Unit: Immune Regulation and Vaccinology - INSERM E 352
Director: LECLERC Claude
The activity of our laboratory is focused on the understanding of the mechanisms that control the activation and regulation of T cell responses and on the development of new strategies of vaccination against tumors and infections. We have recently developed several strategies of activation of CTL responses as well as fully synthetic glycopeptide carrying saccharidic epitopes to induce anti-tumoral immune responses. We are also investigating the biology of dendritic cells, in adults and neonates.
A. The adenylate cyclase toxin (CyaA) of Bordetella pertussis: a new vector targeting dendritic cells (C. Fayolle, P. Berraondo et L. Mascarell, in collaboration with D. Ladant, IP and P. Sebo, Prague)
The adenylate cyclase toxin (CyaA) of Bordetella pertussis is a major virulence factor, which can invade eukaryotic cells. We previously demonstrated that CyaA uses the integrin CD11b/CD18 as a cell receptor. These results will help us to design a highly efficient vector capable of targeting a wide range of antigens to CD11b+ antigen presenting cells, leading to a more efficient immune responses.
The Tat protein from HIV-1 was inserted in recombinant CyaA and its ability to induce specific humoral responses against Tat as well as cellular responses was assessed. Mice immunized with CyaA-Tat developed a Tat specific neutralizing humoral response as well as CD8+ T cell responses. In addition, the induced cellular polarization is of Th1 type. These promising results were extended to non-human primates. Indeed, the injection of CyaA-Tat into green monkeys elicited strong humoral responses capable to neutralize the transactivating activity of Tat and in addition induced a Th1 cellular response.
CyaA targets CD8α-, CD11c+, CD11b+ DC with a high efficiency, and dendritic cells (DC) are the only APC capable of presenting CyaA to CD8+ T and CD4+ T cells. After the encounter with various stimuli, DC increase the expression of MHC molecules and several co-stimulatory molecules, which are necessary to trigger an immune response and in particular, a CTL response. We thus analyzed if this high immunogenicity of CyaA was linked to its capacity to induce DC maturation. We have shown that in vitro very low doses of wild type CyaA, but not a catalytic inactive mutant, trigger DC maturation as probed by increase in surface levels of maturation markers: MHC I and II, costimulatory molecules CD80, CD86 and IL-2 receptor. In parallel, intravenous injection of CyaA, but not its inactive mutant, triggers splenic DC maturation. We are currently investigating the mechanisms responsible for the high immunogenicity of detoxified CyaA.
B. Analysis of immunological properties of viral pseudo-particles (D. Briard and S. Hervas in collaboration with P. Rueda, Madrid)
Chimeric porcine parvovirus virus-like particles (PPV-VLPs), prepared by self-assembly of the VP2 capsid protein of this virus and carrying heterologous epitopes at its N terminus is an efficient antigen delivery system that elicits strong CD4+ and CD8+ T cells responses specific for the foreign epitopes in the absence of adjuvant. As usually exogenous antigens cannot enter into the MHC Class I pathway, PPV-VLPs represent a very interesting antigen carrier to trigger CTL response. We have shown in mice that DC capture these particles very efficiently in vivo and are the only cells capable to present PPV-VLPs to specific CD8+ hybridoma.
We currently analyze the capture of PPV-VLPs in different lymphoid organs either in naive mice or following immunization with these vectors in order to determine if the immunity against a vector affects its capacity to be delivered to DC. We have also deciphered the mechanisms by which the particles induce strong T cell responses.
C. Analysis of CTL and Th responses induced by dendritic cell subpopulations (G. Dadaglio, J. Mouries and M. Rojas)
Plasmacytoid DC (pDC) have recently been identified in mouse lymphoid organs. To assess their in vivo function on adaptive responses, purified immature or activated pDCs were loaded with a synthetic peptide containing a CD8+ T cell epitope and then transferred by i.v. route into naive syngeneic hosts. We show that immature and CpG-activated pDC do not induce specific CTL response nor regulatory activity, suggesting that they cannot prime CD8+ T cells. Nevertheless, CpG-activated pDC were able to recall memory T cells, in contrast to immature pDC. However, after stimulation by heat-inactivated influenza virus, pDC produced IFN-α and induced antigen-specific effector/memory T cells. Thus, pDC differentiate into professional APC in the context of viral infection, demonstrating that they can play a role in both innate and adaptive anti-viral immunity. We are currently investigating the role of pDC in induction of innate and adaptive responses during viral infection using influenza virus as viral model.
Furthermore, we have previously shown that pDC present endogenous antigen, suggesting that pDC have the capacity to induce anti-viral immunity if directly infected. However, induction of protective anti-viral responses may require cross presentation of viral antigen to generate CTL responses against viruses that do not infect pDC. We are currently investigating the ability of pDC to cross present exogenous antigens.
D. Ontogeny, functions and regulation of neonatal dendritic cells (R. Lo-Man and X. Zhang)
We are investigating how the dendritic cell compartment may contribute to the high susceptibility to infections observed in newborns and to biased Th2 responses that are often induced. We recently described the ontogeny of mouse DC and showed that they are fully functional at the level of innate responses and in their capacity to prime T cells in vitro and in vivo. However, if neonatal DC are fully competent, we were able to demonstrate for the first time that B cells can control the capacity of neonatal DC to prime Th1 responses and therefore dampen their immune functions. We are currently investigating the ins and outs of this regulation process.
E. Investigation of mechanisms of anti-mycobacterial immunity (L. Majlessi, C. Nouzé and S. Hervas in collaboration with S. Cole and collaborators)
Induction of Th1 responses to mycobacterial antigens is essential in protection against infection with Mycobacterium tuberculosis, the ethiologic agent of tuberculosis. We study the capacity of recombinant CyaA, bearing mycobacterial antigens, as sub-unit vaccine, to induce T-cell responses and to confer protection against infection with M. tuberculosis. The antigens inserted into CyaA for this investigation are proteins known for their strong immunogenicity in both mice and human or a protein we identified as a potent immunogen for CD4+ or CD8+ T-cell subsets in mice. We currently investigate the potential of these vectors, injected alone or in the presence of appropriate adjuvants, as sub-unit vaccine candidates against infection with M. tuberculosis.
In collaboration with the Unit of Génétique Moléculaire Bactérienne, we recently demonstrated that introduction of a virulence-associated chromosomal region of M. tuberculosis into M. bovis BCG markedly modifies the interactions between the host immune system and mycobacteria. Indeed, BCG complemented with this chromosomal region induces preferential recruitments of dendritic cells and of CD4+ or CD8+ activated/effector T cells. In contrast to the parental BCG, this BCG is able to induce in vivo at the level of dendritic cells an inflammation program as it activates production of inflammatory chemokines and cytokines. More recently, we characterized the impact of the expression of each individual gene of this M. tuberculosis chromosomal region on the immunogenicity of the most promising antigens coded in this region.
Due to generation of CD4+ ou T CD8+ T- cell hybridomas specific to mycobacterial immunogens, we investigate the cellular and molecular mechanisms of the presentation of mycobacterial antigens via MHC-class I or class II pathway.
F. Elaboration of a fully synthetic immunogen bearing a carbohydrate tumor marker for immunotherapy (R. Lo-Man and E. Dériaud in collaboration with S. Bay, T. Freire and S. Vichier-Guerre from Unité de Chimie Organique)
We have developed multiple antigenic glycopeptides (MAG) based on a lysine core extended with peptidic arms displaying a carbohydrate tumor antigen (Tn antigen). The resulting dendrimeric MAGs were able to induce anti-Tn IgG antibodies in a T cell dependent manner that recognized murine as well as human tumor cell lines that express Tn. In mice, therapeutic vaccination using these MAG provided a 70% survival rate of tumor-bearing mice. Together, these results demonstrate that the MAG represents a safe and highly efficient system to induce anti-carbohydrate antibodies and is a potent alternative strategy to the traditional carbohydrate-protein conjugates, which are developed for vaccine and therapeutic purposes. We recently developed new MAGs potentially active in humans and the immunogenicity of these molecules has been demonstrated in non-human primates.
Keywords: Vaccines, CTL, dendritic cells, cancer, T cells, immunotherapy, anti-mycobacterial immunity