidylserine exposure, and decreased mitochondrial transmembrane potential (DeltaPsim). CD47 mAb-induced cell death also occurred in normal resting and activated lymphocytes, with B-CLL cells demonstrating the highest susceptibility. Importantly, iDCs and CD34(+) progenitors were resistant. Structure-function studies in cell lines transfected with various CD47 chimeras demonstrated that killing exclusively required the extracellular and transmembrane domains of the CD47 molecule. Cytochalasin D, an inhibitor of actin polymerization, and antimycin A, an inhibitor of mitochondrial electron transfer, completely suppressed CD47-induced phosphatidylserine exposure. Interestingly, CD47 ligation failed to induce cell death in mononuclear cells isolated from Wiskott-Aldrich syndrome (WAS) patients, suggesting the involvement of Cdc42/WAS protein (WASP) signaling pathway. We propose that CD47-induced caspase-independent cell death be mediated by cytoskeleton reorganization. This form of cell death may be relevant to maintenance of homeostasis and as such might be explored for the development of future therapeutic approaches in lymphoid malignancies.