Cytoskeleton remodeling by storage endosomes – molecular mechanisms and impact on the immune response – CytoEndoStor

We will achieve the objectives of the CytoEndoStor project using in vitro several cell biology and immunology methods, complemented by in vivo studies of the immune response of animals deficient for aminopeptidase IRAP.
The cell biology methods that are implemented in our teams are: state-of-the art cell imaging, cell fractionation followed by biochemical analyzes such as immunoblot, isolation of protein complexes and their identification by mass spectrometry, inactivation of proteins in DC and T cell lines by lentiviral shRNA or more recently by CRISP9 / Cas9 genome editing technology. These cell biology experiments are complemented by in vitro studies of antigen presentation, dendritic cell activation and T cell activation.
The impact of molecular mechanisms discovered by these in vitro approaches will be evaluated on the physiology of immune response in vivo. To this end, our teams obtained by collaboration animal models inactivated for aminopeptidase IRAP (constitutively and conditionally knock-out).

Objective 1: Molecular interactions that control SRSE trafficking.
IRAP deletion in dendritic cells accelerates endosome maturation and increases endosomal TLRs processing and activation. We hypothesized that IRAP interactions with the FHOD formins or vimentin are important for SRSE anchoring to actin cytoskeleton. To test this hypothesis we depleted the formins FHOD and the vimentin in dendritic cells. The depletion of vimentin did not affected SRSE trafficking and endosomal TLRs activation. We found that monocyte-derived DCs (equivalent of bone marrow-derived DCs), express mainly the formin FHOD4. Similar to IRAP deletion, FHOD4 depletion in these cells by lentiviral shRNA destabilized the SRSE compartment, accelerated the trafficking of TLR9 and its ligands to lysosomes and strongly increased TLR9-driven inflammatory response. We have recently published these results in Nat. Immunology 2017.
Objective 2. Characterize SRSE in the cell biology of T cells and their impact on endogenous T cells priming
Our preliminary data suggested a T cell-intrinsic role for IRAP in T cell priming. Co-immunoprecipitations and mass-spectrometry identification of IRAP protein interaction partners showed that IRAP interacts with the TCR (T Cell Receptor). Confocal microscopy experiments demonstrated that the TCR is a cargo of IRAP+/Rab4+ endosomes. In the absence of IRAP, the TCR recycling is compromised and the TCR signaling is decreased, as demonstrated by lack of phosphorylation of CD3, Lck, PLC and ZAP.

In the context of our second objective, we will test the hypothesis that IRAP/Rab4+ endosomes are required for optimal signaling of the TCR using a CD3 reporter molecule in which CD3 is fused to GFP and ZAP70 to cherry. This construct allows to identify the intracellular vesicles for TCR signaling and to quantify by FRET the intensity of TCR signaling (PNAS 2010 by RD Vale’s group). In addition, we will investigate in vivo the importance of IRAP in T cells by immunization of mice that are deficient for IRAP exclusively in T cells. We have obtained IRAP-lox-lox mice from S. Chai (Monash University) and we have crossed them with Lck-Cre mice (obtained from S Lotersztajn, U1149, Bichat). The strain of T-cell specific IRAP deleted mice has been established last month in the laboratory of the P1 and will be used by the P2. A second model for investigation of IRAP function in T cells in vivo has been obtained in the laboratory of P2 (OT1-Rag2ko-IRAPko mice) and will be analyzed by the team of the P2.
In the context of the third objective of our project, we will investigate the function of SRSE in DCs and T cells interactions and migration. We already set-up the experimental methods to analyze IRAP deficient DCs and T cell migration in collagen gels. The role of the protein-interaction partners of IRAP (such as FHODs) will be investigated in cell-lines (DC-like DC2.4 and Jurkat T cells) in which FHOD will be deleted by CrispR/Cas9 method, which has been recently set-up in our teams.

1. Babdor J, Descamps D, Adiko AC, Tohmé M, Maschalidi S, Evnouchidou I, Vasconcellos LR, De Luca M, Mauvais FX, Garfa-Traore M, Brinkmann MM, Chignard M, Manoury B, Saveanu L. IRAP(+) endosomes restrict TLR9 activation and signaling. Nat Immunol. 2017 May;18(5):509-518
2. Nadia Elkhatib, Enzo Bresteau, Francesco Baschieri, Alba L?pez Rioja, Guillaume van Niel, Stéphane Vassilopoulos and Guillaume Montagnac. Tubular clathrin/AP-2 lattices pinch collagen fibers to support 3D cell migration. Science. In press
3. Trinquand A, Dos Santos NR, Tran Quang C, Rocchetti F, Zaniboni B, Belhocine M, da Costa de Jesus C, Lhermitte L, Tesio M, Dussiot M, Cosset FL, Verhoeyen E, Pflumio F, Ifrah N, Dombret H, Spicuglia S, Chatenoud L, Gross DA, Hermine O, Macintyre E, Ghysdael J, Asnafi V. Triggering the TCR developmental checkpoint activates a therapeutically targetable tumor suppressive pathway in T-cell leukemia. Cancer Discov. 2016 Sep;6(9):972-85.
4. Goudin N, Chappert P, Mégret J, Gross DA, Rocha B and Azogui O. Depletion of regulatory T cells induces high numbers of dendritic cells and unmasks a subset of anti-tumor CD8+CD11c+ PD-1lo effector T cells. PLoS One. 2016 Jun 24;11(6):e0157822.

Naive T cells activation by dendritic cells (DCs) is a highly dynamic process in which the cellular cytoskeleton in both cells undergoes coordinated and active rearrangements that are crucial for the immune response. The molecular mechanisms allowing these coordinated rearrangements are still poorly understood. Based on our preliminary data, we propose that a novel class of intracellular vesicles, the slow recycling storage endosomes (SRSE), are involved in the cytoskeleton remodeling required for optimal DC and T cell function.
A highly specific marker of SRSE is the aminopeptidase IRAP (Insulin Regulated AminoPeptidase), a protein with dual function. While the enzymatic activity is required for antigen trimming during MHC class I cross-presentation (Saveanu et al. Science 2009), we have recently found that, independently of its aminopeptidase activity, IRAP regulates endosomal trafficking and specifically SRSE formation and stability. SRSE destabilization affects cell physiology in different ways, depending on the cell type. In DCs, it alters antigen cross-presentation, phagosome maturation and endosomal TLRs signaling (manuscript in preparation) while in CD8+ T cells it modulates their activation and amplification (preliminary results). The observed phenotypes could be the result of a complex interplay between SRSE and cytoskeleton dynamics. If cytoskeleton components are essential for the localization and movement of intracellular vesicles, we have recently reported that conversely, endocytic vesicles control some aspects of cytoskeleton dynamics (Montagnac et al., Nature, 2013). Thus, an appealing hypothesis, supported by our preliminary data, is that SRSE is able to induce local modifications of cytoskeleton.
The main goal of this project is to uncover the molecular mechanism by which SRSE affects cytoskeleton dynamics and estimate their impact in vivo on T cell and DC function. To this end, we propose to: 1. Identify the molecular interactions that control the SRSEs’ trafficking 2. Characterize SRSE in the cell biology of T cells, and their impact on endogenous T cells priming and 3. Investigate the function of SRSE in DCs and T cells interactions and migration.
The results obtained during this project should bring new fundamental knowledge about cellular mechanisms involved in the function of T cells and DCs and their regulatory pathways.