Antigen presentation in dendritic cells

Keywords: dendritic cells, antigen cross presentation, in vivo imaging, phagosomal pH regulation, Nox2, Rab27, CD8, T cell

Read the scientific activity report. (pdf 90Ko, last update 13th, february 2008)

Dendritic cells (DCs) are involved in many pathologies in connection with the immune system, like infectious or autoimmune diseases, and also in cancer. At present, DCs are used to develop vaccines for these pathologies. The aim of our research is to understand the molecular mechanisms at stake in the immune responses against tumours.

Molecular mechanisms involved in the regulation of cross presentation in dendritic cells

Cross presentation is the process by which DCs phagocytose pathogens or dying cell fragments and present proteolytic peptides derived from these antigens in association with MHC class I molecules. This process is a fundamental mechanism of induction of anti tumor immune responses. In the last eight years, we have uncovered some of intracellular pathways that link the antigen internalization pathways and their processing and loading on MHC class I molecules. We have demonstrated that the NADPH oxidase NOX2 is recruited to DC early phagosomes mediating a sustained production of low levels of reactive oxygen species and causing a maintained alkalynization of the phagosomal lumen. Phagosomal alkalinization by NOX2 is controlled by Rab27a, and is required for efficient cross presentation in DCs. We have also recently shown that the same mechanism of phagosome alkalinization through NOX2 activity applies for human DCs. CD8+ DCs cross present antigens more efficiently than other DCs subsets. Because the intracellular mechanisms responsible for this specialization are unknown, we have started to explore if the antigen processing through the control of the phagosomal pH is differently regulated in the “in vivo” subpopulations of DCs. We are also analyzing the mechanism of ER recruitment to phagosomes and of antigen export from phagosomes to the cytosol. Using extensive proteomics approaches and genetics screen, we will attempt unravel the molecular mechanism of cross presentation in dendritic cells.

Dynamic imaging of anti tumor immune responses

The initiation of cytotoxic immune responses requires the direct interaction between naïve CD8+ T lymphocytes and dendritic cells. Multi-photon imaging (Fig. 1) in intact lymph nodes showed that during priming, naïve T cells and DCs establish sequentially brief (i.e. minutes) and long (hours) antigen-specific contacts.

Fig. 1 

We showed that CD8+ T cell responses help the priming of naïve CD8+ T cells through the CCR5-dependent recruitment of polyclonal CD8+ T cells to the dendritic cells that present antigen to cognate CD8+ T cells. In addition, we have demonstrated that the adhesion molecule ICAM-1 is crucial regulator of T-dendritic cells contact duration. Finally, we investigated how regulatory T cells (Tregs) influence CD8+ T cell motility during the initiation of anti-tumor immune responses. We conclude that during anti-tumor immune responses, Tregs prevent the antigen-dependent arrests of CD8+ T cells and effective anti-tumor priming through both dendritic cell-dependent and independent mechanisms.
Although the immune system evolved to fight infections, it may also attack and destroy solid tumors. In most cases, tumor rejection is initiated by CD8+ cytotoxic T lymphocytes (CTLs), which infiltrate solid tumors, recognize tumor antigens and kill tumor cells. However regulatory T cells amplification mediated by tumor growth leads to tumor escape by their powerful ability to inhibit effector T cells. We use a combination of two-photon intravital microscopy and immunofluorescence to analyze the infiltration and destruction of solid tumors by CTLs and the role of regulatory T cells on the effector T cell dynamics. We showed that antigen expression by tumor cells determines both CTL motility within the tumor and profound tumor infiltration. In an other model, our results showed that the functional depletion of regulatory T cells from mice bearing solid MCA101 tumors results in the rejection of the established tumors by antigen-specific CTLs. We also conclude that during anti-tumor immune responses, Tregs prevent the antigen-dependent arrests of CD8+ T cells and effective anti-tumor priming through killing of DCs in tumor draining lymph nodes. Overall these results suggest that efficient T cells effector functions are closely related to the dynamic of their interaction with their environment.

Last update: December 2008

Key publications

2008

• Scholer A, Hugues S, Boissonnas A, Fetler L, Amigorena S.
  Intercellular adhesion molecule-1-dependent stable interactions between T cells and dendritic cells determine CD8+ T cell memory
  Immunity., Feb;28(2):258-70 - Abstract
  The initiation of cytotoxic immune responses requires the direct interaction between naive CD8+ T lymphocytes and dendritic cells (DCs). Multiphoton imaging in intact lymph nodes (LNs) showed that during priming, naive T cells and DCs establish sequentially brief (i.e., minutes) and long (hours) antigen-specific contacts. We show here that the expression of the Intercellular Adhesion Molecule-1 (ICAM-1) by mature DCs is critical for long-lasting contacts with CD8+ T cells but dispensable for short-lived antigen-specific interactions. Serial brief DC-T cell contacts induced early CD8+ T cell activation, proliferation, and differentiation into effector cytotoxic T lymphocytes in the first few days after immunization. ICAM-1-deficient mature DCs, however, failed to induce fully effective priming, because CD8+ T cells produced reduced amounts of interferon gamma and were clonally depleted after 2 weeks. In addition, Icam1(-/-) mice failed to respond to rechallenge. We conclude that ICAM-1-dependent long-lasting interactions between mature DCs and naive CD8+ T cells determine the survival of activated CD8+ T cells and the establishment of effective memory.
• Mantegazza AR, Savina A, Vermeulen M, Perez L, Geffner J, Hermine O, Rosenzweig SD, Faure F, Amigorena S.
  NADPH oxidase controls phagosomal pH and antigen cross- presentation in human dendritic cells
  Blood, 2008 Aug 5 - Abstract
  The phagocyte NADPH oxidase (NOX2) is critical for the bactericidal activity of phagocytic cells and plays a major role in innate immunity. We showed recently that NOX2 activity in mouse dendritic cells (DC) prevents acidification of phagosomes, promoting antigen cross-presentation. In order to investigate the role of NOX2 in the regulation of the phagosomal pH in human DC, we analyzed the production of reactive oxygen species (ROS) and the phagosomal/endosomal pH in monocyte-derived DC and macrophages (M) from healthy donors or chronic granulomatous disease (CGD) patients. As expected, we found that human M acidify their phagosomes more efficiently than human DC. Accordingly, the expression of the vacuolar proton ATPase (V-H+-ATPase) was higher in M than in DC. Phagosomal ROS production, however, was higher in M than in DC, due to higher levels of gp91phox expression and recruitment to phagosomes. In contrast, in the absence of active NOX2, the phagosomal and endosomal pH decreased. Both in the presence of a NOX2 inhibitor and in CGD patients-derived DC, the cross-presentation of two model tumor antigens was impaired. We conclude that NOX2 activity participates in the regulation of the phagosomal and endosomal pH in human DC, and is required for efficient antigen cross-presentation.

2007

• Hugues S, Scholer A, Boissonnas A, Nussbaum A, Combadière C, Amigorena S, Fetler L.
  Dynamic imaging of chemokine-dependent CD8+ T cell help for CD8+ T cell responses
  Nat Immunol., Sep;8(9):921-30. Epub 2007 Jul 29 - Abstract
  Naive T lymphocytes move efficiently in lymphoid tissues while scanning dendritic cells in search of cognate complexes of peptide in major histocompatibility molecules. However, T cell migration ceases after recognition of cognate antigen. We show here that during the initiation of antigen-specific CD8(+) T cell responses, naive CD8(+) polyclonal T cells 'preferentially' interacted in an antigen-independent way with mature dendritic cells competent to present antigen to antigen-specific CD8(+) T cells. These antigen-independent interactions required expression of the chemokine receptor CCR5 on polyclonal T cells and increased the efficiency of the induction of naive, low-precursor-frequency CD8(+) T cell responses. Thus, antigen-specific CD8(+) T cells favor the priming of naive CD8(+) T cells by promoting the CCR5-dependent recruitment of polyclonal CD8(+) T cells to mature dendritic cells.
• Jancic C, Savina A, Wasmeier C, Tolmachova T, El-Benna J, Dang PM, Pascalo S, Gougerot-Pocidalo MA, Raposo G, Seabra MC, Amigorena S
  Rab27 a regulates phagosomal pH and NADPH oxidase recruitment to dendritic cell phagosomes
  Nat Cell Biol, Apr 9 (4) : 367-78 - Abstract
  To prevent excessive degradation of internalized antigens, which could destroy the peptides recognized by T lymphocytes, dendritic cells have developed several strategies that limit proteolytic activity in phagosomes. The recruitment of the NADPH oxidase NOX2 prevents acidification of phagosomes, limiting antigen degradation. Here, we show that dendritic cells derived from Rab27a-deficient ashen mice show increased phagosome acidification and antigen degradation, causing a defect in antigen cross-presentation. Enhanced acidification results from a delay in the recruitment to phagosomes of a subset of lysosome-related organelles containing the membrane subunits of NOX2. The Rab27a-dependent recruitment of these "inhibitory lysosome-related organelles" to phagosomes continuously limits acidification and degradation of ingested particles in dendritic cells, thus promoting antigen cross-presentation.

2006

• Savina A, Jancic C, Hugues S, Guermonprez P, Vargas P, Moura IC, Lennon-Dumenil AM, Seabra MC, Raposo G, Amigorena S
  NOX2 controls phagosomal pH to regulate antigen processing during crosspresentation by dendritic cells
  Cell, Jul 14, 126 (1) : 205-18