Lymphoid EVA1 Expression Is Required for DN1-DN3 Thymocytes Transition

Background: Thymus organogenesis and T lymphocyte development are accomplished together during fetal life. Proper development and maintenance of thymus architecture depend on signals generated by a sustained crosstalk between developing thymocytes and stromal elements. Any maturation impairment occurring in either cellular component leads to an aberrant thymic development. Gene expression occurring during T lymphocyte differentiation must be coordinated in a spatio-temporal fashion; one way in which this is achieved is through the regulation by cell-cell adhesion and interactions. Principal Findings: We examined the role played by Epithelial V-like Antigen 1 (EVA1), an Ig adhesion molecule expressed on thymus epithelial cells (TEC) and immature thymocytes, in T cell development by employing RNA interference in vitro and in vivo models. Fetal liver derived haematopoietic progenitors depleted of Eva1, displayed a delayed DN1-DN3 transition and failed to generate CD4CD8 double positive T cells in OP9-DL1 coculture system. In addition, we could observe a coordinated Eva1 up-regulation in stromal and haematopoietic cells in coculture control experiments, suggesting a possible EVA1 involvement in TEC-haematopoietic cells crosstalk mechanisms. Similarly, Rag2-cc double knock out mice, transplanted with Eva1 depleted haematopoietic progenitors displayed a 10-fold reduction in thymus reconstitution and a time delayed thymocytes maturation compared to controls. Conclusions: Our findings show that modulation of Eva1 expression in thymocytes is crucial for lymphocyte physiological developmental progression and stromal differentiation

Epithelial V-like Antigen (EVA), a Novel Member of the Immunoglobulin Superfamily, Expressed in Embryonic Epithelia with a Potential Role as Homotypic Adhesion Molecule in Thymus Histogenesis

Thymus development depends on a complex series of interactions between thymocytes and the stromal component of the organ. To identify regulated genes during this codependent developmental relationship, we have applied an RNA fingerprinting technique to the analysis of thymus expansion and maturation induced in recombinase-deficient mice injected with anti-CD3 antibodies. This approach led us to the identification of a gene encoding a new member of the immunoglobulin superfamily, named epithelial V-like antigen (EVA), which is expressed in thymus epithelium and strongly downregulated by thymocyte developmental progression. This gene is expressed in the thymus and in several epithelial structures early in embryogenesis. EVA is highly homologous to the myelin protein zero and, in thymus-derived epithelial cell lines, is poorly soluble in nonionic detergents, strongly suggesting an association to the cytoskeleton. Its capacity to mediate cell adhesion through a homophilic interaction and its selective regulation by T cell maturation might imply the participation of EVA in the earliest phases of thymus organogenesis

<i>In vivo</i> T cell development with LSK-EVAi cells.

<p>(A, left panel) <i>Rag2/γc</i> mice were transplanted with either LSK cells infected with a non-interfering (LSK-CT) or with an Eva1-interfering lentivirus (LSK-EVAi). After 8 weeks, thymi were excised and compared: thymus from mice transplanted with LSK-EVAi were comparable to untreated ones for dimension and total cellularity (A, right panel). Untr., untreated control animal. Scale bar, 2 mm. (B) Time course of T cell development of LSK-CT (white bars) or LSK-EVAi (black bars) cells respectively, in recostitution experiments in <i>Rag2/γc</i> mice. Flow cytometric analysis of CD25/CD44 and CD4/CD8 stainings revealed that LSK-EVAi reconstituted thymus had a delay of T cell differentiation with an accumulation of DN cells at 6 weeks after trasplant (left panel). Percentage of DN cells in LSK-EVAi reconstituted thymus persists higher than in LSK-CT reconstituted thymi at 8 weeks after transplant (right panel). Mean and SD of three independent experiments are shown (** p<0.01). (C) Counts of total DN cells generated in the three different mouse groups at six and eight weeks post-treatment.</p

Analysis of <i>Eva1</i> expression.

<p><i>Eva1</i> real-time RT-PCR in fetal thymi (A), adult DN subpopulations (B, left panel) and thymocytes from mutant mice (B, right panel). mRNA from flow cytometrically purified TECs (CD45-) and intrathymic haematopoietic cells (CD45+) or DN1-3 subpopulations was reverse transcribed and used as the template for PCR with <i>Eva1</i>-specific primers. All samples were normalized to the geometric mean of the GAPDH housekeeping gene. NB, newborn; 2mth, two months; WT, wild type; ΔCAM, Tg-Calcineurin; <i>Prkdc</i>^scid, protein kinase, DNA-activated, catalytic polypeptide (C, upper panel) Eva1 interference in LSK cells by lentiviral vector was controlled by real time RT-PCR. LSK-CT cells shown a comparable expression of <i>Eva1</i> while LSK-EVAi cells shown a drastic decrease of <i>Eva1</i> expression, indicating that the interference has occurred. (C, lower panel), fluorescence microscopy analysis confirming <i>Eva1</i> interference in LSK-EVAi cells. LSK-WT, uninfected LSK; LSK-CT, LSK infected with a non-interfering lentiviral vector; LSK-EVAi, LSK infected with a <i>Eva1</i>-interfering lentiviral vector.</p

Mesoangioblast stem cells ameliorate muscle function in dystrophic dogs

Duchenne muscular dystrophy remains an untreatable genetic disease that severely limits motility and life expectancy in affected children. The only animal model specifically reproducing the alterations in the dystrophin gene and the full spectrum of human pathology is the golden retriever dog model. Affected animals present a single mutation in intron 6, resulting in complete absence of the dystrophin protein, and early and severe muscle degeneration with nearly complete loss of motility and walking ability. Death usually occurs at about 1 year of age as a result of failure of respiratory muscles. Here we report that intra-arterial delivery of wild-type canine mesoangioblasts (vessel-associated stem cells) results in an extensive recovery of dystrophin expression, normal muscle morphology and function ( confirmed by measurement of contraction force on single fibres). The outcome is a remarkable clinical amelioration and preservation of active motility. These data qualify mesoangioblasts as candidates for future stem cell therapy for Duchenne patients.status: publishe