Functional properties of skin dendritic cell subsets: their role in Tolerance versus Immunity – SkinDCs

Using the genes defined by transcriptomic analysis, we will generate innovative mouse model combining the gene of interest with a fluorescent protein and a genetic strategy to rather deplete the subset of cell expressing the gene or the active site of the gene in case of enzyme.

According to our results, the first innovative knock-in mice we will do is targetting the ALDH gene which plays a major role in retinoic acid production and T reg induction.

This model should allow us to figure out the role of DC in auto immune syndromes

Langlet C, Tamoutounour S, Henri S, Luche H, Ardouin L, Gregoire C, Malissen B, Guilliams M. CD64 expression distinguishes monocyte-derived and conventional dendritic cells and reveals their distinct role during intramuscular immunization. J Immunol 2012. 188:1751-60.
Crozat K, Tamoutounour S, Manh TP, Fossum E, Luche H, Ardouin L, Guilliams M, Azukizawa H, Bogen B, Malissen B, Henri S, Dalod M. Cutting edge: Expression of XCR1 defines mouse lymphoid-tissue resident and migratory dendritic cells of the CD8alpha+ type. J Immunol 2011. 187:4411-5.
Luche H, Ardouin L, Teo P, See P, Henri S, Merad M, Ginhoux F, Malissen B. The earliest intrathymic precursors of CD8alpha(+) thymic dendritic cells correspond to myeloid-type double-negative 1c cells. Eur J Immunol 2011. 41:2165-75

The skin is a complex tissue, capable of controlling infections through efficient immune responses while maintaining its own integrity. Dendritic cells (DCs) constitute sentinels of the immune system that contribute to elicit and control skin immune responses. Using innovative mouse models (LangEGFP and LangEGFPDTR), we studied the Langerhans cells (LCs) located in the epidermis, we discovered a new DC subset in the dermis (Poulin, 2007) and described five different DC subsets in the skin (LCs, CD207+ CD103-, CD207+ CD103+, CD207- CD11b-, and CD207- CD11b+ dermal DCs (DDCs)). We hypothesized that this phenotypic heterogeneity is associated with functional differences. Accordingly, the main objective of the present project is to understand the biological role of these different skin DC subsets by focussing mainly on tolerance mechanisms such as clonal deletion of self-specific T cells and induction of regulatory T cells (Treg). Four lines of research will be developed :
1. We will take advantage of our ability to discriminate homogeneous skin DC subsets to develop a comparative transcriptomic analysis of such five skin DC subsets. Such approach should provide us important hints on the functional specialization of those various skin DC subsets.
2. Using a transgenic mice where a model antigen (OVA) is expressed in keratinocytes (K5-mOVA), we have showed that the CD207+ CD103+ DDC subset was the only one capable of cross-presenting such self-antigen to OVA-specific OT-I CD8+ T cells (Henri, 2009). One aim of the present proposal is thus to elucidate the mechanism underlying the capacity of the CD207+ CD103+ DDCs to cross-present antigens. More specifically, we will determine whether this cross-presenting capacity is due to their enhanced capacity to encounter OT-I CD8+ T cells via XCR1 chemokine receptor expression. Moreover, the K5-mOVA model should allow us to further understand the mechanism of peripheral tolerance that is based on clonal deletion.
3. Treg cells could be induced by DCs producing retinoic acid (RA) through the oxidation of retinaldehyde by retinaldehyde dehydrogenase (ALDH). We have recently showed that in the skin, only the CD207- CD11b+ DDC was expressing the ALDH at the protein level and was able to induce Treg cells in vitro (Guilliams, in press). We hypothesized that this important property also extends in vivo. To determine if the high frequency of Treg cells found in the skin is the consequence of the inductive function of the RA-producing CD207- CD11b+ skin DDC subset, we will develop an innovative knock-in mouse model allowing to track and ablate RA production by CD207- CD11b+ DCs in vivo.
4. Finally, we will attempt to visualize the interactions of DCs with T cells using confocal and intravital microscopy. To track DDCs both in the dermis and in the CLNs, we intend to use mice that co-express the Cre recombinase under the control of either the CD11c or the Langerin gene and a LoxP-STOP-LoxP-RFP red reporter cassette within the Rosa locus. Adoptive transfer of green (GFP+) OT-I T cells into Langerin-Cre x LoxP-STOP-LoxP-RFP x K5-mOVA recipient will give us the unique possibility to determine by confocal and 2 photon microscopy whether the GFP+ OT-I T cells colocalize with the cross-presenting RFPbright CD207+ DDCs that have captured OVA in the skin. If the expression of XCR1 chemokine receptor is of importance in such cross-talk, developing a K5-mOVA x Lang-Cre x LoxP-STOP-LoxP-RFP x B6.129P2-Xcr1<tm1Dgen>/J mouse line will allow us to visualize whether XCR1-deficient CD207+ DDCs have lost their ability to contact CD8+ T cells. In conclusion, this research proposal aims to unravel if the phenotypic diversity of the skin DCs is associated with specialized functions in vivo. A better understanding of the mechanisms of tolerance contributing to maintain skin homeostasis will allow the selection of the most optimal DC target for therapy intended to intensify or dampen skin immune responses.