Treatments.

<p>m: month. AZA: azathiopurin. 6MP: 6 mercaptopurin. clinical response (+ or-) / mucosal effect (+/-)</p><p>Treatments.</p

Phenotype of lamina propria intestinal lymphocytes.

<p>Phenotype of lamina propria intestinal lymphocytes.</p

Phenotype of peripheral blood lymphocytes.

<p>Phenotype of peripheral blood lymphocytes.</p

H&E staining of the duodenal biopsies of one patient treated with Olmesartan (patient 2) (A, B) and of one patient with AIE (patient 7) (C, D) showing subtotal villous atrophy (A, C: original magnification x 100) with glandular apoptosis (B, D: original magnification x200).

<p>H&E staining of the duodenal biopsies of one patient treated with Olmesartan (patient 2) (A, B) and of one patient with AIE (patient 7) (C, D) showing subtotal villous atrophy (A, C: original magnification x 100) with glandular apoptosis (B, D: original magnification x200).</p

Phenotype of intestinal intraepithelial lymphocytes.

<p>*: excess of CD4+ IEL with onset of CD4 lymphoma after two years treatment with azathioprine (Case published in Malamut et al, ClinGastHepatol 2014); flow cytometry analysis of AIE onset is not available.</p><p>Phenotype of intestinal intraepithelial lymphocytes.</p

Clinical and immune characteristics.

<p>*: detection of serum anti-goblet cells antibodies.</p><p>Ab: antibody. Anti-E: anti-enterocyte Ab. ANA: anti-nuclear Ab. BMI: Body Mass Index. Col: colon. Duod: duodenum. EMA: IgA anti-endomysium. IGA: IgA anti-gliadin. Lymphocytosis: number of intraepithelial lymphocytes for 100 epithelial cells. LC: lymphocytic colitis. LG: lymphocytic gastritis. Sto: stomach. tTG: IgA anti-transglutaminase. VA: villousatrophy. TVA: total villousatrophy. ST VA: sub-totalvillousatrophy. PVA: partial villousatrophy. y: years. Noserum anti-tTG IgG or antigliadin IgG was found. No IgA anti-endomysium was found.</p><p>Clinical and immune characteristics.</p

Semaphorin 3F and Neuropilin-2 Control the Migration of Human T-Cell Precursors

<div><p>Neuropilins and semaphorins are known as modulators of axon guidance, angiogenesis, and organogenesis in the developing nervous system, but have been recently evidenced as also playing a role in the immune system. Here we describe the expression and role of semaphorin 3F (SEMA3F) and its receptor neuropilin-2 (NRP2) in human T cell precursors. NRP2 and SEMA3F are expressed in the human thymus, in both lymphoid and non-lymphoid compartments. SEMA3F have a repulsive effect on thymocyte migration and inhibited CXCL12- and sphingosine-1-phosphate (S1P)-induced thymocyte migration by inhibiting cytoskeleton reorganization prior to stimuli. Moreover, NRP2 and SEMA3F are expressed in human T-cell acute lymphoblastic leukemia/lymphoma primary cells. In these tumor cells, SEMA3F also blocks their migration induced by CXCL12 and S1P. Our data show that SEMA3F and NRP2 are further regulators of human thymocyte migration in physiological and pathological conditions.</p></div

Expression of NRP2 and SEMA3F in the human thymus and thymocytes.

<p><b>a)</b> Upper panels show the expression of NRP2 and SEMA3F in the human thymus <i>in situ</i>, ascertained by immunofluorescence and confocal microscopy analysis. Lower panels show the expression of SEMA3F and cytokeratin, revealing that SEMA3F is expressed in the epithelial as well as in the non-epithelial compartments of the thymus. Colocalization analysis was performed with ImageJ software. Inserts show negative controls for each secondary antibody. C: cortex; M: medulla. Magnification: 400×. <b>b)</b> NRP2 and SEMA3F mRNA expression analyzed by real time quantitative PCR, compared with the control Abelson (Abl) gene in fresh thymocytes and the THPN thymic epithelial cell line. <b>c)</b> Cytofluorometric dot plot depicts the regions used to separate the CD4/CD8-defined thymocyte subpopulations. Graphs represent the expression of NRP2 and SEMA3F in total thymocytes and each subpopulation. n = 3–6. In the case of NRP2, mean fluorescence intensity (MFI) analyses are shown to illustrate differences in the expression among the thymocyte subpopulations. Data are represented as means ± SEM. Selected thymocyte subsets were analyzed by the unpaired Student's <i>t</i> test and differences were considered statistically significant when p<0.05 (*), p<0.01 (**) or p<0.001 (***). DN-1: CD4⁻CD8⁻ cells; DN-2: CD4^lowCD8^low; DP: CD4⁺CD8⁺; CD4-1: CD4^lowCD8⁻; CD4-2: CD4^highCD8⁻; CD8-1: CD4⁻CD8^low; CD8-2: CD4⁻CD8^high.</p

SEMA3F impairs the migratory response of human thymocytes towards S1P.

<p><b>a)</b> S1P₁ expression ascertained by flow cytometry in total human thymocytes and CD4/CD8-defined subsets as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103405#pone-0103405-g001" target="_blank">Figure 1c</a>. DN-1: CD4⁻CD8⁻ cells; DN-2: CD4^lowCD8^low; DP: CD4⁺CD8⁺; CD4-1: CD4^lowCD8⁻; CD4-2: CD4^highCD8⁻; CD8-1: CD4⁻CD8^low; CD8-2: CD4⁻CD8^high. n = 4. <b>b)</b> Bars represent migration of thymocytes in a transwell system. Results are represented by migration ratio, and controls without stimuli were normalized to 1.0. n = 4. Cells migrate towards S1P and when SEMA3F was combined with S1P, it inhibited S1P-induced thymocyte migration. <b>c)</b> Migration response of thymocytes from a representative experiment. Bottom panel show the migration of thymocyte subpopulations based on CD4/CD8 expression, showing that SEMA3F had effect and impaired S1P-induced migration in all thymocyte subpopulations. DP =  double-positive, CD4 =  CD4 single positive, CD8 =  CD8 single-positive. <b>d)</b> Bars show the numbers of migrating thymocytes. The black bar represent thymocyte migration of cells pre-treated with anti-NRP2 blocking antibody which partially abrogated SEMA3F action, as the difference observed between S1P and S1P+SEMA3F+anti-NRP2 is no longer significant (n = 3). <b>e)</b> F-actin modulation of human thymocytes (n = 4) was analyzed by flow cytometry and represented as [(MFI after addition of ligand)/(MFI before addition of ligand)]×100. MFI values obtained before addition of ligand were arbitrarily set at 100% that corresponded to time zero. Data are represented as means ± SEM.Results were analyzed by the unpaired Student's <i>t</i> test, comparing each time point with time zero. Differences were considered statistically significant when p<0.05 (*).</p

SEMA3F is repulsive and impairs the migratory response of human thymocytes towards CXCL12.

<p><b>a)</b> Bars represent the numbers of migrating thymocytes in a transwell system. SEMA3F was added in the upper chambers to evaluate the repulsive response or blocking stimulus of thymocytes. CXCL12 was added in the bottom chambers and induced thymocyte migration. When both stimuli where combined, SEMA3F inhibited CXCL12-induced thymocyte migration (n = 10). <b>b)</b> Migration response of thymocyte subsets (defined by CD4/CD8 expression), showing that SEMA3F has effect in all thymocyte subpopulations. DP = double-positive, CD4 =  CD4 single positive, CD8 =  CD8 single-positive. <b>c)</b> Bars show the numbers of migrating thymocytes. Black bar represent thymocyte migration of cells pre-treated with anti-NRP2 blocking antibody which abrogated SEMA3F action, since the difference observed between CXCL12 and CXCL12+SEMA3F+anti-NRP2 is no longer significant (n = 3). Results were analyzed by the One-way ANOVA analysis of variance and Tukey's multiple comparison post-test. <b>d)</b> F-actin modulation of human thymocytes (n = 4–5) was analyzed by flow cytometry and shown herein as [(MFI after addition of ligand)/(MFI before addition of ligand)]×100. MFI values obtained before addition of ligand were arbitrarily set at 100% that corresponds to time zero. Data are represented as means ± SEM. Results were analyzed by the unpaired Student's <i>t</i> test, comparing each time point compared with time zero. Differences were considered statistically significant when p<0.05 (*), p<0.01 (**) or p<0.001 (***).</p