Unit: Biology of Immune Regulations - 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. 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 particular in neonates.

A. The adenylate cyclase toxin (CyaA) of Bordetella pertussis: a new vector targeting dendritic cells (M. El-Azami El-Idrissi, L. Mascarell, C. Fayolle, et G. Schlecht, 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. Using CyaA mutants and CyaA fragments, we have identified the region of CyaA interacting with CD11b. 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.

An HIV-1 protein was inserted in recombinant CyaA and its ability to induce specific humoral response against viral protein was assessed. Strong humoral response was obtained with CyaA carrying this antigen. In addition, the induced cellular response is of Th1 type. Immune responses were still detected 9 months after immunization demonstrating that CyaA induces long term memory responses.

We also established that CyaA can deliver a CD4 T cell epitope to MHC class II presentation pathway 100 to 1000 more efficiently than the native protein containing this epitope. This potentiation of MHC-II-restricted presentation is blocked when CyaA does not interact with CD11b. The mechanisms of entry and processing of CyaA giving rise to MHC-II-restricted peptides are currently studied.

We have also developed a new approach in which CD8+ T cell epitopes are chemically linked to CyaA. We showed that high CTL responses were induced in mice immunized with CyaA bearing CD8+ T cell epitopes from ovalbumin and LCMV. In addition, we demonstrated that a CD8+ T cell epitope chemically coupled to CyaA is presented to specific T cells by a mechanism requiring proteasome processing, the transporters associated with antigen presentation (TAP molecules) and neosynthesis of MHC class I molecules. The expected advantage of this novel strategy is its versatility as we can easily couple any defined epitope to CyaA.

B. Induction of CTL responses by viral pseudo-particles and mechanism of MHC I presentation (G. Morón and D. Briard in collaboration with P. Rueda, Madrid)

1. Mechanisms of delivery of exogenous viral pseudo-particles into the MHC Class I pathway (G. Morón)

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 can not enter into the MHC Class I pathway, PPV-VLPs represent a very interesting antigen carrier to trigger CTL response. Dendritic cells (DC) capture these particles very efficiently in vivo and are the only cells capable to present PPV-VLPs to specific CD8+ hybridoma. Both CD8α - and CD8α + DC process PPV-VLPs. DC stimulation by PPV-VLPs induces the expression of CD8α and CD205 and of co-stimulatory molecules on both DC subsets but the loss of CD4 molecules in CD8α - DC. Furthermore, DC from mice injected with PPV-VLPs are capable to secreting IL12p70 and IFNγ. After in vivo capture, PPV-VLPs are found in late endosomes. MHC Class I presentation of PPV-VLPs requires intracellular processing, following a non classical pathway involving macropinocytosis, vacuolar acidification and lysosomal proteases, but also processing by the proteasome complex and translocation of epitopes from the cytosol to the endoplasmic reticulum using TAP molecules.

2. Mechanisms of antigen presentation during prime-boost (D. Briard)

We currently analyze the mechanisms of antigen presentation during several schemes of immunization in which two or more immunizations are done using different antigen delivery vectors. These studies are performed using two non replicative delivery vectors developed in our laboratory, CyaA and PPV-VLPs. We also study the capture of PPV-VLPs in different lymphoid organs from naive versus PPV-VLPs-immunized mice.

C. Analysis of CTL and Th responses induced by dendritic cell subpopulations (G. Dadaglio and G. Schlecht)

It is now generally accepted that fully mature DC are the only professional antigen presenting cells (APC) able to induce primary T cell-mediated immune response. The murine equivalent of human plasmacytoid DC (pDC) has 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. Further experiments are currently establishing the capacity of immature and activated pDC to prime naive CD4+ T cells in vivo.

D. Ontogeny and functions of neonatal dendritic cells (CM. Sun and R. Lo-Man)

We investigate whether a developmental immaturity of the dendritic cell compartment could contribute to the high susceptibility to infections observed in newborns. We show that although the ontogeny of DC subsets follows distinct steps, they are functionally capable to produce IL-12 and to prime T cells in vitro and in vivo. However, if neonatal DC are fully competent, the neonatal environment dampens their immune functions. We are currently investigating the parameters of the neonatal environment responsible for this phenomenon.

E. Investigation of mechanisms of anti-mycobacterial immunity (L. Majlessi, S. Hervas et M. Rojas 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 the 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.

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+ T cells of activated and effector phenotype.

Identification of a mycobacterial immunogen with the rare capacity to be recognized by CD8+ CTL of mycobacteria-infected mice and generation of MHC-I-restricted T-cell hybridomas specific to this immunogen enable us to investigate the mechanisms of the presentation of mycobacterial antigens via MHC-class I pathway for the generation of CD8+ T-cell responses.

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 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

Activity Reports 2003 - Institut Pasteur

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