Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with rising incidence and a remarkable resistance to current therapies. The reasons for this therapeutic failure include the tumor's ...extensive infiltration by immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs). By using light sheet fluorescent microscopy, we identified here direct interactions between these major immunoregulatory cells in PDAC. The
depletion of MDSCs led to a significant reduction in Tregs in the pancreatic tumors. Through videomicroscopy and
functional assays we have shown that (i) MDSCs are able to induce Treg cells in a cell-cell dependent manner; (ii) Treg cells affect the survival and/or the proliferation of MDSCs. Furthermore, we have observed contacts between MDSCs and Treg cells at different stages of human cancer. Overall our findings suggest that interactions between MDSCs and Treg cells contribute to PDAC immunosuppressive environment.
Pancreatic adenocarcinoma (PAC) has a poor prognosis. One treatment approach, investigated here, is to reinforce antitumor immunity. Dendritic cells (DCs) are essential for the development and ...regulation of adaptive host immune responses against tumors. A major role for DCs may be as innate tumoricidal effector cells. We explored the efficacy of vaccination with immature (i)DCs, after selecting optimal conditions for generating immunostimulatory iDCs. We used two models, C57BL/6Jrj mice with ectopic tumors induced by the PAC cell line, Panc02, and genetically engineered (KIC) mice developing PAC. Therapeutic iDC-vaccination resulted in a significant reduction in tumor growth in C57BL/6Jrj mice and prolonged survival in KIC mice. Prophylactic iDC-vaccination prevented subcutaneous tumor development. These protective effects were long-lasting in Panc02-induced tumor development, but not in melanoma. iDC-vaccination impacted the immune status of the hosts by greatly increasing the percentage of CD8
+
T-cells, and natural killer (NK)1.1
+
cells, that express granzyme B associated with Lamp-1 and IFN-γ. Efficacy of iDC-vaccination was CD8
+
T-cell-dependent but NK1.1
+
cell-independent. We demonstrated the ability of DCs to produce peroxynitrites and to kill tumor cells; this killing activity involved peroxynitrites. Altogether, these findings make killer DCs the pivotal actors in the beneficial clinical outcome that accompanies antitumor immune responses.
We asked whether efficacy can be improved by combining DC-vaccination with the FOLFIRINOX regimen. Combined treatment significantly increased the lifespan of KIC mice with PAC. Prolonged treatment with FOLFIRINOX clearly augmented this beneficial effect. Combining iDC-vaccination with FOLFIRINOX may therefore represent a promising therapeutic option for patients with PAC.
Oncofetal fucose-rich glycovariants of the pathological bile salt-dependent lipase (pBSDL) appear during human pancreatic oncogenesis and are detected by themonoclonal antibody J28 (mAbJ28). We aimed ...to identify murine counterparts onpancreatic ductal adenocarcinoma (PDAC) cells and tissue and investigate the potential of dendritic cells (DC) loaded with this unique pancreatic tumor antigen to promote immunotherapy in preclinical trials. Pathological BSDLs purified from pancreatic juices of patients with PDAC were cleaved to generate glycosylated C-terminal moieties (C-ter) containing mAbJ28-reactive glycoepitopes. Immunoreactivity of the murine PDAC line Panc02 and tumor tissue to mAbJ28 was detected by immunohistochemistry and flow cytometry. C-ter-J28+ immunization promoted Th1-dominated immune responses. In vitro C-ter-J28+-loaded DCskewed CD3+ T-cells toward Th1 polarization. C-ter-J28+-DC-vaccinations selectively enhanced cell immunoreactivity to Panc02, as demonstrated by CD4+- and CD8+-T-cell activation, increased percentages of CD4+- and CD8+-T-cells and NK1.1+ cells expressing granzyme B, and T-cell cytotoxicity. Prophylactic and therapeutic C-ter-J28+-DC-vaccinations reduced ectopic Panc02-tumor growth, provided long-lasting protection from Panc02-tumor development in 100% of micebut not from melanoma, and attenuated progression of orthotopic tumors as revealed by MRI. Thusmurine DC loaded with pancreatic tumor-specific glycoepitope C-ter-J28+ induce efficient anticancer adaptive immunity and represent a potential adjuvant therapy for patients afflicted with PDAC.
BackgroundDensity and localization of T cells within the tumor microenvironment (TME) is critical to control tumor growth. Immunotherapy with antibodies against immune checkpoint inhibitors (ICI), ...which aim to reinvigorate exhausted T cells, despite inducing long-term response in many cancer types, remains ineffective for most patients. Other factors present in the TME may participate in the control of tumors, among several candidates, the role of B cells has been underestimated. Publications revealed that B-cells are associated with good prognosis in several indications. However, there are no general rules since neutral or even deleterious impact of the presence of B-cells in the tumor was reported. Among other hypotheses, we can suppose that the level of activation and isotype switch will be important to mediate activation of NK cells or ADCC within the tumor and will have a favorable impact on the prognosis. In addition, the spatial distribution of B-cells may be important since, in the TME, they are mostly located in Tertiary Lymphoid Structures (TLS) which are ectopic lymphoid organs. Mature TLSs contain zones where dendritic cells (DC) present antigen to T-cells and others where proliferating B-cells undergo class switch and maturation toward plasma cells. Antigen presentation by B cells to T cells is supported by DCs, therefore, the presence of these three types of cells within the TLS allows T cell activation in the TME. If B-cells are major players in the therapeutic efficacy of ICI antibodies, not all subtypes of tumor infiltrating B-cells are likely to participate in response to immunotherapy.MethodsTo decipher the roles of B cells, new tools are needed to identify the differentiation and activation status of individual B cells. We have developed a 7-plex panel of antibodies against biomarkers that allow the identification of main types of B cells.ResultsOn a single FFPE tissue section: naïve, unswitched memory, switched memory, activated, plasmablast and plasma B cells, as well as T cells and DC are identified. Following images registration, complex cells phenotypes can be detected and quantified. Furthermore, digital pathology tools allow the evaluation of the spatial distribution within the TME of subtypes of B cells especially in association with TLSs.ConclusionsThis new tool unravels the heterogeneity of B cells in TME and could help clinical researchers to understand their contribution to the response to immunotherapy. Integrated into an Immunogram, this new Brightplex® Panel will be critical to understand the immune contexture of the tumor.
Aberrant glycosylation or overexpression of cell-surface glycosylated tumor-associated Ags (TAA) distinguish neoplastic from normal cells. Interactions of TAA MUC1 and HER2/neu with dendritic cells ...(DC) preclude efficient processing, which impairs immune responses. It is thus important to define the mechanisms of interactions between DC and glycosylated TAA and their trafficking and processing for further T cell activation. In this work, we study interactions between DC and the oncofetal fucose-rich glycovariants of bile salt-dependent lipase (BSDL), expressed in pancreatic cancer tissues and referred to as pathological BSDL carrying the fucosylated J28 glycotope (pBSDL-J28) because it is characterized by the mAb J28. The expression of pBSDL-J28 was assessed by immunohistochemistry and quantified by confocal microscopy. Nontumoral pancreatic tissues and cells do not express pBSDL-J28. Using multidisciplinary approaches and functional studies, we provide the first evidence, to our knowledge, that this tumoral glycoprotein is rapidly internalized by human DC through macropinocytosis and endocytosis via mannose receptors and then transported to late endosomes for processing. Interestingly, pBSDL-J28 per se induced DC maturation with increased expression of costimulatory and CD83 molecules associated with cytokine secretion (IL-8 and IL-6). Surprisingly, DC retained their full ability to internalize Ags, making this maturation atypical. Finally, the allogeneic pBSDL-J28-treated DC stimulated lymphocyte proliferation. Besides, pulsing DC with pBSDL-J28 C-terminal glycopolypeptide and maturation with CD40L triggered CD4(+) and CD8(+) T cell proliferation. Therefore, interactions of pBSDL-J28, expressed on tumoral pancreatic tissue, with DC may lead to adequate Ag trafficking and processing and result in T cell activation.
Glycoproteins, as valuable targets for dendritic cell (DC)-vaccination in cancers, remain an open question. Glycosylated structures, which are aberrantly modified during cancerisation, impact ...positively or negatively on glycoprotein immunogenicity. Here is presented an oncofetal glycovariant of bile-salt-dependent-lipase, expressed on human tumoral pancreas and efficiently processed by DC's, inducing T-lymphocyte activation.
BackgroundTumor-infiltrating immune cells play an important role against cancer and are critical to controlling tumor growth and spread. Immunotherapy drugs such as immune checkpoint inhibitors, ...despite inducing long-term responses in many cancer types, remain ineffective for a majority of patients. A better understanding of the tumor microenvironment (TME), and the characterization of populations of immune cells, their activation status, spatial distribution, and relationship, along with cytokine signaling, may help to better stratify patients and explain mechanisms of resistance to immunotherapy.Veracyte’s Brightplex® is a chromogenic multiplex immunohistochemistry (IHC) technology that leverages digital pathology enhanced by artificial intelligence. It allows the detection of up to eight biomarkers on a single FFPE slide to identify cellular subpopulations. However, to characterize the exact role of an immune cell it is often necessary to determine if it expresses soluble proteins which cannot be detected by IHC, such as cytokines or activation factors. In that case, the detection of the corresponding RNA transcripts by in situ hybridization (ISH) can be used as a substitute for protein detection.MethodsHere, we propose a multiplex technology automated on the Leica Bond RX platform which combines ISH and IHC staining on a single FFPE tissue section. In brief, that tissue section is sequentially stained to detect biomarkers of interest either with antibodies for proteins or nucleic probes for transcripts. Each round of staining is followed by the digitization of the slide. Whole slide images are fused to create a virtual multi-channel image where biomarkers are detected by digital pathology and combined to identify immune cell populations. The spatial distribution and cell-to-cell interactions within a slide or between multiple adjacent slides are assessed by combining multiplex ISH/IHC panels.ResultsThis technology allowed us to quantify different sub-populations of tumor-infiltrating-lymphocytes (activated, cytotoxic and exhausted), interferon-γ producing cells, and tumor-associated-macrophages expressing the chemokine CXCL9. We investigated the spatial distribution of these immune cells within the TME. We also assessed the cell-to-cell proximity between these populations to assess their interactions.ConclusionsIntegrated into an Immunogram, an analytics platform that integrates multi-omics datasets from Veracyte Biopharma Atlas, this new tool could be a powerful solution to decipher the tumor landscape and predict response to immunotherapy and patient outcome.
Pancreatic adenocarcinomas and diabetes mellitus are responsible for the deaths of around two million people each year worldwide. Patients with chronic pancreatitis do not die directly of this ...disease, except where the pathology is hereditary. Much current literature supports the involvement of bile salt-dependent lipase (BSDL), also known as carboxyl ester lipase (CEL), in the pathophysiology of these pancreatic diseases. The purpose of this review is to shed light on connections between chronic pancreatitis, diabetes, and pancreatic adenocarcinomas by gaining an insight into BSDL and its variants. This enzyme is normally secreted by the exocrine pancreas, and is diverted within the intestinal lumen to participate in the hydrolysis of dietary lipids. However, BSDL is also expressed by other cells and tissues, where it participates in lipid homeostasis. Variants of BSDL resulting from germline and/or somatic mutations (nucleotide insertion/deletion or nonallelic homologous recombination) are expressed in the pancreas of patients with pancreatic pathologies such as chronic pancreatitis, MODY-8, and pancreatic adenocarcinomas. We discuss the possible link between the expression of BSDL variants and these dramatic pancreatic pathologies, putting forward the suggestion that BSDL and its variants are implicated in the cell lipid metabolism/reprogramming that leads to the dyslipidemia observed in chronic pancreatitis, MODY-8, and pancreatic adenocarcinomas. We also propose potential strategies for translation to therapeutic applications.
BackgroundCancer immunotherapy reinvigorates tumor-specific T cell responses of CD8+ cytotoxic T lymphocytes that detect intracellular antigens that are presented by MHC class I molecules expressed ...by all tumor cell types. Because most tumors do not express MHC class II, the potential antitumor protective role of CD4 T cells, which bind MHC class II molecules on target cells, has been less studied. However, CD4+ T cells are also required for efficacious antitumor immunity; they are core components of adaptive immunity that differentiate into lineages responsible for effector activities. Both TH1 and TH2 cell types mediate antitumor immunity, although TH1 cells may be more potent due to the production of large amounts of IFN-γ, as well as chemokines that enhance the priming and expansion of CD8 cells. TH1 cells help in recruiting natural killer cells and type I macrophages to tumor sites, which can act in concert toward tumor eradication. The ability of TH2 cells to mobilize innate cells, may represent a general pathway for their impact on the host antitumor response. Tumor infiltrating TFH cells play a key role in immune cell recruitment to the tumor and in the formation of intratumoral follicular structures, which correlate with a positive prognosis. On the contrary, cells from the TH17 subset induces inflammatory responses resulting in a tumor-promoting environment. CD4+ Tregs which are critically important for the maintenance of self-tolerance, impede effective immunity against the tumor when they are present in the tumor microenvironment (TME).Therefore, beyond the detection of total CD4+ T cells within the TME, it is of critical importance to determine to which subpopulation each CD4+ T cell belongs to decipher their roles in tumor rejection.MethodsWe have developed a multiplex 7-plex panel of of antibodies against biomarkers to identify main types of CD4+ T cellsResultsOn a single FFPE tissue section, main types of CD4+ T cells: TH1, TH2, TFH, TH17 and Tregs are identified by a combination of antibodies against transcription factors and membrane proteins. Following images registration, complex cells phenotypes can be detected and quantified. Furthermore, digital pathology tools allow the evaluation of the spatial distribution of CD4+ T cells within the TME.ConclusionsThis new tool unravels the diversity of CD4+ T cells in TME and could help clinical researchers to design more effective immunotherapies in cancer treatment. Integrated into an Immunogram, this new Brightplex® Panel will also be critical to understand the immune contexture of tumors.
Le cancer du pancréas exocrine est un cancer très agressif associé à un diagnostic tardif et une résistance aux traitements conventionnels. L'identification de nouveaux marqueurs spécifiques est ...nécessaire afin de développer des outils diagnostiques ainsi que des traitements innovants. La lipase sels biliaires-dépendante pathologique (BSDLp), une glycoforme tumorale de la BSDL, se caractérise par l'apparition d'épitopes glycosylés reconnus par les anticorps monoclonaux J28 et 16D10. Leur expression spécifique par certaines lignées et tissus pancréatiques tumoraux humains nous permet d'envisager leur utilisation comme cible d'immunothérapie anti-tumorale par les cellules dendritiques (DC). Notre objectif est d'explorer la capacité de la BSDLp à induire une immunité cellulaire et d'apporter une preuve de concept de vaccination par les DC dans un modèle expérimental. Chez l'Homme, nous montrons que les DC chargées avec la partie C-terminale de la BSDLpJ28 (C-ter-J28) induisent l'activation des lymphocytes T. Dans le modèle murin, nous mettons en évidence l'expression de l'épitope J28 à la surface des cellules d'adénocarcinome pancréatique Panc02, utilisées pour induire des tumeurs. Nous montrons que la glycoprotéine BSDLpJ28 est immunogène chez la souris C57BL/6J. De plus, les DC chargées avec le C-ter-J28 puis soumises à maturation sont capables d'induire une réponse cellulaire T. Enfin, les DC, chargées et soumises à maturation ou non, testées en traitement prophylactique instaurent une protection substantielle, de longue durée, chez les souris vaccinées. Dans ces conditions, les DC, immatures lors de l'injection, jouent un rôle important dans la protection anti-tumorale.
Pancreatic adenocarcinoma is an aggressive cancer associated to late diagnosis and resistance to conventional treatments. Identification of novel specific markers is necessary to develop diagnostic tools and innovative treatments. Pathological bile salt-dependent lipase (pBSDL), a tumoral glycovariant of BSDL, is characterized by the appearance of glycosylated epitopes recognized by J28 and 16D10 monoclonal antibodies. Their expression specific to some human tumor pancreatic cell lines and tissues led us to consider their use as targets for dendritic cell (DC) antitumor immunotherapy. Our aim is to explore the ability of pBSDL to induce cellular mediated immunity and to provide a proof of concept of DC vaccination in an experimental model. In humans, we show that DC pulsed with C-ter moiety of pBSDL-J28 (C-ter-J28) can induce T-cell activation. In mouse model, we demonstrate the expression of J28 epitope on pancreatic adenocarcinoma Panc02 cells, used to induce tumors in C57Bl/6 mice. We show that glycoprotein pBSDL (pBSDL-J28) is immunogenic in mice. Moreover, DC pulsed with C-ter-J28 and matured, are able to induce T-cell response. Finally, DC pulsed and matured or not, tested in prophylactic treatment provide long-term substantial protection in vaccinated mice. In these conditions, DC, immature at the time of the injection, play an important role in antitumor protection.
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