Chemotherapy pretreatment sensitizes solid tumor-derived cell lines to Vα24+ NKT cell-mediated cytotoxicity

There is an increasing awareness of the therapeutic potential for combining immune-based therapies with chemotherapy in the treatment of malignant diseases, but few published studies evaluate possible cytotoxic synergies between chemotherapy and cytotoxic immune cells. Human Vα24 +/Vβ11+ NKT cells are being evaluated for use in cell-based immunotherapy of malignancy because of their immune regulatory functions and potent cytotoxic potential. In this study, we evaluated the cytotoxicity of combinations of chemotherapy and NKT cells to determine whether there is a potential to combine these treatment modalities for human cancer therapy. The cytotoxicity of NKT cells was tested against solid-tumor derived cell lines NCI-H358, DLD-1, HT-29, DU-145, TSU-Pr1 and MDA-MB231, with or without prior treatment of these target cells, with a range of chemotherapy agents. Low concentrations of chemotherapeutic agents led to sensitization of cell lines to NKT-mediated cytotoxicity, with the greatest effect being observed for prostate cancer cells. Synergistic cytotoxicity occurred in an NKT cell in a dose-dependent manner. Chemotherapy agents induced upregulation of cell surface TRAIL-R2 (DR5) and Fas (CD95) expression, increasing the capacity for NKT cells to recognize and kill via TRAIL- and FasL-mediated pathways. We conclude that administration of cytotoxic immune cells after chemotherapy may increase antitumor activities in comparison with the use of either treatment alone.

A kinetic analysis of the tumor-associated galactopyranosyl-(1→3)-2-acetamido-2-deoxy-α-d-galactopyranoside antigen—lectin interaction

A kinetic study of the tumor-associated galactopyranosyl-(1→3)-2-acetamido-2-deoxy-α-d-galactopyranoside (T-antigen) with lectin peanut agglutinin is described. The disaccharide antigen was synthesized by chemical methods and was functionalized suitably for immobilization onto a carboxy-methylated sensor chip. The ligand immobilized surface was allowed interaction with the lectin peanut agglutinin, which acted as the analyte and the interaction was studied by the surface plasmon resonance method. The ligand—lectin interaction was characterized by the kinetic on-off rates and a bivalent analyte binding model was found to describe the observed kinetic constants. It was identified that the antigen-lectin interaction had a faster association rate constant (k a1) and a slower dissociation rate constant (k d1) in the initial binding step. The subsequent binding step showed much reduced kinetic rates. The antigen-lectin interaction was compared with the kinetic rates of the interaction of a galactopyranosyl-(1→4)-β-d-galactopyranoside derivative and a mannopyranoside derivative with the lectin.

Quantifying Cell Binding Kinetics Mediated By Surface-Bound Blood Type B Antigen To Immobilized Antibodies

Cell adhesion is crucial to many biological processes, such as inflammatory responses, tumor metastasis and thrombosis formation. Recently a commercial surface plasmon resonance (SPR)-based BIAcore biosensor has been extended to determine cell binding mediated by surface-bound biomolecular interactions. How such cell binding is quantitatively governed by kinetic rates and regulating factors, however, has been poorly understood. Here we developed a novel assay to determine the binding kinetics of surface-bound biomolecular interactions using a commercial BIAcore 3000 biosensor. Human red blood cells (RBCs) presenting blood group B antigen and CM5 chip bearing immobilized anti-B monoclonal antibody (mAb) were used to obtain the time courses of response unit, or sensorgrams, when flowing RBCs over the chip surface. A cellular kinetic model was proposed to correlate the sensorgrams with kinetic rates. Impacts of regulating factors, such as cell concentration, flow duration and rate, antibody-presenting level, as well as pH value and osmotic pressure of suspending medium were tested systematically, which imparted the confidence that the approach can be applied to kinetic measurements of cell adhesion mediated by surface-bound biomolecular interactions. These results provided a new insight into quantifying cell binding using a commercial SPR-based BIAcore biosensor.

Antigen-specific responses assessment for the evaluation of Bordetella pertussis T cell immunity in humans.

Measurement of antigen-specific T cell responses is an adjunctive parameter to evaluate protection induced by a previous Bordetella pertussis infection or vaccination. The assessment of T cell responses is technically complex and usually performed on fresh peripheral blood mononuclear cells (PBMC). The objective of this study was to identify simplified methods to assess pertussis specific T cell responses and verify if these assays could be performed using frozen/thawed (frozen) PBMC. Three read-outs to measure proliferation were compared: the fluorescent dye 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) dilution test, the number of blast cells defined by physical parameters, and the incorporation of (3)H-thymidine. The results of pertussis-specific assays performed on fresh PBMC were compared to the results on frozen PBMC from the same donor. High concordance was obtained when the results of CFSE and blast read-outs were compared, an encouraging result since blast analysis allows the identification of proliferating cells and does not require any use of radioactive tracer as well as any staining. The results obtained using fresh and frozen PBMC from the same donor in the different T cell assays, including IFNγ and TNFα cytokine production, did not show significant differences, suggesting that a careful cryopreservation process of PBMC would not significantly influence T cell response evaluation. Adopting blast analysis and frozen PBMC, the possibility to test T cell responses is simplified and might be applied in population studies, providing for new instruments to better define correlates of protection still elusive in pertussis.

Phenotypic characteristics of human type II alveolar epithelial cells suitable for antigen presentation to T lymphocytes.

Type II alveolar epithelial cells (AECII) are well known for their role in the innate immune system. More recently, it was proposed that they could play a role in the antigen presentation to T lymphocytes but contradictory results have been published both concerning their surface expressed molecules and the T lymphocyte responses in mixed lymphocyte cultures. The use of either AECII cell line or fresh cells could explain the observed discrepancies. Thus, this study aimed at defining the most relevant model of accessory antigen presenting cells by carefully comparing the two models for their expression of surface molecules necessary for efficient antigen presentation.

Desmoplastic Small Round Cell Tumor of Major Salivary Glands: Report of 1 Case and a Review of the Literature

Desmoplastic small round cell tumor is a rare malignant neoplasm mostly occurring in the vicinity of or within the peritoneal cavity, and is uncommon in the head and neck region. Tumor location within a major salivary gland is exceptional. We report a case of a 41-year-old Chinese man with a history of diabetes mellitus and end-stage renal failure on peritoneal dialysis with a desmoplastic small round cell tumor occurring in the left submandibular gland. Fine-needle aspiration cytology showed variably cohesive clusters of small cells with hyperchromatic nuclei and fine granular chromatin. On histology the neoplasm displayed classic features of a desmoplastic small round cell tumor with angulated nests of small round blue cells in a fibromyxoid/desmoplastic stroma. Neoplastic cells were immunoreactive for cytokeratins (AE1/3), desmin (paranuclear dot-like), WT-1 (nuclear), epithelial membrane antigen, and CD56. EWS gene translocation and EWS-WT1 gene fusion were detected by fluorescence in situ hybridization and reverse transcriptase polymerase chain reaction, respectively. The case presented is the sixth case of and the oldest reported patient with a desmoplastic small round cell tumor occurring in a major salivary gland to date. Desmoplastic small round cell tumor should be considered in the differential diagnosis of a salivary gland neoplasm with a basaloid or small cell pattern on fine-needle aspiration cytology.

CD69, CD25, and HLA-DR activation antigen expression on CD3+ lymphocytes and relationship to serum TNF-alpha, IFN-gamma, and sIL-2R levels in aging

Aging is associated with changes in lymphocyte subsets and unexplained HLA-DR upregulation on T-lymphocytes. We further investigated this activation, by measuring early (CD69), middle (CD25), and late (HLA-DR) T-lymphocyte activation markers on CD3+ lymphocytes, across subjects (20-100 years) together with serum tumor necrosis factor (TNF-alpha), interferon-gamma (IFN-gamma), and soluble interleukin-2 receptor (sIL-2R). HLA-DR was present as a CD3+ HLA-DR+ subset that constituted 8% of total lymphocytes, increased twofold with age and included CD4+, CD8+, and CD45RA+ phenotypes. HLA-DR was also expressed on a CD8+ CD57+ subset. The CD3+ CD25+ subset constituted 13% of lymphocytes, fell with age but was weakly associated with the CD3+ HLA-DR+ subset especially in older subjects. A small 3-5% CD3+ CD69+ subsets showed no age effect. Serum sIL-2R, TNF-alpha, but not IFN-gamma, were associated with CD3+ HLA-DR+ lymphocytes, TNF-alpha with CD8+ CD57+ count and sIL-2R and IFN-gamma with the CD3+ CD25+/CD3+ CD4+ ratio. The study confirms age-related upregulation of HLA-DR on CD3+ lymphocytes, shows some evidence for associated upregulation of CD25 on CD3+ cells in older subjects, and links serum TNF-alpha, IFN-gamma, and sIL2-R to T-lymphocyte activation.

A selective antagonist of histamine H₄ receptors prevents antigen-induced airway inflammation and bronchoconstriction in guinea pigs:involvement of lipocortin-1

BACKGROUND AND PURPOSE: Among the pathogenic mechanisms of asthma, a role for oxidative/nitrosative stress has been well documented. Recent evidence suggests that histamine H₄ receptors play a modulatory role in allergic inflammation. Here we report the effects of compound JNJ 7777120 (JNJ), a selective H4 receptor antagonist, on antigen-induced airway inflammation, paying special attention to its effects on lipocortin-1 (LC-1/annexin-A1), a 37 kDA anti-inflammatory protein that plays a key role in the production of inflammatory mediators.

EXPERIMENTAL APPROACH: Ovalbumin (OA)-sensitized guinea pigs placed in a respiratory chamber were challenged with antigen. JNJ (5, 7.5 and 10 mg.kg⁻¹) was given i.p. for 4 days before antigen challenge. Respiratory parameters were recorded. Bronchoalveolar lavage (BAL) fluid was collected and lung specimens taken for further analyses 1 h after antigen challenge. In BAL fluid, levels of LC-1, PGD2 , LTB4 and TNF-α were measured. In lung tissue samples, myeloperoxidase, caspase-3 and Mn-superoxide dismutase activities and 8-hydroxy-2-deoxyguanosine levels were measured.

KEY RESULTS: OA challenge decreased LC-1 levels in BAL fluid, induced cough, dyspnoea and bronchoconstriction and increased PGD2 , LTB4 and TNF-α levels in lung tissue. Treatment with JNJ dose-dependently increased levels of LC-1, reduced respiratory abnormalities and lowered levels of PGD2 , LTB4 and TNF-α in BAL fluid.

CONCLUSIONS AND IMPLICATIONS: Antigen-induced asthma-like reactions in guinea pigs decreased levels of LC-1 and increased TNF-α and eicosanoid production. JNJ pretreatment reduced allergic asthmatic responses and airway inflammation, an effect associated with LC-1 up-regulation.

Infrequent Expression of the Cancer-Testis Antigen, PASD1, in Ovarian Cancer

Ovarian cancer is very treatable in the early stages of disease; however, it is usually detected in the later stages, at which time, treatment is no longer as effective. If discovered early (Stage I), there is a 90% chance of five-year survival. Therefore, it is imperative that early-stage biomarkers are identified to enhance the early detection of ovarian cancer. Cancer-testis antigens (CTAs), such as Per ARNT SIM (PAS) domain containing 1 (PASD1), are unique in that their expression is restricted to immunologically restricted sites, such as the testis and placenta, which do not express MHC class I, and cancer, making them ideally positioned to act as targets for immunotherapy as well as potential biomarkers for cancer detection where expressed. We examined the expression of PASD1a and b in a number of cell lines, as well as eight healthy ovary samples, eight normal adjacent ovarian tissues, and 191 ovarian cancer tissues, which were predominantly stage I (n = 164) and stage II (n = 14) disease. We found that despite the positive staining of skin cancer, only one stage Ic ovarian cancer patient tissue expressed PASD1a and b at detectable levels. This may reflect the predominantly stage I ovarian cancer samples examined. To examine the restriction of PASD1 expression, we examined endometrial tissue arrays and found no expression in 30 malignant tumor tissues, 23 cases of hyperplasia, or 16 normal endometrial tissues. Our study suggests that the search for a single cancer-testes antigen/biomarker that can detect early ovarian cancer must continue.

Disseminated tumor cells and their prognostic significance in nonmetastatic prostate cancer patients

Detection of pretreatment disseminated cells (pre-DTC) reflecting its homing to bone marrow (BM) in prostate cancer (PCa) might improve the current model to predict recurrence or survival in men with nonmetastatic disease despite of primary treatment. Thereby, pre-DTC may serve as an early prognostic biomarker. Post-treatment DTCs (post-DTC) finding may supply the clinician with additional predictive information about the possible course of PCa. To assess the prognostic impact of DTCs in BM aspirates sampled before initiation of primary therapy (pre-DTC) and at least 2 years after (post-DTC) to established prognostic factors and survival in patients with PCa. Available BM of 129 long-term follow-up patients with T1-3N0M0 PCa was assessed in addition to 100 BM of those in whom a pretreatment BM was sampled. Patients received either combined therapy [n = 81 (63%)], radiotherapy (RT) with different duration of hormone treatment (HT) or monotherapy with RT or HT alone [n = 48 (37%)] adapted to the criteria of the SPCG-7 trial. Mononuclear cells were deposited on slides according to the cytospin methodology and DTCs were identified by immunocytochemistry using the pancytokeratin antibodies AE1/AE3. The median age of men at diagnosis was 64.5 years (range 49.5-73.4 years). The median long-term follow-up from first BM sampling to last observation was 11 years. Categorized clinically relevant factors in PCa showed only pre-DTC status as the statistically independent parameter for survival in the multivariate analysis. Pre-DTCs homing to BM are significantly associated with clinically relevant outcome independent to the patient's treatment at diagnosis with nonmetastatic PCa.

Concentration of tumor biomarkers using aqueous biphasic systems

According to the World Health Organization, around 8.2 million people die each year with cancer. Most patients do not perform routine diagnoses and the symptoms, in most situations, occur when the patient is already at an advanced stage of the disease, consequently resulting in a high cancer mortality. Currently, prostate cancer is the second leading cause of death among males worldwide. In Portugal, this is the most diagnosed type of cancer and the third that causes more deaths. Taking into account that there is no cure for advanced stages of prostate cancer, the main strategy comprises an early diagnosis to increase the successful rate of the treatment. The prostate specific antigen (PSA) is an important biomarker of prostate cancer that can be detected in biological fluids, including blood, urine and semen. However, the commercial kits available are addressed for blood samples and the commonly used analytical methods for their detection and quantification requires specialized staff, specific equipment and extensive sample processing, resulting in an expensive process. Thus, the aim of this MSc thesis consisted on the development of a simple, efficient and less expensive method for the extraction and concentration of PSA from urine samples using aqueous biphasic systems (ABS) composed of ionic liquids. Initially, the phase diagrams of a set of aqueous biphasic systems composed of an organic salt and ionic liquids were determined. Then, their ability to extract PSA was ascertained. The obtained results reveal that in the tested systems the prostate specific antigen is completely extracted to the ionic-liquid-rich phase in a single step. Subsequently, the applicability of the investigated ABS for the concentration of PSA was addressed, either from aqueous solutions or urine samples. The low concentration of this biomarker in urine (clinically significant below 150 ng/mL) usually hinders its detection by conventional analytical techniques. The obtained results showed that it is possible to extract and concentrate PSA, up to 250 times in a single-step, so that it can be identified and quantified using less expensive techniques.

Concentration of tumor biomarkers using aqueous biphasic systems

According to the World Health Organization, around 8.2 million people die each year with cancer. Most patients do not perform routine diagnoses and the symptoms, in most situations, occur when the patient is already at an advanced stage of the disease, consequently resulting in a high cancer mortality. Currently, prostate cancer is the second leading cause of death among males worldwide. In Portugal, this is the most diagnosed type of cancer and the third that causes more deaths. Taking into account that there is no cure for advanced stages of prostate cancer, the main strategy comprises an early diagnosis to increase the successful rate of the treatment. The prostate specific antigen (PSA) is an important biomarker of prostate cancer that can be detected in biological fluids, including blood, urine and semen. However, the commercial kits available are addressed for blood samples and the commonly used analytical methods for their detection and quantification requires specialized staff, specific equipment and extensive sample processing, resulting in an expensive process. Thus, the aim of this MSc thesis consisted on the development of a simple, efficient and less expensive method for the extraction and concentration of PSA from urine samples using aqueous biphasic systems (ABS) composed of ionic liquids. Initially, the phase diagrams of a set of aqueous biphasic systems composed of an organic salt and ionic liquids were determined. Then, their ability to extract PSA was ascertained. The obtained results reveal that in the tested systems the prostate specific antigen is completely extracted to the ionic-liquid-rich phase in a single step. Subsequently, the applicability of the investigated ABS for the concentration of PSA was addressed, either from aqueous solutions or urine samples. The low concentration of this biomarker in urine (clinically significant below 150 ng/mL) usually hinders its detection by conventional analytical techniques. The obtained results showed that it is possible to extract and concentrate PSA, up to 250 times in a single-step, so that it can be identified and quantified using less expensive techniques.

Development of a bifunctional CD1d-anti-HER2 scFv fusion molecule to redirect the iNKT cell-mediated immune response to the tumor site

Abstract : Invariant natural killer T lymphocytes (iNKT) are a unique subpopulation of T lymphocytes recognizing glycolipid antigens in the context of the MHC class I-like molecule CD1d. Upon activation with the high affinity ligand α-galactosylceramide (αGalCer), iNKT cells rapidly produce large amounts of the pro-inflammatory cytokine interferon gamma (IFN-γ) and potently activate cells of the innate and adaptive immune response, such as dendritic cells (DCs), NK and T cells. In this context, iNKT cells have been shown to efficiently mediate antitumor activity, and recent research has focused on the manipulation of these cells for antitumor therapies. However, a major drawback of αGalCer as a free drug is that a single injection of this ligand leads to a short-lived iNKT cell activation followed by a long-term anergy, limiting its therapeutic use. In contrast, we demonstrate here that when αGalCer is loaded on a recombinant soluble CD1d molecule (αGalCer/sCD1d), repeated injections lead to a sustained iNKT and NK cell activation associated with IFN-γ secretion as well as with DC maturation. Most importantly, when the αGalCer/sCD1d is fused to an anti-HER2 scFv antibody fragment, potent inhibition of experimental lung metastasis and established subcutaneous tumors is obtained when systemic treatment is started two to seven days after the injection of HER2-expressing B16 melanoma cells, whereas at this time free αGalCer has no effect. The antitumor activity of the sCD1d-anti-HER2 fusion protein is associated with HER2-specific tumor localization and accumulation of iNKT, NK and T cells at the tumor site. Importantly, active T cell immunization combined with the sCD1d-anti-HER2 treatment leads to the accumulation of antigen-specific CD8 T cells exclusively in HER2-expressing tumors, resulting in potent tumor inhibition. In conclusion, sustained activation and tumor targeting of iNKT cells by recombinant αGalCer/sCD1d molecules thus may promote a combined innate and adaptive immune response at the tumor site that may prove to be effective in cancer immunotherapy. RESUME : Les lymphocytes «invariant Natural Killer T » (iNKT) forment une sous-population particulière de lymphocytes T reconnaissant des antigènes glycolipidiques présentés sur la molécule non-polymorphique CD1d, analogue aux protéines du complexe majeur d'histocompatibilité de classe I. Après activation avec le ligand de haute affinité α-galactosylceramide (αGalCer), les cellules iNKT produisent des grandes quantités de la cytokine pro-inflammatoire interferon gamma (IFN-γ) et activent les cellules du système immunitaire inné et acquis, telles que les cellules dendritiques (DC), NK et T. En conséquence, on a montré que les cellules iNKT exercent des activités anti-tumorales et la recherche s'est intéressée à la manipulation de ces cellules pour développer des thérapies anti-tumorales. Néanmoins, le désavantage majeur de l'αGalCer, injecté seul, est qu'une seule dose de ce ligand aboutit à une activation des cellules iNKT de courte durée suivie par un état anergique prolongé, limitant l'utilisation thérapeutique de ce glycolipide. En revanche, l'étude présentée ici démontre que, si l'αGalCer est chargé sur des molécules récombinantes soluble CD1d (αGalCer/sCDld), des injections répétées aboutissent à une activation prolongée des cellules iNKT et NK associée avec la sécrétion d'IFN-γ et la maturation des cellules DC. Plus important, si on fusionne la molécule αGalCer/sCD1d avec un fragment single-chain (scFv) de l'anticorps anti-HER2, on observe une importante inhibition de métastases expérimentales aux poumons et de tumeurs sous-cutanées même lorsque le traitement systémique est commencé 2 à 7 jours après la greffe des cellules de mélanome B16 transfectées avec l'antigène HER2. Dans les mêmes conditions le traitement avec l'αGalCer seul est inefficace. L'activité anti-tumorale de la protéine sCDld-anti-HER2 est associée à son accumulation spécifique dans des tumeurs exprimant le HER2 ainsi qu'avec une accumulation des cellules iNKT, NK et T à la tumeur. De plus, une immunisation active combinée avec le traitement sCD1d-anti-HER2 aboutit à une accumulation des lymphocytes T CD8 spécifiques de l'antigène d'immunisation, ceci exclusivement dans des tumeurs qui expriment l'antigène HER2. Cette combinaison résulte dans une activité anti-tumeur accrue. En conclusion, l'activation prolongée des cellules iNKT redirigées à la tumeur par des molécules recombinantes αGalCer/sCDld conduit à l'activation de la réponse innée et adaptative au site tumoral, offrant une nouvelle stratégie prometteuse d'immunothérapie contre le cancer. RESUME POUR UN LARGE PUBLIC : Le cancer est une cause majeure de décès dans le monde. Sur un total de 58 millions de décès enregistrés au niveau mondial en 2005, 7,6 millions (soit 13%) étaient dus au cancer. Les principaux traitements de nombreux cancers sont la chirurgie, en association avec la radiothérapie et la chimiothérapie. Néanmoins, ces traitements nuisent aussi aux cellules normales de notre corps et parfois, ils ne suffisent pas pour éliminer définitivement une tumeur. L'immunothérapie est l'une des nouvelles approches pour la lutte contre le cancer et elle vise à exploiter la spécificité du système immunitaire qui peut distinguer des cellules normales et tumorales. Une cellule exprimant un marqueur tumoral (antigène) peut être reconnue par le système immunitaire humoral (anticorps) et/ou cellulaire, induisant une réponse spécifique contre la tumeur. L'immunothérapie peut s'appuyer alors sur la perfusion d'anticorps monoclonaux dirigés contre des antigènes tumoraux, par exemple les anticorps dirigés contre les protéines oncogéniques Her-2/neu dans le cancer du sein. Ces anticorps ont le grand avantage de spécifiquement se localiser à la tumeur et d'induire la lyse ou d'inhiber la prolifération des cellules tumorales exprimant l'antigène. Aujourd'hui, six anticorps monoclonaux non-conjugés sont approuvés en clinique. Cependant l'efficacité de ces anticorps contre des tumeurs solides reste limitée et les traitements sont souvent combinés avec de la chimiothérapie. L'immunothérapie spécifique peut également être cellulaire et exploiter par immunisation active le développement de lymphocytes T cytotoxiques (CTL) capables de détruire spécifiquement les cellules malignes. De telles «vaccinations »sont actuellement testées en clinique, mais jusqu'à présent elles n'ont pas abouti aux résultats satisfaisants. Pour obtenir une réponse lymphocytaire T cytotoxique antitumorale, la cellule T doit reconnaître un antigène associé à la tumeur, présenté sous forme de peptide dans un complexe majeur d'histocompatibilité de classe I (CHM I). Cependant les cellules tumorales sont peu efficace dans la présentation d'antigène, car souvent elles se caractérisent par une diminution ou une absence d'expression des molécules d'histocompatibilité de classe I, et expriment peu ou pas de molécules d'adhésion et de cytokines costimulatrices. C'est en partie pourquoi, malgré l'induction de fortes réponses CTL spécifiquement dirigés contre des antigènes tumoraux, les régressions tumorales obtenus grâce à ces vaccinations sont relativement rares. Les lymphocytes «invariant Natural Killer T » (iNKT) forment une sous-population particulière de lymphocytes T reconnaissant des antigènes glycolipidiques présentés sur la molécule non-polymorphique CD1d, analogue aux protéines CMH I. Après activation avec le ligand de haute affinité α-galactosylceramide (αGalCer), les cellules iNKT produisent des grandes quantités de la cytokine pro-inflammatoire interferon gamma (IFN-γ) et activent les cellules du système immunitaire inné et acquis, telles que les cellules dendritiques (DC), NK et T. En conséquence, on a montré que les cellules iNKT exercent des activités anti-tumorales et la recherche s'est intéressée à la manipulation de ces cellules pour développer des thérapies anti-tumorales. Néanmoins, le désavantage majeur de l'αGalCer, injecté seul, est qu'une seule dose de ce ligand aboutit à une activation des cellules iNKT de courte durée suivie par un état anergique prolongé, limitant l'utilisation thérapeutique de ce glycolipide. Notre groupe de recherche a donc eu l'idée de développer une nouvelle approche thérapeutique où la réponse immunitaire des cellules iNKT serait prolongée et redirigée vers la tumeur par des anticorps monoclonaux. Concrètement, nous avons produit des molécules récombinantes soluble CD1d (sCD1d) qui, si elles sont chargés avec l'αGalCer (αGalCer/sCDld), aboutissent à une activation prolongée des cellules iNKT et NK associée avec la sécrétion d'IFN-γ et la maturation des cellules DC. Plus important, si la molécule αGalCer/sCD1d est fusionnée avec un fragment single-chain (scFv) de l'anticorps anti-HER2, la réponse immunitaire est redirigée à la tumeur pour autant que les cellules cancéreuses expriment l'antigène HER2. Les molécules αGalCer/sCDld ainsi présentées activent les lymphocytes iNKT. Avec cette stratégie, on observe une importante inhibition de métastases expérimentales aux poumons et de tumeurs sous-cutanées, même lorsque le traitement systémique est commencé 2 à 7 jours après la greffe des cellules de mélanome B16 transfectées avec l'antigène HER2. Dans les mêmes conditions le traitement avec l'αGalCer seul est inefficace. L'activité anti-tumorale de la protéine sCDld-anti-HER2 est associée à son accumulation spécifique dans des tumeurs exprimant le HER2 ainsi qu'avec une accumulation des cellules iNKT, NK et T à la tumeur. En conclusion, l'activation prolongée des cellules iNKT redirigées à la tumeur par des molécules récombinantes αGalCer/sCD1d conduit à l'activation de la réponse innée et adaptative au site tumoral, offrant une nouvelle stratégie prometteuse d'immunothérapie contre le cancer.

Combined clonotyping and gene signatures of individual tumor-reactive CD8 T-cells define their functional relevance following peptide vaccination in melanoma patients

Novel cancer vaccines are capableto efficiently induce and boost humantumor antigen specific T-cells. However,the properties of these CD8T-cells are only partially characterized.For in depth investigation ofT-cells following Melan-A/MART-1peptide vaccination in melanoma patients,we conducted a detailed prospectivestudy at the single cell level.We first sorted individual human naiveand effector CD8 T-cells from peripheralblood by flow cytometry, andtested a modified RT-PCR protocolincluding a global amplification ofexpressed mRNAs to obtain sufficientcDNAfromsingle cells.We successfullydetected the expression ofseveral specific genes of interest evendown to 106-fold dilution (equivalentto 10-5 cell). We then analyzed tumor-specific effector memory (EM)CD8T-cell subpopulations ex vivo, assingle cells from vaccinated melanomapatients. To elucidate the hallmarksof effective immunity the genesignatures were defined by a panel ofgenes related to effector functions(e.g. IFN-, granzyme B, perforin),and individual clonotypes were identifiedaccording to the expression ofdistinct T-cell receptors (TCR). Usingthis novel single cell analysis approach,we observed that T-cell differentiationis clonotype dependent,with a progressive restriction in TCRBV clonotype diversity from EMCD28pos to EMCD28neg subsets. However,the effector function gene imprintingis clonotype-independent,but dependent on differentiation,since it correlates with the subset oforigin (EMCD28pos or EMCD28neg). We also conducted a detailedcomparative analysis after vaccinationwith natural vs. analog Melan-Apeptide. We found that the peptideused for vaccination determines thefunctional outcome of individualT-cell clonotypes, with native peptideinducing more potent effector functions.Yet, selective clonotypic expansionwith differentiation was preservedregardless of the peptide usedfor vaccination. In summary, the exvivo single cell RT-PCR approach ishighly sensitive and efficient, andrepresents a reliable and powerfultool to refine our current view of molecularprocesses taking place duringT-cell differentiation.

HARNESSING iNKT CELLS WITH RECOMBINANT CD1d FUSION PROTEINS TO REDIRECT INNATE AND ADAPTIVE IMMUNITY TO THE TUMOR

Les thérapies du cancer, comme la radiothérapie et la chimiothérapie, sont couramment utilisées mais ont de nombreux effets secondaires. Ces thérapies invasives pour le patient nécessitent d'être améliorées et de nombreuses avancées ont été faites afin d'adapter et de personnaliser le traitement du cancer. L'immunothérapie a pour but de renforcer le système immunitaire du patient et de le rediriger de manière spécifique contre la tumeur. Dans notre projet, nous activons les lymphocytes Invariant Natural Killer T (iNKT) afin de mettre en place une immunothérapie innovatrice contre le cancer. Les cellules iNKT sont une unique sous-population de lymphocytes T qui ont la particularité de réunir les propriétés de l'immunité innée ainsi qu'adaptative. En effet, les cellules iNKT expriment à leur surface des molécules présentes aussi sur les cellules tueuses NK, caractéristique de l'immunité innée, ainsi qu'un récepteur de cellules T (TCR) qui représente l'immunité adaptative. Les cellules iNKT reconnaissent avec leur TCR des antigènes présentés par la molécule CD1d. Les antigènes sont des protéines, des polysaccharides ou des lipides reconnus par les cellules du système immunitaire ou les anticorps pour engendrer une réponse immunitaire. Dans le cas des cellules iNKT, l'alpha-galactosylceramide (αGC) est un antigène lipidique fréquemment utilisé dans les études cliniques comme puissant activateur. Après l'activation des cellules iNKT avec l'αGC, celles-ci produisent abondamment et rapidement des cytokines. Ces cytokines sont des molécules agissant comme des signaux activateurs d'autres cellules du système immunitaire telles que les cellules NK et les lymphocytes T. Cependant, les cellules iNKT deviennent anergiques après un seul traitement avec l'αGC c'est à dire qu'elles ne peuvent plus être réactivées, ce qui limite leur utilisation dans l'immunothérapie du cancer. Dans notre groupe, Stirnemann et al ont publié une molécule recombinante innovante, composée de la molécule CD1d soluble et chargée avec le ligand αGC (αGC/sCD1d). Cette protéine est capable d'activer les cellules iNKT tout en évitant l'anergie. Dans le système immunitaire, les anticorps sont indispensables pour combattre une infection bactérienne ou virale. En effet, les anticorps ont la capacité de reconnaître et lier spécifiquement un antigène et permettent l'élimination de la cellule qui exprime cet antigène. Dans le domaine de l'immunothérapie, les anticorps sont utilisés afin de cibler des antigènes présentés seulement par la tumeur. Ce procédé permet de réduire efficacement les effets secondaires lors du traitement du cancer. Nous avons donc fusionné la protéine recombinante αGC/CD1d à un fragment d'anticorps qui reconnaît un antigène spécifique des cellules tumorales. Dans une étude préclinique, nous avons démontré que la protéine αGC/sCD1d avec un fragment d'anticorps dirigé contre la tumeur engendre une meilleure activation des cellules iNKT et entraîne un effet anti-tumeur prolongé. Cet effet anti-tumeur est augmenté comparé à une protéine αGC/CD1d qui ne cible pas la tumeur. Nous avons aussi montré que l'activation des cellules iNKT avec la protéine αGC/sCD1d-anti-tumeur améliore l'effet anti- tumoral d'un vaccin pour le cancer. Lors d'expériences in vitro, la protéine αGC/sCD1d-anti- tumeur permet aussi d'activer les cellules humaines iNKT et ainsi tuer spécifiquement les cellules tumorales humaines. La protéine αGC/sCD1d-anti-tumeur représente une alternative thérapeutique prometteuse dans l'immunothérapie du cancer. - Les cellules Invariant Natural Killer T (iNKT), dont les effets anti-tumoraux ont été démontrés, sont de puissants activateurs des cellules Natural Killer (NK), des cellules dendritiques (DC) et des lymphocytes T. Cependant, une seule injection du ligand de haute affinité alpha-galactosylceramide (αGC) n'induit une forte activation des cellules iNKT que durant une courte période. Celle-ci est alors suivie d'une longue phase d'anergie, limitant ainsi leur utilisation pour la thérapie. Comme alternative prometteuse, nous avons montré que des injections répétées d'αGC chargé sur une protéine recombinante de CD1d soluble (αGC/sCD1d) chez la souris entraînent une activation prolongée des cellules iNKT, associée à une production continue de cytokine. De plus, le maintien de la réactivité des cellules iNKT permet de prolonger l'activité anti-tumorale lorsque la protéine αGC/sCD1d est fusionnée à un fragment d'anticorps (scFv) dirigé contre la tumeur. L'inhibition de la croissance tumorale n'est optimale que lorsque les souris sont traitées avec la protéine αGC/sCD1d-scFv ciblant la tumeur, la protéine αGC/sCD1d-scFv non-appropriée étant moins efficace. Dans le système humain, les protéines recombinantes αGC/sCD1d-anti-HER2 et anti-CEA sont capables d'activer et de faire proliférer des cellules iNKT à partir de PBMCs issues de donneurs sains. De plus, la protéine αGC/sCD1d-scFv a la capacité d'activer directement des clones iNKT humains en l'absence de cellules présentatrices d'antigènes (CPA), contrairement au ligand αGC libre. Mais surtout, la lyse des cellules tumorales par les iNKT humaines n'est obtenue que lorsqu'elles sont incubées avec la protéine αGC/sCD1d-scFv anti- tumeur. En outre, la redirection de la cytotoxicité des cellules iNKT vers la tumeur est supérieure à celle obtenue avec une stimulation par des CPA chargées avec l'αGC. Afin d'augmenter les effets anti-tumoraux, nous avons exploité la capacité des cellules iNKT à activer l'immunité adaptive. Pour ce faire, nous avons combiné l'immunothérapie NKT/CD1d avec un vaccin anti-tumoral composé d'un peptide OVA. Des effets synergiques ont été obtenus lorsque les traitements avec la protéine αGC/sCD1d-anti-HER2 étaient associés avec le CpG ODN comme adjuvant pour la vaccination avec le peptide OVA. Ces effets ont été observés à travers l'activation de nombreux lymphocytes T CD8+ spécifique de la tumeur, ainsi que par la forte expansion des cellules NK. Les réponses, innée et adaptive, élevées après le traitement avec la protéine αGC/sCD1d-anti-HER2 combinée au vaccin OVA/CpG ODN étaient associées à un fort ralentissement de la croissance des tumeurs B16- OVA-HER2. Cet effet anti-tumoral corrèle avec l'enrichissement des lymphocytes T CD8+ spécifiques observé à la tumeur. Afin d'étendre l'application des protéines αGC/sCD1d et d'améliorer leur efficacité, nous avons développé des fusions CD1d alternatives. Premièrement, une protéine αGC/sCD1d dimérique, qui permet d'augmenter l'avidité de la molécule CD1d pour les cellules iNKT. Dans un deuxième temps, nous avons fusionné la protéine αGC/sCD1d avec un scFv dirigé contre le récepteur 3 du facteur de croissance pour l'endothélium vasculaire (VEGFR-3), afin de cibler l'environnement de la tumeur. Dans l'ensemble, ces résultats démontrent que la thérapie médiée par la protéine recombinante αGC/sCD1d-scFv est une approche prometteuse pour rediriger l'immunité innée et adaptive vers le site tumoral. - Invariant Natural Killer T cells (iNKT) are potent activators of Natural Killer (NK), dendritic cells (DC) and T lymphocytes, and their anti-tumor activities have been well demonstrated. However, a single injection of the high affinity CD1d ligand alpha-galactosylceramide (αGC) leads to a strong but short-lived iNKT cell activation followed by a phase of long-term anergy, limiting the therapeutic use of this ligand. As a promising alternative, we have demonstrated that when αGC is loaded on recombinant soluble CD1d molecules (αGC/sCD1d), repeated injections in mice led to the sustained iNKT cell activation associated with continued cytokine secretion. Importantly, the retained reactivity of iNKT cell led to prolonged antitumor activity when the αGC/sCD1d was fused to an anti-tumor scFv fragments. Optimal inhibition of tumor growth was obtained only when mice were treated with the tumor-targeted αGC/CD1d-scFv fusion, whereas the irrelevant αGC/CD1d-scFv fusion was less efficient. When tested in a human system, the recombinant αGC/sCD1d-anti-HER2 and -anti-CEA fusion proteins were able to expand iNKT cells from PBMCs of healthy donors. Furthermore, the αGC/sCD1d-scFv fusion had the capacity to directly activate human iNKT cells clones without the presence of antigen-presenting cells (APCs), in contrast to the free αGC ligand. Most importantly, tumor cell killing by human iNKT cells was obtained only when co- incubated with the tumor targeted sCD1d-antitumor scFv, and their direct tumor cytotoxicity was superior to the bystander killing obtained with αGC-loaded APCs stimulation. To further enhance the anti-tumor effects, we exploited the ability of iNKT cells to transactivate the adaptive immunity, by combining the NKT/CD1d immunotherapy with a peptide cancer vaccine. Interestingly, synergistic effects were obtained when the αGC/sCD1d- anti-HER2 fusion treatment was combined with CpG ODN as adjuvant for the OVA peptide vaccine, as seen by higher numbers of activated antigen-specific CD8 T cells and NK cells, as compared to each regimen alone. The increased innate and adaptive immune responses upon combined tumor targeted sCD1d-scFv treatment and OVA/CpG vaccine were associated with a strong delay in B16-OVA-HER2 melanoma tumor growth, which correlated with an enrichment of antigen-specific CD8 cells at the tumor site. In order to extend the application of the CD1d fusion, we designed alternative CD1d fusion proteins. First, a dimeric αGC/sCD1d-Fc fusion, which permits to augment the avidity of the CD1d for iNKT cells and second, an αGC/sCD1d fused to an anti vascular endothelial growth factor receptor-3 (VEGFR-3) scFv, in order to target tumor stroma environment. Altogether, these results demonstrate that the iNKT-mediated immunotherapy via recombinant αGC/sCD1d-scFv fusion is a promising approach to redirect the innate and adaptive antitumor immune response to the tumor site.