Thèse Mise au Point de Nouveaux Adjuvants Immunitaires Ciblant les Cellules Dendritiques Conventionnelles Humaines H/F - 69

Établissement : Université Claude Bernard Lyon 1
École doctorale : CanBioS - Cancérologie, Biologie, Santé de Lyon
Laboratoire de recherche : CRCL - CENTRE DE RECHERCHE EN CANCÉROLOGIE DE LYON
Direction de la thèse : Jenny VALLADEAU-GUILEMOND ORCID 0000000341608867
Début de la thèse : 2026-10-01
Date limite de candidature : 2026-05-11T23:59:59

Les inhibiteurs de points de contrôle immunitaires (ICB) ont profondément transformé l'immunothérapie du cancer en réactivant l'immunité antitumorale. Toutefois, leur efficacité reste limitée dans les tumeurs « froides » dépourvues d'infiltrat immunitaire. L'association avec des vaccins thérapeutiques pourrait lever cette résistance en déclenchant une réponse CD8 + T élargie (epitope spreading) grâce à la capture et la présentation des antigènes tumoraux par les cellules dendritiques (DC).

Parmi les DC humaines, les cellules conventionnelles de type 1 (cDC1) - exprimant Clec9A, TLR3 et XCR1 - sont les plus efficaces pour la crossprésentation des antigènes exogènes. Leur présence dans les tumeurs est corrélée à la réponse aux ICB et au rejet tumoral. Notre laboratoire ValladeauGuilemond a montré que les cDC1 tumorales produisent de l'IFN de type III (IFN) après activation par un ligand TLR3, favorisant l'expression de chimiokines CXCR3ligands indispensables au recrutement des lymphocytes CD8 +, confirmant leur rôle clé dans l'immunité antitumorale.

Les mécanismes de la crossprésentation restent mal définis chez l'humain. Deux voies principales sont décrites : l'une cytosolique (dégradation au protéasome puis chargement dans le réticulum endoplasmique), l'autre vacuolaire, contrôlée notamment par Sec22b. Faute de modèles disponibles, les régulations spécifiques des cDC1 humaines demeurent largement inexplorées, alors que la forme de l'antigène (soluble, cellulaire, vésiculaire) influence fortement leur capacité de présentation.

Hypothèse : renforcer la crossprésentation des antigènes par les cDC1 pourrait restaurer l'immunité dans les tumeurs résistantes et accroître l'efficacité des vaccins anticancéreux combinés aux ICB.

Objectifs : identifier des petites molécules et formulations antigéniques capables d'augmenter la crossprésentation par les DC humaines, définies comme de nouveaux « adjuvants de crossprésentation ».

Données préliminaires :
(1) Mise au point d'un système produisant des cDC1 et cDC2 humaines à partir de CD34 +, phénotypiquement et fonctionnellement comparables aux DC sanguines.
(2) Validation d'un test de crossprésentation utilisant les cDC1 et le peptide MART1, mesurant l'activation spécifique de lymphocytes CD8 + via la production d'IFN.

Plan de recherche :
Tâche 1 : miniaturiser le test de crossprésentation pour le rendre compatible avec un criblage à haut débit. Une lecture fluorescente (GFP/NFAT) remplacera la cytométrie classique, permettant d'évaluer rapidement différentes formes d'antigènes (peptides longs, lysats tumoraux, ARNm).
Tâche 2 : cribler, avec la plateforme C3D du CRCL, des bibliothèques de molécules afin d'identifier des composés augmentant la crossprésentation par cDC1/cDC2.
Tâche 3 : caractériser les voies moléculaires ciblées par les composés identifiés à l'aide d'analyses d'expression, d'imagerie confocale et de cytométrie spectrale, en conditions pro et antiinflammatoires.

Méthodologie :
Le test miniaturisé reposera sur des cellules Jurkat rapportant l'activation NFAT par GFP et exprimant un TCR MART1spécifique, cocultivées avec des cDC présentant différentes formes d'antigène (protéines solubles, lysats de MELXY3/SKmel5, ou ARNm MART1 dans SKBR3 et OVCAR3). L'activation T sera mesurée par fluorescence (lecteur Tecan M200, Incucyte).

Les molécules identifiées serviront à décrypter les voies clés de la crossprésentation humaine et à identifier de nouvelles cibles thérapeutiques. Ce projet, à l'interface entre immunologie fondamentale, criblage pharmacologique et immunothérapie translationnelle, vise à découvrir des modulateurs capables d'améliorer les vaccins anticancéreux de nouvelle génération.

Cancer immunotherapy has been revolutionized in the past decade by the development of immune checkpoint blockers (ICBs), able to restore anti-tumor immunity suppressed by the tumor microenvironment. However, ICBs have had very little efficacy in immunologically cold tumors, where immune infiltration is absent, thus another strategy needs to be proposed such as the combination of ICBs with a vaccine(1).
The success of a vaccine strategy relies on the amplification of the anti-tumor immunity and epitope spreading. It has now been established in various anti-tumour vaccine approaches that the therapeutic response in patients is linked to the reactivation of cytotoxic T cells (CTL) recognising not only the vaccine antigens, but also multiple other endogenous tumour antigens. This epitope spreading results from the capture and presentation by tumour-infiltrating dendritic cells (DCs) of tumour antigens released by lysed tumour cells. Some DCs are specialized in processing internalized exogenous antigens into the MHC class I pathway for presentation to CD8+ T cells, a process called cross-presentation (2). In this context, J. Valladeau-Guilemond lab and others have shown in humans that type 1 conventional dendritic cells (cDC1), characterized by the expression of Clec9A, TLR3 and XCR1 3, are the most efficient for cross-presentation 4-7. Moreover, several studies in mice have highlighted the importance of cDC1 in the rejection of transplanted tumours, in the efficacy of adoptive transfer of anti-tumor effector CD8+ T cells 8 and in the response to anti-PD-1 and anti-CD137 9-12.
J. Valladeau-Guilemond lab has also shown that cDC1 infiltrating breast and ovarian tumors can produce type III interferon (IFN) after reactivation by a TLR3 ligand and that the presence of this type III IFN alone in the tumor microenvironment correlates with the presence of CXCR3 ligands, which are essential for the recruitment of cytotoxic CD8+T lymphocytes, confirming the essential role of this immune population 13.
Antigen cross-presentation has been studied using different cell types and different forms of antigen, but not all results have been confirmed in vivo 14. There are two main models of cross-presentation: one involving transport of the exogenous antigen to the cytosolic proteasome prior to loading of the peptide into the endoplasmic reticulum (ER), and another where peptide loading occurs directly into phagosome/endosome fusion compartments, the latter pathway possibly regulated by the SNARE Sec22b family member 15In murine cDC1, many proteins may allow cDC1 cross-presentation such as WDFY412 or RAB 16. Nevertheless, few studies have focused on cDC1 in humans due to their rarity, despite their key role in the anti-tumour response. The form of the antigen must also be taken into account as cross-presentation will be impacted depending on the state of the antigen: cellular, soluble or vesicular (liposomes, exosomes, etc.). In this context, others DC such as cDC2 will be also able to cross-present some antigen depending on their formulation 17,18.

Internalisation and priming against tumour antigens by cDC1 and their level of activation are essential to the success of a vaccine 19. Therefore, our hypothesis is that:
Therapies that increase antigen cross-presentation by cDC1 could provide new ways to restore anti-tumour immunity, increase efficacy of cancer vaccine and/or overcome resistance to ICBs.
Our objective is thus to develop new targets or adjuvants that will increase anti-cancer vaccine efficacy. This includes screening small-molecules for modulators of cross-presentation by human DC and testing different antigen forms compatible with vaccine development.

Research plan and experimental strategy of the project:
Task 1: Set up of a fluorescent miniaturized cross-presention assay using different antigen formulation: This task will consist of miniaturizing the previously developed cross-presentation assay to make it compatible with large screening of small-molecule libraries. Instead of monitoring specific T cell activation by flow cytometry, which requires too many cells, the activation will be quantified by fluorescence, which is compatible with an automated evaluation of many co-cultured cells. Different antigen formulation (long/synthetic peptides, whole tumor lysates, mRNA) which are compatible with clinical vaccine formulation will be tested.
Task 2 : Screening of small-molecule libraries for unbiased identification of novel adjuvants : Once optimized, the miniaturized assay will be used to test thousands of small-molecule using libraries provided by the C3D platform of the CRCL. The aim is the identification of novel small molecules capable of enhancing cDC1 and/or cDC2-mediated cross-presentation that will be thus nammed « cross-presentation adjuvants ».

Task 3 : Decipher molecular mechanism of antigen presentation by cDC based on the activity of target identified. The identified « cross-presentating adjuvants » will help elucidate key mechanisms and signaling pathways involved in human dendritic cells cross-presentation.

Methodology for carrying out the project :
Task 1: To miniaturize the cross-presentation assay, Jurkat cells expressing the GFP under the control of NFAT, will be used. Jurkat cells will be co-transduced with a lentiviral construct encoding a MART1-specific TCR 22. To ensure relevance and compatibility in vaccine formulation, we will compare different antigen forms: soluble long protein, MART1-coated beads, tumor cell lysate from MART1-expressing cells (MELXY3 and SKmel5 UVB irradiated), and in vitro antigen mRNA delivery with cationic lipopolymer reagents (tebubio) in various cancer cell lines, including SKBR3, and OVCAR3, representing breast, and ovarian cancer respectively. Human cDC cross-presenting the MART1 epitope will be co-cultured with the co-transduced Jurkat T cells. T cell activation, which reflects the efficiency of antigen cross-presentation will be monitored by quantifying the fluorescence with the Infinite M200 Tecan (C3D platform), or using the Incucyte live-cell analysis system (available at the CRCL
Task 2: In collaboration with the C3D platform at the CRCL, we will evaluate the impact of small molecule libraries on cross presentation efficiency using the optimized assay systems based on distinct antigen forms.
Task 3: The identified targets will help elucidate key mechanisms and signaling pathways involved in cross-presentation. We will further characterize these pathways by analyzing the expression of their main components, the intracellular localization of the corresponding proteins (confocal microscopie), and their regulation by the tumor microenvironment (spectral flow cytometry) as well as by pro and anti inflammatory cytokines. Because cDC1 are rare, we plan to analyse cDC2 which were also decscribed to perform antigen cross-presentation depending of the antigen form.