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Bone regeneration relies on the activation of skeletal stem cells (SSCs) that still remain poorly characterized. Here, we show that periosteum contains SSCs with high bone regenerative potential compared to bone marrow stromal cells/skeletal stem cells (BMSCs) in mice. Although periosteal cells (PCs) and BMSCs are derived from a common embryonic mesenchymal lineage, postnatally PCs exhibit greater clonogenicity, growth and differentiation capacity than BMSCs. During bone repair, PCs can efficiently contribute to cartilage and bone, and integrate long-term after transplantation. Molecular profiling uncovers genes encoding Periostin and other extracellular matrix molecules associated with the enhanced response to injury of PCs. Periostin gene deletion impairs PC functions and fracture consolidation. Periostin-deficient periosteum cannot reconstitute a pool of PCs after injury demonstrating the presence of SSCs within periosteum and the requirement of Periostin in maintaining this pool. Overall our results highlight the importance of analyzing periosteum and PCs to understand bone phenotypes.

T cell–dependent immune responses develop soon after birth, whereas it takes 2 yr for humans to develop T cell–independent responses. We used this dissociation to analyze the repertoire diversification of IgM+IgD+CD27+ B cells (also known as “IgM memory” B cells), comparing these cells with switched B cells in children <2 yr of age, with the aim of determining whether these two subsets are developmentally related. We show that the repertoire of IgM+IgD+CD27+ B cells in the spleen and blood displays no sign of antigen-driven activation and expansion on H-CDR3 spectratyping, despite the many antigenic challenges provided by childhood vaccinations. This repertoire differed markedly from those of switched B cells and splenic germinal center B cells, even at the early stage of differentiation associated with μ heavy chain expression. These data provide evidence for the developmental diversification of IgM+IgD+CD27+ B cells, at least in very young children, outside of T cell–dependent and –independent immune responses.

Abstract.Maturation of functional neuronal circuits during central nervous system development relies on sophisticated mechanisms. First, axonal and dendritic growth should reach appropriate targets for correct synapse elaboration. Second, pruning and neuronal death are required to eliminate redundant or inappropriate neuronal connections. Serotonin, in addition to its role as a neurotransmitter, actively participates in postnatal establishment and refinement of brain wiring in mammals. Brain resident macrophages, i.e. microglia, also play an important role in developmentally-regulated neuronal death as well as in synaptic maturation and elimination. Here, we tested the hypothesis of cross-regulation between microglia and serotonin during postnatal brain development in a mouse model of synaptic refinement. We found expression of the serotonin 5-HT 2B receptor on postnatal microglia, suggesting that serotonin could participate in temporal and spatial synchronization of microglial functions. Using two-photon microscopy, acute brain slices and local delivery of serotonin, we observed that microglial processes moved rapidly toward the source of serotonin in Htr 2B +/+ mice, but not in Htr 2B-/-mice lacking the 5-HT 2B receptor. We then investigated whether some developmental steps known to be controlled by serotonin, could potentially result from microglia sensitivity to serotonin. Using an in vivo model of synaptic refinement during early brain development, we investigated the maturation of the retinal projections to the thalamus and observed that Htr 2B -/-mice present anatomical alterations of the ipsilateral projecting area of retinal axons into the thalamus. In addition, activation markers were upregulated in microglia from Htr 2B -/-compared to control neonates, in the absence of apparent morphological modifications. These results support the hypothesis that serotonin interacts with microglial cells and these interactions participate in brain maturation.

Regulatory T cells (Treg) are commonly identified by CD25 (IL-2Ra) surface expression and/or intracellular expression of the FOXP3 transcription factor. In addition, Treg are also characterized by low CD127 (IL-7Ra) expression when compared to conventional T cells and their biology in the periphery is considered essentially independent of IL-7. We further investigated CD127 expression on Treg and we demonstrated differential CD127 expression depending on Treg subsets considered. Notably, we observed high CD127 expression on inducible costimulatory molecule (ICOS)-and CD103-expressing Treg subsets. Since these two markers reflect activation status, we addressed whether Treg activation modulated CD127 expression. We demonstrated that in contrast to conventional T cells, Treg significantly upregulated CD127 expression during in vitro and in vivo activation using adoptive transfer and contact dermatitis models. High CD127 expression on Treg was also predominantly detected ex vivo in some specific sites, notably bone marrow and skin. Importantly, higher CD127 expression on Treg correlated with higher phosphorylation of STAT5 upon IL-7 exposure. High CD127 expression on Treg also provided survival advantage upon in vitro incubation with IL-7. We thus demonstrated that low CD127 expression is not an intrinsic characteristic of Treg and we identified activated Treg as a potential target of endogenous or therapeutic IL-7.

Thymic export of cells is believed to be restricted to mature T cells. Here we show that the thymus also exports fully committed T cell precursors that colonize primary lymphoid organs. These precursor cells exited the thymus before T cell receptor rearrangements and colonized lymphoid organs such as the thymus and the gut. Migration of the thymic T cell-committed precursors led to permanent colonization of the gut precursor compartment, improved the capacity of gut precursors to further differentiate into T cells and was sufficient for the generation of 'euthymic like' CD8alphaalpha(+) intraepithelial lymphocytes. These data demonstrate a new function for the thymus in peripheral seeding with T cell precursors that become long lived after thymus export.

We characterized CD8+ T cells constitutively expressing CD25 in mice lacking the expression of MHC class II molecules. We showed that these cells are present not only in the periphery but also in the thymus. Like CD4+CD25+ T cells, CD8+CD25+ T cells appear late in the periphery during ontogeny. Peripheral CD8+CD25+ T cells from MHC class II-deficient mice also share phenotypic and functional features with regulatory CD4+CD25+ T cells: in particular, they strongly express glucocorticoid-induced TNFR family-related gene, CTLA-4 and Foxp3, produce IL-10, and inhibit CD25− T cell responses to anti-CD3 stimulation through cell contacts with similar efficiency to CD4+CD25+ T cells. However, unlike CD4+CD25+ T cells CD8+CD25+ T cells from MHC class II-deficient mice strongly proliferate and produce IFN-γ in vitro in response to stimulation in the absence of exogenous IL-2.

Myotonic dystrophy type 1 is a neuromuscular affection associated with the expansion of an unstable CTG repeat in the DM protein kinase gene. The disease is characterized by somatic tissue-specific mosaicism and very high intergenerational instability with a strong bias towards expansions. We used transgenic mice carrying more than 300 unstable CTG repeats within their large human genomic environment to investigate the dynamics of CTG repeat germinal mosaicism in males. Germinal mosaicism towards expansions was already present in spermatozoa at 7 weeks of age and continued to increase with age, suggesting that expansions are continuously produced throughout life. To determine the precise stage at which germinal expansions occur during spermatogenesis, we sorted and collected the different germ cell types produced during spermatogenesis from males of different ages and analyzed the CTG repeat mosaicism in each fraction. Strong mosaicisms towards expansions were already observed in spermatogonia before meiosis. In transgenic Msh2-deficient mice, germinal instability of the CTG repeats (only contractions) also occurs premeiotically. No significant difference in mosaicism was detected between spermatogonia and spermatozoa, arguing against continued expansions during postmeiotic stages. This indicates that germinal expansions are produced at the beginning of spermatogenesis, in spermatogonia, by a meiosis-independent mechanism involving MSH2.Myotonic dystrophy type 1 (DM1) is associated with the expansion of a CTG trinucleotide repeat located in the 3Ј untranslated region of the DM protein kinase (DMPK) gene at 19q13. 3 (3, 7, 17, 27). In normal subjects, there are usually between 5 and 37 copies of this repeat, which remains stable following intergenerational transmissions. In DM1 patients, more than 50 CTG repeats are typically present, and the repeat is highly unstable and increases with each generation. The number of CTG repeats is positively correlated with the severity of symptoms and is negatively correlated with age at onset, resulting in an anticipation phenomenon that is particularly obvious in DM1 (19). The behavior of the CTG repeat between generations appears to depend on the sex of the transmitting parent. Paternal transmissions lead to larger expansions for Ͻ100 CTG repeats, whereas maternal transmissions lead to larger expansions when the CTG repeat tract contains Ͼ500 repeats in the transmitting parent (2, 24). In between, both paternal and maternal alleles expand. In addition to intergenerational instability, somatic CTG repeat-length mosaicism is also found in DM1 patients. Inter-and intratissue mosaicisms increasing with age are observed, with a strong bias towards expansions (1,22). DM1 is one of the growing group of diseases caused by dynamic mutations. This group currently comprises more than a dozen diseases, including Huntington's disease (HD), spinocerebellar ataxias, and fragile X syndrome, which are generally associated with CNG trinucleotide repeats (11, 46). The dynamics of the different...