Local production of T cells in the FOXN1^−/− human intestine.

<p>(A) Quantitative real-time PCR showing the expression of mRNAs encoding RAG1, RAG2 and pTα (relative to β-actin) in skin fibroblasts (negative control), thymus (positive control) and intestinal tissue of control and FOXN1^−/− fetuses (16 weeks of gestation). (B) Comparison of TCR Vβ-region usage between intestinal lymphocytes (black bars) and CBMCs (white bars) from the FOXN1^−/− fetus. Experiments were repeated three times in (A) and two times in (B).</p

Detection of extrathymically derived T lymphocytes in the cord blood of FOXN1^−/− human fetus.

<p>(A) Flow cytometry analysis of CBMCs from WT (left dot plots) or FOXN1^−/− (right dot plots) fetuses (16 weeks of gestation). CD7 and CD2 together with the CD8α and CD8β expression patterns for the gated CD45⁺CD3⁻ cells are shown. CD8α and CD4 expression is shown for the gated CD45⁺CD3⁺ cells. Numbers indicate the frequency of the cells within the gate. Experiment was repeated two times. Data were obtained by gating first on viable cells and later on CD45⁺ cells. (B) RT-PCR analysis of CD3ε expression in CBMCs. The expression of CD3ε transcript in human skin fibroblasts (negative control), human thymus (positive control), CBMCs from WT or FOXN1^−/− fetuses is shown. Blanck, no cDNA. β-actin was used as loading control. Representative results from three independent experiments are shown. (C) Quantitative real-time PCR showing the expression of mRNAs encoding CD3ε (relative to β-actin) in skin fibroblasts (negative control), thymus (positive control) and CBMCs from WT or FOXN1^−/− fetuses (16 weeks of gestation). Representative results from two independent experiments are shown.</p

Lymphocytes with naive phenotype in cord blood and intestine.

<p>Flow cytometry of CBMCs from normal and FOXN1^−/− fetuses matched for gestational age (16 weeks of gestation). Dot plots show the expression pattern of the naïve cell markers. (A) Frequencies of CBMCs expressing both CD45RA and CD3 markers. (B) Frequencies of CBMCs expressing both CD62L and CD3 markers. (C) Frequencies of CBMCs coexpressing CD45RA and CD62L markers. (D) Frequencies of CBMCs coexpressing CD45RA and CD27 markers. Experiment in (A), (B), (C) and (D) was repeated two times. Data were obtained by gating first on viable cells and later on CD45⁺ cells (A and B) or finally also on CD3⁺ (C and D). (E) Confocal microscopy of fetal intestinal sections labeled with anti-human CD3 (red) and anti-human CD45RA (green). Representative results from three independent experiments with two samples are shown.</p

Identification of lymphocytes at extrathymic sites of differentiation in a Nude/SCID human fetus.

<p>(A, B) Immunohistochemical detection of lymphocytes at extrathymic sites of differentiation in FOXN1^−/− human fetus (16 weeks of gestation). (A) Stem cells, B cells and NK cells were detected by immunohistochemical stain for CD34 (brown), CD20 (brown) and CD56 (brown) in intestinal and liver sections obtained from a FOXN1^−/− human fetus (16 weeks of gestation) or an aged-matched control fetus. In CD34 and CD20 stained intestine sections from WT and FOXN1^−/− fetuses original magnification was 100x. (B) As negative control, intestinal sections from FOXN1^−/− fetus were counterstained with hematoxylin and with the isotype control (primary antibody omitted) by DAB. As positive control, hyperplastic lymph node sections were stained for CD3 (brown) by DAB. Intestinal or liver sections of FOXN1^−/− human fetus were counterstained with hematoxylin and eosin (H&E). Immunohistochemical analysis of intestinal or liver sections of a FOXN1^−/− human fetus and an aged-matched control fetus using anti-CD3 staining to mark T cells, anti-CD4 staining to mark T helper cells and anti-CD8 staining to mark cytotoxic T cells. DAB, 200x. Representative results from two independent experiments with two distinct samples are shown. (C, D) Confocal microscopy of FOXN1^−/− intestinal sections. (C) Labeling with anti-human CD4 (red) and anti-human CD8 (green). (D) Labeling with anti-human CD7 (green) and anti-human CD3 (red). Representative results from three independent experiments are shown. (E) RT-PCR analysis of CD3ε intestinal expression. CD3ε transcript expression in human skin fibroblasts (negative control), intestinal lymphocytes from WT or FOXN1^−/− fetuses is shown. Blanck, no cDNA. β-actin was used as loading control. Representative results from three independent experiments are shown. (F) Quantitative real-time PCR showing the expression of mRNAs encoding CD3ε (relative to β-actin) in skin fibroblasts (negative control), thymus (positive control) and intestinal tissue of control and FOXN1^−/− fetuses (16 weeks of gestation). Representative results from two independent experiments are shown.</p