Dendritic Cell Biology
Dendritic cells (DCs) have the unique capacity to initiate primary immune responses by stimulating naive T cells. Our group is specialized in the cell biology of this important cell type and is expert at growing both mouse and human DC in vitro.
We are interested in the fundamental changes occurring during the activation of DCs by pathogens (maturation) and how these biochemical events might contribute to chronic inflammation or autoimmunity, understanding DC maturation will provide new insights to how the immune response can be modulated.
Our aim is to elucidate the molecular mechanisms by which DCs control antigen processing and cytokine production, particularly during pathogen detection.
We have been focusing on protein translation and quality control and their relationship to autophagy and MHC restricted antigen presentation. We are also searching for molecules regulating membrane traffic in conventional DCs and Interferon producing cells (plasmacytoid DCs), thus likely to also control the exogenous antigen processing and presentation capability of these cells.
The laboratory is specialized in interdisciplinary approaches using cell biological techniques and bio-imaging tools to try to meet the challenge of understanding the massive molecular changes occurring during DC activation.
Control of antigen processing and presentation by dendritic cells
In order to trigger an immune response, protein antigens are converted to short peptides, loaded on MHC molecules and presented at the surface of the APCs. MHC class I molecules interact mostly with cytosolic self or viral peptides, while MHC class II are most often associated with foreign peptides generated in the endocytic pathway.
In APCs, newly synthesized misfolded proteins (defective ribosomal products, or DRiPs) provide the bulk of MHC class I restricted peptides. DRiPs, which can represent up to 30% of synthesized proteins, are ubiquitinated and degraded rapidly by the proteasome. A faithful representation of cellular protein synthesis is therefore displayed by surface MHC class I molecules independently of the relative half-life of each individual protein produced by APCs. Our laboratory has found that maturing DCs accumulate ubiquitinated proteins in large cytosolic aggregates. These DC specific Aggresome Like Induced Structures (DALIS) are transient, require continuous protein synthesis and are totally dependent on microbe detection.
The majority of the proteins forming DALIS are newly synthesized DRiPs, which are poorly degraded. DRiPs storage into DALIS is strongly correlated with the inhibition of the autophagic flux. DRiPs are therefore also autophagic substrates and can have access to the endosomal MHC II pathway in non activated DCs. Both MHC class I and class II endogenous antigen presentations are connected to DALIS formation and are regulated during DC maturation.
Our laboratory is therefore interested in studying all aspect of this regulation including: antigen translation and degradation, autophagy regulation and MHC I and II intracellular transport.
We have demonstrated (Head of Project Dr. Evelina Gatti )that ubiquitination of the cytoplasmic tail of HLA-DR ß-chain is key for the accumulation and degradation of peptide-loaded MHC II in the lysosomes of immature in human monocyte-derived DCs and this ubiquitination ceases upon DC maturation.
We have further demonstrated that MARCH I, a membrane associated RING-CH ubiquitin E3 ligase, promotes the ubiquitination of peptide-loaded HLA-DR molecules, inducing their internalization from the cell surface and impairing antigen presentation in the absence of an appropriate stimuli.
Upon Toll-like receptor (TLR) engagement, MARCH I expression is lost, allowing HLA-DR molecules stabilization at plasma membrane of DCs and productive interaction with T cell receptors. MARCH I is therefore a key player in the acquisition of the potent immuno-stimulatory properties of activated human DCs. Interestingly Interleukin-10 (IL-10), a potent immunosuppressive cytokine known to mediate MHC II intracellular sequestration and degradation, was further shown to control MARCH I transcription, inducing its up-regulation and subsequent ubiquitination of peptide-loaded HLA-DR complexes in monocytes.
Thus, both in immunogenic or tolerogenic contexts, regulation of antigen presentation is a MARCH I-dependent phenomenon. Ubiquitination is a pivotal mechanism of regulation for many fundamental cellular processes, which has been only recently implicated in the control of the immune response and we are currently investigating the details of this regulation .
Control of translation, stress and immunity
We have launched an experimental strategy mostly based on gene microarrays analysis to characterize the relevance of protein synthesis regulation and microRNAs during DC maturation.
Our work performed on Human monocyte derived-DCs in collaboration with several international teams (M. Santos, Aveiro, Portugal, W. Reth, Geneva, CH, and D. Chaussabel, Dallas, USA) has led to the identification of the TAB2 molecule, an adaptor in the TLR/IL-1 signaling cascade, as a direct target of miR-155, one the microRNAs most prominently induced during DC activation. miR-155 plays an essential role in controlling the intensity of the inflammatory response to microbes in human DCs (Ceppi et al., PNAS, 2009).
In addition, a systematic analysis of the mRNAs being translationally regulated at various stages of DC activation was performed using translational profiling, which combines sucrose gradient fractionation of polysomal-bound mRNAs with microarray analysis. We have demonstrated that dsRNA detection by DCs trigger a specific integrated stress response (ISR). This ISR in activated DCs limit minimize the impact of stressful events on DCs and allow them to function in conditions in which other cells will be anergized (e.g. aa depletion).
Upon ds-RNA exposure, DCs and also mouse embryonic fibroblasts (MEF) produce large amounts of cytokines as part as an antiviral response. We have also shown that the ISR in both cell types is absolutely required for the production of several cytokines and resistance to Chikunguya virus infection.
All these findings have revealed many novel aspects of DC function, which can all be linked through the interaction of the ubiquitin, stress and pathogen detection biochemical pathways.