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Identification of a thymic epithelial cell subset sharing expression of the class Ib HLA-G molecule with fetal trophoblasts.

Crisa L, McMaster MT, Ishii JK, Fisher SJ, Salomon DR - J. Exp. Med. (1997)

Bottom Line: Thymic epithelial cell lines were generated and shown to express three alternatively spliced HLA-G transcripts, previously identified in human trophoblasts.Sequencing of HLA-G1 transcripts revealed a few nucleotide changes resulting in amino acid substitutions, all clustered within exon 3 of HLA-G, encoding for the alpha2 domain of the molecule.Our findings raise the possibility that maternal unresponsiveness to HLA-G-expressing fetal tissues may be shaped in the thymus by a previously unrecognized central presentation of this MHC molecule on the medullary epithelium.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Experimental Medicine and Immunology, The Scripps Research Institute, La Jolla, California 92037, USA. crisa@scripps.edu

ABSTRACT
HLA-G is the only class I determinant of the major histocompatibility complex (MHC) expressed by the trophoblasts, the fetal cells invading the maternal decidua during pregnancy. A unique feature of this nonclassical HLA molecule is its low polymorphism, a property that has been postulated to play an important role in preventing local activation of maternal alloreactive T and natural killer cells against the fetus. Yet, the mechanisms by which fetal HLA-G can be recognized as a self-MHC molecule by the maternal immune system remain unclear. Here we report the novel observation that HLA-G is expressed in the human thymus. Expression is targeted to the cell surface of thymic medullary and subcapsular epithelium. Thymic epithelial cell lines were generated and shown to express three alternatively spliced HLA-G transcripts, previously identified in human trophoblasts. Sequencing of HLA-G1 transcripts revealed a few nucleotide changes resulting in amino acid substitutions, all clustered within exon 3 of HLA-G, encoding for the alpha2 domain of the molecule. Our findings raise the possibility that maternal unresponsiveness to HLA-G-expressing fetal tissues may be shaped in the thymus by a previously unrecognized central presentation of this MHC molecule on the medullary epithelium.

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Surface expression of HLA-G on  freshly isolated epithelial cells. A cell suspension enriched for HLA-G+ cells was obtained  by collagenase digestion and nonenzymatic  dissociation of thymic fragments, followed by  positive selection of IB8-labeled cells using an  immunomagnetic bead separation technique.  (A) Flow cytometric analysis of a single cell  suspension stained for HLA-G before (left) and  after (right) enrichment by positive selection  on the magnetic column. The vertical line  marks the upper limit of the background  intensity of fluorescence obtained using a  control IgM. (B) This HLA-G–enriched cell  population was further probed for the  expression of the surface epithelial marker EpCAM using an anti-EpCAM FITC-labeled  mAb (clone 323A3) or a FITC-labeled isotype-matching control IgG, and analyzed by  flow cytometry. The horizontal line marks  the upper limit of the background fluorescence obtained with the control IgG (left).  HLA-Ghigh cells (A, R3) comprise cells with a  large FSC and coexpress high levels of  EpCAM, supporting their epithelial nature. HLA-Gint cells (A, R2) comprise EpCAMint-high cells with an intermediate FSC as well as EpCAMlow cells  with a large FSC. HLA-Gneg cells are EpCAMlow-neg.
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Figure 3: Surface expression of HLA-G on freshly isolated epithelial cells. A cell suspension enriched for HLA-G+ cells was obtained by collagenase digestion and nonenzymatic dissociation of thymic fragments, followed by positive selection of IB8-labeled cells using an immunomagnetic bead separation technique. (A) Flow cytometric analysis of a single cell suspension stained for HLA-G before (left) and after (right) enrichment by positive selection on the magnetic column. The vertical line marks the upper limit of the background intensity of fluorescence obtained using a control IgM. (B) This HLA-G–enriched cell population was further probed for the expression of the surface epithelial marker EpCAM using an anti-EpCAM FITC-labeled mAb (clone 323A3) or a FITC-labeled isotype-matching control IgG, and analyzed by flow cytometry. The horizontal line marks the upper limit of the background fluorescence obtained with the control IgG (left). HLA-Ghigh cells (A, R3) comprise cells with a large FSC and coexpress high levels of EpCAM, supporting their epithelial nature. HLA-Gint cells (A, R2) comprise EpCAMint-high cells with an intermediate FSC as well as EpCAMlow cells with a large FSC. HLA-Gneg cells are EpCAMlow-neg.

Mentions: To determine whether HLA-G is expressed on the cell surface, a suspension of thymic stromal cells was prepared in two steps, a collagenase digestion followed by a nonenzymatic dissociation of the thymic tissue. Cells were then stained with the IB8 mAb and enriched by positive selection with a magnetic bead–column technique. Flow cytometric analysis of the cells before purification on the column demonstrated a low percentage (∼1%) of HLA-G+ cells (Fig. 3 A, left). However, two discrete populations of cells expressing intermediate and high levels of HLA-G (HLA-Gint and HLA-Ghigh, respectively) on the cell surface could be detected after enrichment on the magnetic column (Fig. 3 A, right). HLA-Gint and HLA-Ghigh cells represent 33 and 17% of the enriched population, respectively.


Identification of a thymic epithelial cell subset sharing expression of the class Ib HLA-G molecule with fetal trophoblasts.

Crisa L, McMaster MT, Ishii JK, Fisher SJ, Salomon DR - J. Exp. Med. (1997)

Surface expression of HLA-G on  freshly isolated epithelial cells. A cell suspension enriched for HLA-G+ cells was obtained  by collagenase digestion and nonenzymatic  dissociation of thymic fragments, followed by  positive selection of IB8-labeled cells using an  immunomagnetic bead separation technique.  (A) Flow cytometric analysis of a single cell  suspension stained for HLA-G before (left) and  after (right) enrichment by positive selection  on the magnetic column. The vertical line  marks the upper limit of the background  intensity of fluorescence obtained using a  control IgM. (B) This HLA-G–enriched cell  population was further probed for the  expression of the surface epithelial marker EpCAM using an anti-EpCAM FITC-labeled  mAb (clone 323A3) or a FITC-labeled isotype-matching control IgG, and analyzed by  flow cytometry. The horizontal line marks  the upper limit of the background fluorescence obtained with the control IgG (left).  HLA-Ghigh cells (A, R3) comprise cells with a  large FSC and coexpress high levels of  EpCAM, supporting their epithelial nature. HLA-Gint cells (A, R2) comprise EpCAMint-high cells with an intermediate FSC as well as EpCAMlow cells  with a large FSC. HLA-Gneg cells are EpCAMlow-neg.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198976&req=5

Figure 3: Surface expression of HLA-G on freshly isolated epithelial cells. A cell suspension enriched for HLA-G+ cells was obtained by collagenase digestion and nonenzymatic dissociation of thymic fragments, followed by positive selection of IB8-labeled cells using an immunomagnetic bead separation technique. (A) Flow cytometric analysis of a single cell suspension stained for HLA-G before (left) and after (right) enrichment by positive selection on the magnetic column. The vertical line marks the upper limit of the background intensity of fluorescence obtained using a control IgM. (B) This HLA-G–enriched cell population was further probed for the expression of the surface epithelial marker EpCAM using an anti-EpCAM FITC-labeled mAb (clone 323A3) or a FITC-labeled isotype-matching control IgG, and analyzed by flow cytometry. The horizontal line marks the upper limit of the background fluorescence obtained with the control IgG (left). HLA-Ghigh cells (A, R3) comprise cells with a large FSC and coexpress high levels of EpCAM, supporting their epithelial nature. HLA-Gint cells (A, R2) comprise EpCAMint-high cells with an intermediate FSC as well as EpCAMlow cells with a large FSC. HLA-Gneg cells are EpCAMlow-neg.
Mentions: To determine whether HLA-G is expressed on the cell surface, a suspension of thymic stromal cells was prepared in two steps, a collagenase digestion followed by a nonenzymatic dissociation of the thymic tissue. Cells were then stained with the IB8 mAb and enriched by positive selection with a magnetic bead–column technique. Flow cytometric analysis of the cells before purification on the column demonstrated a low percentage (∼1%) of HLA-G+ cells (Fig. 3 A, left). However, two discrete populations of cells expressing intermediate and high levels of HLA-G (HLA-Gint and HLA-Ghigh, respectively) on the cell surface could be detected after enrichment on the magnetic column (Fig. 3 A, right). HLA-Gint and HLA-Ghigh cells represent 33 and 17% of the enriched population, respectively.

Bottom Line: Thymic epithelial cell lines were generated and shown to express three alternatively spliced HLA-G transcripts, previously identified in human trophoblasts.Sequencing of HLA-G1 transcripts revealed a few nucleotide changes resulting in amino acid substitutions, all clustered within exon 3 of HLA-G, encoding for the alpha2 domain of the molecule.Our findings raise the possibility that maternal unresponsiveness to HLA-G-expressing fetal tissues may be shaped in the thymus by a previously unrecognized central presentation of this MHC molecule on the medullary epithelium.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Experimental Medicine and Immunology, The Scripps Research Institute, La Jolla, California 92037, USA. crisa@scripps.edu

ABSTRACT
HLA-G is the only class I determinant of the major histocompatibility complex (MHC) expressed by the trophoblasts, the fetal cells invading the maternal decidua during pregnancy. A unique feature of this nonclassical HLA molecule is its low polymorphism, a property that has been postulated to play an important role in preventing local activation of maternal alloreactive T and natural killer cells against the fetus. Yet, the mechanisms by which fetal HLA-G can be recognized as a self-MHC molecule by the maternal immune system remain unclear. Here we report the novel observation that HLA-G is expressed in the human thymus. Expression is targeted to the cell surface of thymic medullary and subcapsular epithelium. Thymic epithelial cell lines were generated and shown to express three alternatively spliced HLA-G transcripts, previously identified in human trophoblasts. Sequencing of HLA-G1 transcripts revealed a few nucleotide changes resulting in amino acid substitutions, all clustered within exon 3 of HLA-G, encoding for the alpha2 domain of the molecule. Our findings raise the possibility that maternal unresponsiveness to HLA-G-expressing fetal tissues may be shaped in the thymus by a previously unrecognized central presentation of this MHC molecule on the medullary epithelium.

Show MeSH
Related in: MedlinePlus