Réponses immunitaires et cancer

Publications de l’équipe

Année de publication : 2017

Florence Faure, Mabel Jouve, Isabelle Lebhar-Peguillet, Charlotte Sadaka, Fernando Sepulveda, Olivier Lantz, Stefano Berre, Raphael Gaudin, Silvia Sánchez-Ramón, Sebastian Amigorena (2017 Sep 9)

Blood monocytes sample MelanA/MART1 antigen for long-lasting cross-presentation to CD8(+) T cells after differentiation into dendritic cells.

International journal of cancer : DOI : 10.1002/ijc.31037 En savoir plus
Résumé

Human blood monocytes are very potent to take up antigens. Like macrophages in tissue, they efficiently degrade exogenous protein and are less efficient than dendritic cells (DCs) at cross-presenting antigens to CD8(+) T cells. Although it is generally accepted that DCs take up tissue antigens and then migrate to lymph nodes to prime T cells, the mechanisms of presentation of antigens taken up by monocytes are poorly documented so far. In the present work, we show that monocytes loaded in vitro with MelanA long peptides retain the capacity to stimulate antigen-specific CD8(+) T cell clones after 5 days of differentiation into monocytes-derived dendritic cells (MoDCs). Tagged-long peptides can be visualized in electron-dense endocytic compartments distinct from lysosomes, suggesting that antigens can be protected from degradation for extended periods of time. To address the pathophysiological relevance of these findings, we screened blood monocytes from 18 metastatic melanoma patients and found that CD14(+) monocytes from two patients effectively activate a MelanA-specific CD8 T cell clone after in vitro differentiation into MoDCs. This in vivo sampling of tumor antigen by circulating monocytes might alter the tumor-specific immune response and should be taken into account for cancer immunotherapy.

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Andrés Alloatti, Derek C Rookhuizen, Leonel Joannas, Jean-Marie Carpier, Salvador Iborra, Joao G Magalhaes, Nader Yatim, Patrycja Kozik, David Sancho, Matthew L Albert, Sebastian Amigorena (2017 Jul 1)

Critical role for Sec22b-dependent antigen cross-presentation in antitumor immunity.

The Journal of experimental medicine : 2231-2241 : DOI : 10.1084/jem.20170229 En savoir plus
Résumé

CD8(+) T cells mediate antigen-specific immune responses that can induce rejection of solid tumors. In this process, dendritic cells (DCs) are thought to take up tumor antigens, which are processed into peptides and loaded onto MHC-I molecules, a process called « cross-presentation. » Neither the actual contribution of cross-presentation to antitumor immune responses nor the intracellular pathways involved in vivo are clearly established because of the lack of experimental tools to manipulate this process. To develop such tools, we generated mice bearing a conditional DC-specific mutation in the sec22b gene, a critical regulator of endoplasmic reticulum-phagosome traffic required for cross-presentation. DCs from these mice show impaired cross-presentation ex vivo and defective cross-priming of CD8(+) T cell responses in vivo. These mice are also defective for antitumor immune responses and are resistant to treatment with anti-PD-1. We conclude that Sec22b-dependent cross-presentation in DCs is required to initiate CD8(+) T cell responses to dead cells and to induce effective antitumor immune responses during anti-PD-1 treatment in mice.

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Eik Hoffmann, Anne-Marie Pauwels, Andrés Alloatti, Fiorella Kotsias, Sebastian Amigorena (2017 Feb 28)

Analysis of Phagosomal Antigen Degradation by Flow Organellocytometry.

Bio-protocol : DOI : e2014 En savoir plus
Résumé

Professional phagocytes internalize self and non-self particles by phagocytosis to initiate innate immune responses. After internalization, the formed phagosome matures through fusion and fission events with endosomes and lysosomes to obtain a more acidic, oxidative and hydrolytic environment for the degradation of its cargo. Interestingly, phagosome maturation kinetics differ between cell types and cell activation states. This protocol allows to quantify phagosome maturation kinetics on a single organelle level in different types of phagocytes using flow cytometry. Here, ovalbumin (OVA)-coupled particles are used as phagocytosis model system in dendritic cells (DC), which are internalized by phagocytosis. After different time points, phagosome maturation parameters, such as phagosomal degradation of OVA and acquisition of lysosomal proteins (like LAMP-1), can be measured simultaneously in a highly quantitative manner by flow organellocytometry. These read-outs can be correlated to other phagosomal functions, for example antigen degradation, processing and loading in DC.

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Andrés Alloatti, Fiorella Kotsias, Eik Hoffmann, Sebastian Amigorena (2017 Feb 28)

Evaluation of Cross-presentation in Bone Marrow-derived Dendritic Cells in vitro and Splenic Dendritic Cells ex vivo Using Antigen-coated Beads.

Bio-protocol : DOI : e2015 En savoir plus
Résumé

Antigen presentation by MHC class I molecules, also referred to as cross-presentation, elicits cytotoxic immune responses. In particular, dendritic cells (DC) are the most proficient cross-presenting cells, since they have developed unique means to control phagocytic and degradative pathways. This protocol allows the evaluation of antigen cross-presentation both in vitro (by using bone marrow-derived DC) and ex vivo (by purifying CD8(+) DC from spleen after incorporation of particulate antigen) using ovalbumin (OVA)-coupled particles. Cross-presentation efficiency is measured by three different readouts: the B3Z hybridoma T cell line (Karttunen et al., 1992) and stimulation of antigen-specific CD8(+) T cells (OT-I) (Kurts et al., 1996), either analyzing OT-I activation by CD69 expression or OT-I proliferation after labeling them with carboxyfluorescein succinimidyl ester (CFSE). By using this approach, we could show recently that DCs are able to increase cross-presentation efficiency transiently upon engagement of TLR4 (Alloatti et al., 2015).

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Marianne Burbage, Sebastian Amigorena (2017 Feb 24)

Antigen-Primed CD8(+) T Cells Call DCs for Back Up.

Immunity : 163-164 : DOI : S1074-7613(17)30035-3 En savoir plus
Résumé

Encounters between naive T lymphocytes and dendritic cells (DCs) bearing adequate co-stimulatory signals are rare. In this issue of Immunity, Brewitz et al. (2017) show that chemokines secreted by CD8(+) T cells recruit myeloid and plasmacytoid DCs that in turn boost CD8(+) T cell activation.

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Ronan Crépin, David Gentien, Angeline Duché, Audrey Rapinat, Cecile Reyes, Fariba Némati, Gérald Massonnet, Didier Decaudin, Selma Djender, Sandrine Moutel, Klervi Desrumeaux, Nathalie Cassoux, Sophie Piperno-Neumann, Sebastian Amigorena, Franck Perez, Sergio Roman Roman, Ario de Marco (2017 Feb 1)

Nanobodies against surface biomarkers enable the analysis of tumor genetic heterogeneity in uveal melanoma Patient Derived Xenografts.

Pigment cell & melanoma research : DOI : 10.1111/pcmr.12577 En savoir plus
Résumé

Monoclonal antibodies specific for biomarkers expressed on the surface of uveal melanoma (UM) cells would simplify the immune-capture and genomic characterization of heterogeneous tumor cells originated from patient derived xenografts (PDXs). Antibodies against four independent tumor antigens were isolated by panning a nanobody synthetic library. Such antibodies enabled flow-cytometry-based sorting of distinct cell sub-populations from UM PDXs and to analyze their genomic features. The complexity and specificity of the biochemical and genomic biomarker combinations mirrored the UM tumor polyclonality. The data showed that MUC18 is highly and universally displayed at the surface of UM cells with different genetic background and consequently represents a reliable pan-biomarker for their identification and purification. In contrast, the other three biomarkers were detected in very variable combinations in UM PDX cells. The availability of the identified nanobodies will be instrumental in developing clone-specific drug evaluation and rational clinical strategies based on accurate genomic profiling. This article is protected by copyright. All rights reserved.

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Année de publication : 2016

Martijn H den Brok, Christian Büll, Melissa Wassink, Annemarie M de Graaf, Jori A Wagenaars, Marthe Minderman, Mayank Thakur, Sebastian Amigorena, Eric O Rijke, Carla C Schrier, Gosse J Adema (2016 Nov 8)

Saponin-based adjuvants induce cross-presentation in dendritic cells by intracellular lipid body formation.

Nature communications : 13324 : DOI : 10.1038/ncomms13324 En savoir plus
Résumé

Saponin-based adjuvants (SBAs) are being used in animal and human (cancer) vaccines, as they induce protective cellular immunity. Their adjuvant potency is a factor of inflammasome activation and enhanced antigen cross-presentation by dendritic cells (DCs), but how antigen cross-presentation is induced is not clear. Here we show that SBAs uniquely induce intracellular lipid bodies (LBs) in the CD11b+ DC subset in vitro and in vivo. Using genetic and pharmacological interference in models for vaccination and in situ tumour ablation, we demonstrate that LB induction is causally related to the saponin-dependent increase in cross-presentation and T-cell activation. These findings link adjuvant activity to LB formation, aid the application of SBAs as a cancer vaccine component, and will stimulate development of new adjuvants enhancing T-cell-mediated immunity.

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Christine Sedlik, Adèle Heitzmann, Sophie Viel, Rafik Ait Sarkouh, Cornélie Batisse, Frédéric Schmidt, Philippe De La Rochere, Nathalie Amzallag, Eduardo Osinaga, Pablo Oppezzo, Otto Pritsch, Xavier Sastre-Garau, Pascale Hubert, Sebastian Amigorena, Eliane Piaggio (2016 Sep 14)

Effective antitumor therapy based on a novel antibody-drug conjugate targeting the Tn carbohydrate antigen.

Oncoimmunology : e1171434 : DOI : 10.1080/2162402X.2016.1171434 En savoir plus
Résumé

Antibody-drug conjugates (ADC), combining the specificity of tumor recognition by monoclonal antibodies (mAb) and the powerful cytotoxicity of anticancer drugs, are currently under growing interest and development. Here, we studied the potential of Chi-Tn, a mAb directed to a glyco-peptidic tumor-associated antigen, to be used as an ADC for cancer treatment. First, we demonstrated that Chi-Tn specifically targeted tumor cells in vivo. Also, using flow cytometry and deconvolution microscopy, we showed that the Chi-Tn mAb is rapidly internalized – condition necessary to ensure the delivery of conjugated cytotoxic drugs in an active form, and targeted to early and recycling endosomes. When conjugated to saporin (SAP) or to auristatin F, the Chi-Tn ADC exhibited effective cytotoxicity to Tn-positive tumor cells in vitro, which correlated with the level of tumoral Tn expression. Furthermore, the Chi-Tn mAb conjugated to auristatin F also exhibited efficient antitumor activity in vivo, validating for the first time the use of an anti-Tn antibody as an effective ADC.

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Marie Gosset, Anne-Sophie Hamy, Peter Mallon, Myriam Delomenie, Delphine Mouttet, Jean-Yves Pierga, Marick Lae, Alain Fourquet, Roman Rouzier, Fabien Reyal, Jean-Guillaume Feron (2016 Aug 6)

Prognostic Impact of Time to Ipsilateral Breast Tumor Recurrence after Breast Conserving Surgery.

PloS one : e0159888 : DOI : 10.1371/journal.pone.0159888 En savoir plus
Résumé

The poor prognosis of patients who experience ipsilateral breast tumor recurrence (IBTR) after breast conserving surgery (BCS) is established. A short time between primary cancer and IBTR is a prognostic factor but no clinically relevant threshold was determined. Classification of IBTR may help tailor treatment strategies.

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Delphine Knittel, Adeline Gadzinski, Stéphane Hua, Jordan Denizeau, Alexandra Savatier, Philippe de la Rochère, Jean-Claude Boulain, Sebastian Amigorena, Eliane Piaggio, Christine Sedlik, Michel Léonetti (2016 May 8)

Heparan sulfates targeting increases MHC class I- and MHC class II-restricted antigen presentation and CD8(+) T-cell response.

Vaccine : 3093-101 : DOI : 10.1016/j.vaccine.2016.04.073 En savoir plus
Résumé

Heparan sulfates (HS) are carbohydrate moieties of HS proteoglycans (HSPGs). They often represent alternative attachment points for proteins or microorganisms targeting receptors. HSPGs, which are ubiquitously expressed, thereby participate in numerous biological processes. We previously showed that MHC class II-restricted antigen presentation is increased when antigens are coupled to HS ligands, suggesting that HSPGs might contribute to adaptive immune responses. Here, we examined if HSPG targeting influences other aspects of immune responses. We found that coupling of an HS ligand to the antigen increases antigen presentation to CD4(+) and CD8(+) T-cells after antigen targeting to membrane immunoglobulins or to MHC-II molecules. Moreover, this increased stimulating capacity correlates with an enhanced CD8(+) immune response in mice. Last, animals control more effectively the growth of Ova-expressing tumour cells when they are immunized with an Ova construct targeting HSPGs and MHC-II molecules. Our results indicate that ubiquitous molecules can influence both MHC class I- and MHC class II-restricted antigen presentation and behave as co-receptors during T-cell stimulation. Moreover, they suggest that tumour-antigens endowed with the ability to target both HSPGs and MHC-II molecules could be of value to increase CD8(+) immune response and control tumour-growth, opening new perspectives for the design of highly immunogenic protein-based vaccines.

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Omar I Vivar, Joao G Magalhaes, Sebastian Amigorena (2016 May 5)

Measurement of Export to the Cytosol in Dendritic Cells Using a Cytofluorimetry-Based Assay.

Methods in molecular biology (Clifton, N.J.) : 183-8 : DOI : 10.1007/978-1-4939-3606-9_13 En savoir plus
Résumé

The presentation of exogenous antigens on MHC class I molecules, known as cross-presentation, is a key function of dendritic cells (DCs). Cross-presentation via the cytosolic pathway involves antigen export from endocytic compartments to the cytosol. We have recently developed a cytofluorimetry-based assay to examine the kinetics and the efficiency of antigen export to the cytosol in DC populations. In this assay, DCs are loaded with a FRET-sensitive cytosolic substrate of β-lactamase, CCF4. Following uptake of β-lactamase by the DCs, the enzyme undergoes export to the cytosol leading to cleavage of the FRET dye. This cleavage and switch of fluorescence are analyzed by flow cytometry, allowing a quantitative measurement of this event.

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Daphné Laubreton, Sylvie Bay, Christine Sedlik, Cécile Artaud, Christelle Ganneau, Edith Dériaud, Sophie Viel, Anne-Laure Puaux, Sebastian Amigorena, Catherine Gérard, Richard Lo-Man, Claude Leclerc (2016 Feb 6)

The fully synthetic MAG-Tn3 therapeutic vaccine containing the tetanus toxoid-derived TT830-844 universal epitope provides anti-tumor immunity.

Cancer immunology, immunotherapy : CII : 315-25 : DOI : 10.1007/s00262-016-1802-0 En savoir plus
Résumé

Malignant transformations are often associated with aberrant glycosylation processes that lead to the expression of new carbohydrate antigens at the surface of tumor cells. Of these carbohydrate antigens, the Tn antigen is particularly highly expressed in many carcinomas, especially in breast carcinoma. We designed MAG-Tn3, a fully synthetic vaccine based on three consecutive Tn moieties that are O-linked to a CD4(+) T cell epitope, to induce anti-Tn antibody responses that could be helpful for therapeutic vaccination against cancer. To ensure broad coverage within the human population, the tetanus toxoid-derived peptide TT830-844 was selected as a T-helper epitope because it can bind to various HLA-DRB molecules. We showed that the MAG-Tn3 vaccine, which was formulated with the GSK proprietary immunostimulant AS15 and designed for human cancer therapy, is able to induce an anti-Tn antibody response in mice of various H-2 haplotypes, and this response correlates with the ability to induce a specific T cell response against the TT830-844 peptide. The universality of the TT830-844 peptide was extended to new H-2 and HLA-DRB molecules that were capable of binding this T cell epitope. Finally, the MAG-Tn3 vaccine was able to induce anti-Tn antibody responses in cynomolgus monkeys, which targeted Tn-expressing tumor cells and mediated tumor cell death both in vitro and in vivo. Thus, MAG-Tn3 is a highly promising anticancer vaccine that is currently under evaluation in a phase I clinical trial.

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Année de publication : 2015

Omar I Vivar, Giulia Masi, Jean-Marie Carpier, Joao G Magalhaes, Donatella Galgano, Gregory J Pazour, Sebastian Amigorena, Claire Hivroz, Cosima T Baldari (2015 Dec 31)

IFT20 controls LAT recruitment to the immune synapse and T-cell activation in vivo.

Proceedings of the National Academy of Sciences of the United States of America : 386-91 : DOI : 10.1073/pnas.1513601113 En savoir plus
Résumé

Biogenesis of the immune synapse at the interface between antigen-presenting cells and T cells assembles and organizes a large number of membrane proteins required for effective signaling through the T-cell receptor. We showed previously that the intraflagellar transport protein 20 (IFT20), a component of the intraflagellar transport system, controls polarized traffic during immune synapse assembly. To investigate the role of IFT20 in primary CD4(+) T cells in vitro and in vivo, we generated mice bearing a conditional defect of IFT20 expression in T cells. We show that in the absence of IFT20, although cell spreading and the polarization of the centrosome were unaffected, T-cell receptor (TCR)-mediated signaling and recruitment of the signaling adaptor LAT (linker for activation of T cells) at the immune synapse were reduced. As a consequence, CD4(+) T-cell activation and proliferation were also defective. In vivo, conditional IFT20-deficient mice failed to mount effective antigen-specific T-cell responses, and their T cells failed to induce colitis after adoptive transfer to Rag(-/-) mice. IFT20 is therefore required for the delivery of the intracellular pool of LAT to the immune synapse in naive primary T lymphocytes and for effective T-cell responses in vivo.

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Alloatti A1, Kotsias F2, Pauwels AM3, Carpier JM, Jouve M, Timmerman E, Pace L, Vargas P, Maurin M, Gehrmann U, Joannas L, Vivar OI, Lennon-Duménil AM, Savina A, Gevaert K, Beyaert R, Hoffmann E, Amigorena S. (2015 Dec 15)

Toll-like Receptor 4 Engagement on Dendritic Cells Restrains Phago-Lysosome Fusion and Promotes Cross-Presentation of Antigens.

Immunity : DOI : 10.1016/j.immuni.2015.11.006 En savoir plus
Résumé

The initiation of cytotoxic immune responses by dendritic cells (DCs) requires the presentation of antigenic peptides derived from phagocytosed microbes and infected or dead cells to CD8(+) T cells, a process called cross-presentation. Antigen cross-presentation by non-activated DCs, however, is not sufficient for the effective induction of immune responses. Additionally, DCs need to be activated through innate receptors, like Toll-like receptors (TLRs). During DC maturation, cross-presentation efficiency is first upregulated and then turned off. Here we show that during this transient phase of enhanced cross-presentation, phago-lysosome fusion was blocked by the topological re-organization of lysosomes into perinuclear clusters. LPS-induced lysosomal clustering, inhibition of phago-lysosome fusion and enhanced cross-presentation, all required expression of the GTPase Rab34. We conclude that TLR4 engagement induces a Rab34-dependent re-organization of lysosomal distribution that delays antigen degradation to transiently enhance cross-presentation, thereby optimizing the priming of CD8(+) T cell responses against pathogens.

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Sebastian Amigorena (2015 Sep 12)

Helping the Help for CD8+ T Cell Responses.

Cell : 1210-2 : DOI : 10.1016/j.cell.2015.08.051 En savoir plus
Résumé

Eickhoff et al. and Hor et al. use time-lapse intravital microscopy to show an unexpected choreography of CD4+ and CD8+ T cells « dancing » between different dendritic cell sub-populations during priming of cytotoxic immune responses to viruses.

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