Gilles Dadaglio

Plasmacytoid dendritic cells efficiently cross-prime naive T cells in vivo after TLR activation

Cross-presentation is a crucial mechanism in tumoral and microbial immunity because it allows internalized cell associated or exogenous antigens (Ags) to be delivered into the major histocompatibility complex I pathway. This pathway is important for the development of CD8(+) T-cell responses and for the induction of tolerance. In mice, cross-presentation is considered to be a unique property of CD8alpha+ conventional dendritic cells (DCs). Here we show that splenic plasmacytoid DCs (pDCs) efficiently capture exogenous Ags in vivo but are not able to cross-present these Ags at steady state. However, in vitro and in vivo stimulation by Toll-like receptor-7, or -9 or viruses licenses pDCs to cross-present soluble or particulate Ags by a transporter associated with antigen processing-dependent mechanism. Induction of cross-presentation confers to pDCs the ability to generate efficient effector CD8+ T-cell responses against exogenous Ags in vivo, showing that pDCs may play a crucial role in induction of adaptive immune responses against pathogens that do not infect tissues of hemopoietic origin. This study provides the first evidence for an in vivo role of splenic pDCs in Ag cross-presentation and T-cell cross-priming and suggests that pDCs may constitute an attractive target to boost the efficacy of vaccines based on cytotoxic T lymphocyte induction.

Efficient In Vivo Priming of Specific Cytotoxic T Cell Responses by Neonatal Dendritic Cells

In early life, a high susceptibility to infectious diseases as well as a poor capacity to respond to vaccines are generally observed as compared with observations in adults. The mechanisms underlying immune immaturity have not been fully elucidated and could be due to the immaturity of the T/B cell responses and/or to a defect in the nature and quality of Ag presentation by the APC. This prompted us to phenotypically and functionally characterize early life murine dendritic cells (DC) purified from spleens of 7-day-old mice. We showed that neonatal CD11c+ DC express levels of costimulatory molecules and MHC molecules similar to those of adult DC and are able to fully maturate after LPS activation. Furthermore, we demonstrated that neonatal DC can efficiently take up, process, and present Ag to T cells in vitro and induce specific CTL responses in vivo. Although a reduced number of these cells was observed in the spleen of neonatal mice as compared with adults, this study clearly shows that neonatal DC have full functional capacity and may well prime Ag-specific naive T cells in vivo.

Induction of a Polarized Th1 Response by Insertion of Multiple Copies of a Viral T-Cell Epitope into Adenylate Cyclase of Bordetella pertussis

ABSTRACT The adenylate cyclase (CyaA) of Bordetella pertussisdelivers the N-terminal catalytic domain into the cytosol of a large number of eukaryotic cells, in particular, professional antigen-presenting cells. This allows the delivery of CD8+T-cell epitopes to the major histocompatibility complex class I presentation pathway. We have previously shown that immunization of mice with CyaA carrying a single CD8+ T-cell epitope leads to antiviral protection as well as to protective and therapeutic antitumor immunity associated with the induction of specific cytotoxic T-lymphocyte (CTL) responses. Here, we evaluated the capacity of CyaA carrying one to four copies of the CD8+ CD4+T-cell epitope from the nucleoprotein of the lymphocytic choriomeningitis virus to induce T-cell responses. Both CTL and Th1-like specific responses were detected in mice immunized with recombinant CyaA with or without adjuvant. Although the insertion of the larger peptides resulted in partial loss of the invasive capacity of recombinant CyaA, insertion of several copies of the same epitope led to a strong enhancement of Th1 responses and, to a lesser degree, CTL responses. These results underscore the potency of CyaA for vaccine design with a new impact on diseases in which the Th1 response has been described to have a beneficial effect.

Potential deleterious effect of anti-viral cytotoxic lymphocyte through the CD95 (FAS/APO-1)-mediated pathway during chronic HIV infection.

The potential deleterious effect through a CD95-based pathway of anti-viral cytotoxic lymphocyte (CTL) during HIV-infection was studied. The present paper reports that a Nef specific CTL line derived from an HIV-infected person is able to kill not only Nef-expressing target cells but also CD95+ compliant Jurkat cells. The two mechanisms of cytotoxicity, i.e. perforin-vs-CD95-dependent were differentiated according to their respective Ca(2+)-dependence. The existence of the dual killing machinery in the anti-HIV CTL line was correlated with the coexpression in these cells of perforin and CD95-L molecules. A model of AIDS pathogenesis involving the deleterious effect through the CD95 pathway of the viral specific CTL response is discussed.

Induction of CTL and nonpolarized Th cell responses by CD8alpha(+) and CD8alpha(-) dendritic cells

Two distinct dendritic cell (DC) subpopulations have been evidenced in mice on the basis of their differential CD8α expression and their localization in lymphoid organs. Several reports suggest that CD8α+ and CD8α− DC subsets could be functionally different. In this study, using a panel of MHC class I- and/or class II-restricted peptides, we analyzed CD4+ and CD8+ T cell responses obtained after i.v. injection of freshly purified peptide-pulsed DC subsets. First, we showed that both DC subsets efficiently induce specific CTL responses and Th1 cytokine production in the absence of CD4+ T cell priming. Second, we showed that in vivo activation of CD4+ T cells by CD8α+ or CD8α− DC, injected i.v., leads to a nonpolarized Th response with production of both Th1 and Th2 cytokines. The CD8α− subset induced a higher production of Th2 cytokines such as IL-4 and IL-10 than the CD8α+ subset. However, IL-5 was produced by CD4+ T cells activated by both DC subsets. When both CD4+ and CD8+ T cells were primed by DC injected i.v., a similar pattern of cytokines was observed, but, under these conditions, Th1 cytokines were mainly produced by CD8+ T cells, while Th2 cytokines were produced by CD4+ T cells. Thus, this study clearly shows that CD4+ T cell responses do not influence the development of specific CD8+ T cell cytotoxic responses induced either by CD8α+ or CD8α− DC subsets.

Enhancement of HIV-specific cytotoxic T lymphocyte responses by zidovudine (AZT) treatment

Zidovudine or 3′‐azido‐2′‐3′‐dideoxy‐thymidine (AZT) is an antiviral drug widely used to treat HIV‐infected patients. Because cytotoxic T lymphocytes (CTL) are thought to contribute actively to resistance against HIV‐induced disease, we studied sequentially 10 HIV‐infected individuals under zidovudine treatment for a period of 6–12 months. For a given patient all lymphocyte suspensions corresponding to the complete zidovudine therapy period were tested on the same day and on the same target cells. Patients were selected for expression of HLA‐A2 and/or HLA‐A3 class I transplantation antigen. HLA‐restricted cytotoxicity specific for env, gag and nef HIV proteins was quantified for each patient at 6 week intervals. The data clearly indicated that zidovudine has a beneficial effect on the CTL response during the first 6–12 weeks of treatment, inducing cytotoxicity levels up to 100‐fold stronger than base line. This effect was usually short lived. However, patients who maintained strong levels of cytotoxicity had better clinical and survival outlook than patients who had lost all detectable cytotoxic lymphocytes. It is proposed that AZT, among other effects, delays the onset of disease in HIV‐infected patients by contributing to the stimulation of the HIV specific CTL response.

Antigen Targeting to CD11b+ Dendritic Cells in Association with TLR4/TRIF Signaling Promotes Strong CD8+ T Cell Responses

Deciphering the mechanisms that allow the induction of strong immune responses is crucial to developing efficient vaccines against infectious diseases and cancer. Based on the discovery that the adenylate cyclase from Bordetella pertussis binds to the CD11b/CD18 integrin, we developed a highly efficient detoxified adenylate cyclase-based vector (CyaA) capable of delivering a large variety of Ags to the APC. This vector allows the induction of protective and therapeutic immunity against viral and tumoral challenges as well as against transplanted tumors in the absence of any added adjuvant. Two therapeutic vaccine candidates against human papilloma viruses and melanoma have been developed recently, based on the CyaA vector, and are currently in clinical trials. We took advantage of one of these highly purified vaccines, produced under good manufacturing practice–like conditions, to decipher the mechanisms by which CyaA induces immune responses. In this study, we demonstrate that CyaA binds both human and mouse CD11b+ dendritic cells (DCs) and induces their maturation, as shown by the upregulation of costimulatory and MHC molecules and the production of proinflammatory cytokines. Importantly, we show that DCs sense CyaA through the TLR4/Toll/IL-1R domain–containing adapter-inducing IFN-β pathway, independent of the presence of LPS. These findings show that CyaA possesses the intrinsic ability to not only target DCs but also to activate them, leading to the induction of strong immune responses. Overall, this study demonstrates that Ag delivery to CD11b+ DCs in association with TLR4/Toll/IL-1R domain–containing adapter-inducing IFN-β activation is an efficient strategy to promote strong specific CD8+ T cell responses.

B cells promote tumor progression in a mouse model of HPV‐mediated cervical cancer

Enhancing anti‐tumor immunity and preventing tumor escape are efficient strategies to increase the efficacy of therapeutic cancer vaccines. However, the treatment of advanced tumors remains difficult, mainly due to the immunosuppressive tumor microenvironment. Regulatory T cells and myeloid‐derived suppressor cells have been extensively studied, and their role in suppressing tumor immunity is now well established. In contrast, the role of B lymphocytes in tumor immunity remains unclear because B cells can promote tumor immunity or display regulatory functions to control excessive inflammation, mainly through IL‐10 secretion. Here, in a mouse model of HPV‐related cancer, we demonstrate that B cells accumulated in the draining lymph node of tumor‐bearing mice, due to a prolonged survival, and showed a decreased expression of MHC class II and CD86 molecules and an increased expression of Ly6A/E, PD‐L1 and CD39, suggesting potential immunoregulatory properties. However, B cells from tumor‐bearing mice did not show an increased ability to secrete IL‐10 and a deficiency in IL‐10 production did not impair tumor growth. In contrast, in B cell‐deficient μMT mice, tumor rejection occurred due to a strong T cell‐dependent anti‐tumor response. Genetic analysis based on single nucleotide polymorphisms identified genetic variants associated with tumor rejection in μMT mice, which could potentially affect reactive oxygen species production and NK cell activity. Our results demonstrate that B cells play a detrimental role in anti‐tumor immunity and suggest that targeting B cells could enhance the anti‐tumor response and improve the efficacy of therapeutic cancer vaccines.

Induction of HLA-A2-restricted CTL responses by a tubular structure carrying human melanoma epitopes.

Epitope-based vaccination strategies designed to induce strong tumor-specific CD8(+) T cell responses are being widely considered for cancer immunotherapy. Here, two recombinant tubular structures, NS1-Mela 1 and NS1-Mela 2, carrying, respectively two HLA-A2 epitopes derived from human melanoma antigens were constructed and their capability to induce CTL responses in vivo were studied in HLA-A2 transgenic mice. Strong CTL responses specific for GnT-V/NA 17-A and gp100 (154-162) epitopes were generated in HLA-A2 transgenic mice immunized by the construct NS1-Mela l carrying these two epitopes. The second construct NS1-Mela 2 carrying both Tyrosinase (369-377Da) and Melan-A/Mart-1 (27-35) epitopes induced a weak Tyrosinase-specific CTL response in mice but failed to induce specific CTL responses against the Melan-A/Mart-1 (27-35) epitope in the tested mice. Thus, recombinant tubular structures containing multiple tumoral epitopes may lead to new strategies for the induction of strong tumor-specific CTL responses in cancer patients.

Pore-formation by adenylate cyclase toxoid activates dendritic cells to prime CD8 + and CD4 + T cells

The adenylate cyclase toxin‐hemolysin (CyaA) of Bordetella pertussis is a bi‐functional leukotoxin. It penetrates myeloid phagocytes expressing the complement receptor 3 and delivers into their cytosol its N‐terminal adenylate cyclase enzyme domain (~400 residues). In parallel, ~1300 residue‐long RTX hemolysin moiety of CyaA forms cation‐selective pores and permeabilizes target cell membrane for efflux of cytosolic potassium ions. The non‐enzymatic CyaA‐AC− toxoid, has repeatedly been successfully exploited as an antigen delivery tool for stimulation of adaptive T‐cell immune responses. We show that the pore‐forming activity confers on the CyaA‐AC− toxoid a capacity to trigger Toll‐like receptor and inflammasome signaling‐independent maturation of CD11b‐expressing dendritic cells (DC). The DC maturation‐inducing potency of mutant toxoid variants in vitro reflected their specifically enhanced or reduced pore‐forming activity and K+ efflux. The toxoid‐induced in vitro phenotypic maturation of DC involved the activity of mitogen activated protein kinases p38 and JNK and comprised increased expression of maturation markers, interleukin 6, chemokines KC and LIX and granulocyte‐colony‐stimulating factor secretion, prostaglandin E2 production and enhancement of chemotactic migration of DC. Moreover, i.v. injected toxoids induced maturation of splenic DC in function of their cell‐permeabilizing capacity. Similarly, the capacity of DC to stimulate CD8+ and CD4+ T‐cell responses in vitro and in vivo was dependent on the pore‐forming activity of CyaA‐AC−. This reveals a novel self‐adjuvanting capacity of the CyaA‐AC− toxoid that is currently under clinical evaluation as a tool for delivery of immunotherapeutic anti‐cancer CD8+ T‐cell vaccines into DC.