A Coronin 1-Dependent Decision Switch in Juvenile Mice Determines the Population of the Peripheral Naive T Cell Compartment.

Following thymic maturation, T cells egress as recent thymic emigrants to peripheral lymphoid organs where they undergo an additional maturation step to mature naive T cells that circulate through secondary lymphoid organs ready to be activated upon pathogenic challenges. Thymic maturation and peripheral T cell survival depend on several signaling cascades, but whether a dedicated mechanism exists that exclusively regulates homeostasis of mature naive T cells without affecting thymocytes and/or recent thymic emigrants remains unknown. In this article, we provide evidence for a specific and exclusive role of the WD repeat containing protein coronin 1 in the maintenance of naive T cells in peripheral lymphoid organs. We show that coronin 1 is dispensable for thymocyte survival and development, egress from the thymus, and survival of recent thymic emigrants. Importantly, coronin 1-deficient mice possessed comparable levels of peripheral T cells within the first 2 wk after birth but failed to populate the peripheral T cell compartment at later stages. Furthermore, dendritic cell- and IL-2/7-dependent T cell survival was found to be independent of coronin 1. Together, these results suggest the existence of a hitherto unrecognized coronin 1-dependent decision switch early during life that is responsible for peripheral naive T cell survival and homeostasis.

An evolutionarily conserved coronin-dependent pathway defines cell population size.

Maintenance of cell population size is fundamental to the proper functioning of multicellular organisms. Here, we describe a cell-intrinsic cell density-sensing pathway that enabled T cells to reach and maintain an appropriate population size. This pathway operated "kin-to-kin" or between identical or similar T cell populations occupying a niche within a tissue or organ, such as the lymph nodes, spleen, and blood. We showed that this pathway depended on the cell density-dependent abundance of the evolutionarily conserved protein coronin 1, which coordinated prosurvival signaling with the inhibition of cell death until the cell population reached threshold densities. At or above threshold densities, coronin 1 expression peaked and remained stable, thereby resulting in the initiation of apoptosis through kin-to-kin intercellular signaling to return the cell population to the appropriate cell density. This cell population size-controlling pathway was conserved from amoeba to humans, thus providing evidence for the existence of a coronin-regulated, evolutionarily conserved mechanism by which cells are informed of and coordinate their relative population size.

Suppression of caspase 8 activity by a coronin 1-PI3Kδ pathway promotes T cell survival independently of TCR and IL-7 signaling.

The control of T cell survival is crucial for defense against infectious pathogens or emerging cancers. Although the survival of peripheral naïve T cells has been proposed to be controlled by interleukin-7 (IL-7) signaling and T cell receptor (TCR) activation by peptide-loaded major histocompatibility complexes (pMHC), the essential roles for these pathways in thymic output and T cell proliferation have complicated the analysis of their contributions to T cell survival. Here, we showed that the WD repeat­containing protein coronin 1, which is dispensable for thymic selection and output, promoted naïve T cell survival in the periphery in a manner that was independent of TCR and IL-7 signaling. Coronin 1 was required for the maintenance of the basal activity of phosphoinositide 3-kinase δ (PI3Kδ), thereby suppressing caspase 8­mediated apoptosis. These results therefore reveal a coronin 1­dependent PI3Kδ pathway that is independent of pMHC:TCR and IL-7 signaling and essential for peripheral T cell survival.

Skin Transplantation and Lymphoid Organ Analysis in Mice.

Skin transplantation in mice is an important procedure to evaluate immune responses generated against heterologous grafts, especially given its highly immunogenic nature. In fact, skin is one of the most challenging organs in terms of allograft retention. In this protocol, we provide a detailed procedure for skin grafting using the tail skin as donor organ that is grafted on the dorsal site of thoracic cage in a recipient mouse. We also provide protocols for the systematic analysis of lymphoid organ analysis in transplanted mice. Together these protocols may be valuable for evaluation of parameters that affect skin grafting, including genetic factors, immune cell activation as well as the analysis of compounds that may be useful in allowing graft tolerance.

Assigning mitochondrial localization of dual localized proteins using a yeast Bi-Genomic Mitochondrial-Split-GFP.

A single nuclear gene can be translated into a dual localized protein that distributes between the cytosol and mitochondria. Accumulating evidences show that mitoproteomes contain lots of these dual localized proteins termed echoforms. Unraveling the existence of mitochondrial echoforms using current GFP (Green Fluorescent Protein) fusion microscopy approaches is extremely difficult because the GFP signal of the cytosolic echoform will almost inevitably mask that of the mitochondrial echoform. We therefore engineered a yeast strain expressing a new type of Split-GFP that we termed Bi-Genomic Mitochondrial-Split-GFP (BiG Mito-Split-GFP). Because one moiety of the GFP is translated from the mitochondrial machinery while the other is fused to the nuclear-encoded protein of interest translated in the cytosol, the self-reassembly of this Bi-Genomic-encoded Split-GFP is confined to mitochondria. We could authenticate the mitochondrial importability of any protein or echoform from yeast, but also from other organisms such as the human Argonaute 2 mitochondrial echoform.

Trib1 Is Overexpressed in Systemic Lupus Erythematosus, While It Regulates Immunoglobulin Production in Murine B Cells.

Systemic lupus erythematosus (SLE) is a severe and heterogeneous autoimmune disease with a complex genetic etiology, characterized by the production of various pathogenic autoantibodies, which participate in end-organ damages. The majority of human SLE occurs in adults as a polygenic disease, and clinical flares interspersed with silent phases of various lengths characterize the usual evolution of the disease in time. Trying to understand the mechanism of the different phenotypic traits of the disease, and considering the central role of B cells in SLE, we previously performed a detailed wide analysis of gene expression variation in B cells from quiescent SLE patients. This analysis pointed out an overexpression of TRIB1. TRIB1 is a pseudokinase that has been implicated in the development of leukemia and also metabolic disorders. It is hypothesized that Trib1 plays an adapter or scaffold function in signaling pathways, notably in MAPK pathways. Therefore, we planned to understand the functional significance of TRIB1 overexpression in B cells in SLE. We produced a new knock-in model with B-cell-specific overexpression of Trib1. We showed that overexpression of Trib1 specifically in B cells does not impact B cell development nor induce any development of SLE symptoms in the mice. By contrast, Trib1 has a negative regulatory function on the production of immunoglobulins, notably IgG1, but also on the production of autoantibodies in an induced model. We observed a decrease of Erk activation in BCR-stimulated Trib1 overexpressing B cells. Finally, we searched for Trib1 partners in B cells by proteomic analysis in order to explore the regulatory function of Trib1 in B cells. Interestingly, we find an interaction between Trib1 and CD72, a negative regulator of B cells whose deficiency in mice leads to the development of autoimmunity. In conclusion, the overexpression of Trib1 could be one of the molecular pathways implicated in the negative regulation of B cells during SLE.

Overexpression of Fkbp11, a feature of lupus B cells, leads to B cell tolerance breakdown and initiates plasma cell differentiation.

Systemic Lupus Erythematosus (SLE) is a severe systemic autoimmune disease, characterized by multi-organ damages, triggered by an autoantibody-mediated inflammation, and with a complex genetic influence. It is today accepted that adult SLE arises from the building up of many subtle gene variations, each one adding a new brick on the SLE susceptibility and contributing to a phenotypic trait to the disease. One of the ways to find these gene variations consists in comprehensive analysis of gene expression variation in a precise cell type, which can constitute a good complementary strategy to genome wide association studies. Using this strategy, and considering the central role of B cells in SLE, we analyzed the B cell transcriptome of quiescent SLE patients, and identified an overexpression of FKBP11, coding for a cytoplasmic putative peptidyl-prolyl cis/trans isomerase and chaperone enzyme. To understand the consequences of FKBP11 overexpression on B cell function and on autoimmunity's development, we created lentiviral transgenic mice reproducing this gene expression variation. We showed that high expression of Fkbp11 reproduces by itself two phenotypic traits of SLE in mice: breakdown of B cell tolerance against DNA and initiation of plasma cell differentiation by acting upstream of Pax5 master regulator gene.

B cell signature during inactive systemic lupus is heterogeneous: toward a biological dissection of lupus.

Systemic lupus erythematosous (SLE) is an autoimmune disease with an important clinical and biological heterogeneity. B lymphocytes appear central to the development of SLE which is characterized by the production of a large variety of autoantibodies and hypergammaglobulinemia. In mice, immature B cells from spontaneous lupus prone animals are able to produce autoantibodies when transferred into immunodeficient mice, strongly suggesting the existence of intrinsic B cell defects during lupus. In order to approach these defects in humans, we compared the peripheral B cell transcriptomas of quiescent lupus patients to normal B cell transcriptomas. When the statistical analysis is performed on the entire group of patients, the differences between patients and controls appear quite weak with only 14 mRNA genes having a false discovery rate ranging between 11 and 17%, with 6 underexpressed genes (PMEPA1, TLR10, TRAF3IP2, LDOC1L, CD1C and EGR1). However, unforced hierarchical clustering of the microarrays reveals a subgroup of lupus patients distinct from both the controls and the other lupus patients. This subgroup has no detectable clinical or immunological phenotypic peculiarity compared to the other patients, but is characterized by 1/an IL-4 signature and 2/the abnormal expression of a large set of genes with an extremely low false discovery rate, mainly pointing to the biological function of the endoplasmic reticulum, and more precisely to genes implicated in the Unfolded Protein Response, suggesting that B cells entered an incomplete BLIMP1 dependent plasmacytic differentiation which was undetectable by immunophenotyping. Thus, this microarray analysis of B cells during quiescent lupus suggests that, despite a similar lupus phenotype, different biological roads can lead to human lupus.