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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: 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.

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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Related in: MedlinePlus

PCR amplification and sequence of HLA-G transcripts expressed by thymic epithelial cell lines. (A) Agarose gel electrophoresis of HLA-G  alternatively spliced transcripts amplified by RT-PCR from mRNA obtained from a primary human thymic epithelial cell line. 1,200-, 900- and 500-bp  bands are detected (lane 1), consistent with the presence of the three main alternatively spliced forms of HLA-G, namely HLA-G1, -G2, and -G3 described  by Ishitani et al. (32). In contrast, mRNA from freshly isolated thymocytes (lane 2) demonstrates no HLA-G–specific transcripts. A positive control PCR  reaction for thymocyte mRNA showing a 237-bp DNA fragment of cyclophillin is displayed in lane 3. A negative control PCR reaction run using HLA-G–specific primers and no cDNA template is shown in lane 4. Molecular weight markers corresponding to a 100-bp DNA ladder (GIBCO BRL, Gaithersburg, MD) are shown in the left lane (MW). (B) Primary structure and sequence variations identified within the HLA-G1 and -G2 cDNAs amplified  by PCR from two of the three TEC lines that were sequenced, e.g., FT6 and HTS 2.900. Sequence variations are displayed with respect to their location  within each exon and aligned with the published sequence of a full-length HLA-G cDNA expressed by placental trophoblasts reported by Shukla et al.  (35). SP, signal peptide; TM, transmembrane domain. Stretches of nucleotide sequences are identified by base pair numbers for orientation. Dashes represent sequence identity; stars represent gaps. Codons containing point mutations are boxed. Mutations resulting in amino acid variations are also shadowed. Note the prevalence of point mutations resulting in amino acid changes within exon 4. The CTC→ ATC (leu→ ile) substitution corresponding to  amino acid position 110 of the HTS 2.900 line has been previously reported (35). Though the GGC→ GAC (gly→ asp) substitution at amino acid position 120 of the FT6 line has not been reported, a variety of other amino acid substitutions have been identified at the very same position (36).
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Figure 5: PCR amplification and sequence of HLA-G transcripts expressed by thymic epithelial cell lines. (A) Agarose gel electrophoresis of HLA-G alternatively spliced transcripts amplified by RT-PCR from mRNA obtained from a primary human thymic epithelial cell line. 1,200-, 900- and 500-bp bands are detected (lane 1), consistent with the presence of the three main alternatively spliced forms of HLA-G, namely HLA-G1, -G2, and -G3 described by Ishitani et al. (32). In contrast, mRNA from freshly isolated thymocytes (lane 2) demonstrates no HLA-G–specific transcripts. A positive control PCR reaction for thymocyte mRNA showing a 237-bp DNA fragment of cyclophillin is displayed in lane 3. A negative control PCR reaction run using HLA-G–specific primers and no cDNA template is shown in lane 4. Molecular weight markers corresponding to a 100-bp DNA ladder (GIBCO BRL, Gaithersburg, MD) are shown in the left lane (MW). (B) Primary structure and sequence variations identified within the HLA-G1 and -G2 cDNAs amplified by PCR from two of the three TEC lines that were sequenced, e.g., FT6 and HTS 2.900. Sequence variations are displayed with respect to their location within each exon and aligned with the published sequence of a full-length HLA-G cDNA expressed by placental trophoblasts reported by Shukla et al. (35). SP, signal peptide; TM, transmembrane domain. Stretches of nucleotide sequences are identified by base pair numbers for orientation. Dashes represent sequence identity; stars represent gaps. Codons containing point mutations are boxed. Mutations resulting in amino acid variations are also shadowed. Note the prevalence of point mutations resulting in amino acid changes within exon 4. The CTC→ ATC (leu→ ile) substitution corresponding to amino acid position 110 of the HTS 2.900 line has been previously reported (35). Though the GGC→ GAC (gly→ asp) substitution at amino acid position 120 of the FT6 line has not been reported, a variety of other amino acid substitutions have been identified at the very same position (36).

Mentions: Amplification of HLA-G–specific sequences from mRNA of TEC lines by RT-PCR demonstrated 1,200-, 900-, and 500-bp DNA fragments (Fig. 5 A) consistent with mRNA transcripts for the membrane-bound HLA-G1, -G2, and -G3 alternatively spliced isoforms, respectively (25, 32). Amplification of these transcripts was consistently obtained in six independent TEC lines. Semiquantitative analysis of these PCR products obtained in the linear range of amplification from mRNA of three TEC lines revealed that the HLA-G2 and -G3 transcripts represent 20.5 ± 3.9% and 4.4 ± 3.5% (mean ± SEM) of the HLA-G1 mRNA, respectively. PCR amplification of HLA-G transcripts from mRNA of fresh thymic tissue obtained from three separate donors revealed a similar pattern of frequency of the HLA-G2 and -G3 transcripts relative to the HLA-G1 transcript (e.g., 29.8 ± 4.1% and 0.57 ± 0.2%, respectively). Sequence analysis of HLA-G1 and -G2 cDNAs isolated from two pediatric thymic epithelial cell lines confirmed an intact primary structure and correct splicing of the corresponding transcripts. In particular, HLA-G1 cDNAs revealed a nucleotide sequence consistent with the splicing of exons 1, 2, 3, 4, 5, 6, and 8 of the HLA-G gene (21; Fig. 5 B). In contrast, HLA-G2 cDNAs revealed a sequence consistent with the splicing of exons 1, 2, 4, 5, 6, and 8. Similar analysis of HLA-G1 and -G2 cDNAs obtained from a fetal thymic epithelial cell line revealed an intact primary structure of the HLA-G1 transcript, but an HLA-G2 mRNA containing a 33-nucleotide deletion within exon 4. Fig. 5 B shows the location of the sequence variations identified by our analysis, as compared to the cDNA sequence of the full-length HLA-G reported by Shukla et al. (35). Notably, within the HLA-G1 transcripts, most nucleotide variations resulting in amino acid changes are clustered within exon 3, encoding for the α2 domain, located between the β-pleated sheet and the start of the α2 helix. Interestingly, such amino acid changes fall into the same stretch of sequence (e.g., amino acids 101–138) previously identified as a mutational hot spot by genomic sequence analysis of HLA-G alleles (36). The data indicate that, unlike classical HLA class I molecules in which polymorphic residues restricting peptide binding are present in both the α1 and α2 domains, polymorphism of HLA-G appears to be largely restricted to the α2 domain. The deletion which we report within HLA-G2 cDNA expressed by the fetal cell line includes the loss of cysteine residue 259 located in the α3 domain. It is conceivable that this sequence change results in a gross alteration of protein structure, suggesting that this transcript may either yield a protein with an altered function or behave as a pseudogene.


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)

PCR amplification and sequence of HLA-G transcripts expressed by thymic epithelial cell lines. (A) Agarose gel electrophoresis of HLA-G  alternatively spliced transcripts amplified by RT-PCR from mRNA obtained from a primary human thymic epithelial cell line. 1,200-, 900- and 500-bp  bands are detected (lane 1), consistent with the presence of the three main alternatively spliced forms of HLA-G, namely HLA-G1, -G2, and -G3 described  by Ishitani et al. (32). In contrast, mRNA from freshly isolated thymocytes (lane 2) demonstrates no HLA-G–specific transcripts. A positive control PCR  reaction for thymocyte mRNA showing a 237-bp DNA fragment of cyclophillin is displayed in lane 3. A negative control PCR reaction run using HLA-G–specific primers and no cDNA template is shown in lane 4. Molecular weight markers corresponding to a 100-bp DNA ladder (GIBCO BRL, Gaithersburg, MD) are shown in the left lane (MW). (B) Primary structure and sequence variations identified within the HLA-G1 and -G2 cDNAs amplified  by PCR from two of the three TEC lines that were sequenced, e.g., FT6 and HTS 2.900. Sequence variations are displayed with respect to their location  within each exon and aligned with the published sequence of a full-length HLA-G cDNA expressed by placental trophoblasts reported by Shukla et al.  (35). SP, signal peptide; TM, transmembrane domain. Stretches of nucleotide sequences are identified by base pair numbers for orientation. Dashes represent sequence identity; stars represent gaps. Codons containing point mutations are boxed. Mutations resulting in amino acid variations are also shadowed. Note the prevalence of point mutations resulting in amino acid changes within exon 4. The CTC→ ATC (leu→ ile) substitution corresponding to  amino acid position 110 of the HTS 2.900 line has been previously reported (35). Though the GGC→ GAC (gly→ asp) substitution at amino acid position 120 of the FT6 line has not been reported, a variety of other amino acid substitutions have been identified at the very same position (36).
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Figure 5: PCR amplification and sequence of HLA-G transcripts expressed by thymic epithelial cell lines. (A) Agarose gel electrophoresis of HLA-G alternatively spliced transcripts amplified by RT-PCR from mRNA obtained from a primary human thymic epithelial cell line. 1,200-, 900- and 500-bp bands are detected (lane 1), consistent with the presence of the three main alternatively spliced forms of HLA-G, namely HLA-G1, -G2, and -G3 described by Ishitani et al. (32). In contrast, mRNA from freshly isolated thymocytes (lane 2) demonstrates no HLA-G–specific transcripts. A positive control PCR reaction for thymocyte mRNA showing a 237-bp DNA fragment of cyclophillin is displayed in lane 3. A negative control PCR reaction run using HLA-G–specific primers and no cDNA template is shown in lane 4. Molecular weight markers corresponding to a 100-bp DNA ladder (GIBCO BRL, Gaithersburg, MD) are shown in the left lane (MW). (B) Primary structure and sequence variations identified within the HLA-G1 and -G2 cDNAs amplified by PCR from two of the three TEC lines that were sequenced, e.g., FT6 and HTS 2.900. Sequence variations are displayed with respect to their location within each exon and aligned with the published sequence of a full-length HLA-G cDNA expressed by placental trophoblasts reported by Shukla et al. (35). SP, signal peptide; TM, transmembrane domain. Stretches of nucleotide sequences are identified by base pair numbers for orientation. Dashes represent sequence identity; stars represent gaps. Codons containing point mutations are boxed. Mutations resulting in amino acid variations are also shadowed. Note the prevalence of point mutations resulting in amino acid changes within exon 4. The CTC→ ATC (leu→ ile) substitution corresponding to amino acid position 110 of the HTS 2.900 line has been previously reported (35). Though the GGC→ GAC (gly→ asp) substitution at amino acid position 120 of the FT6 line has not been reported, a variety of other amino acid substitutions have been identified at the very same position (36).
Mentions: Amplification of HLA-G–specific sequences from mRNA of TEC lines by RT-PCR demonstrated 1,200-, 900-, and 500-bp DNA fragments (Fig. 5 A) consistent with mRNA transcripts for the membrane-bound HLA-G1, -G2, and -G3 alternatively spliced isoforms, respectively (25, 32). Amplification of these transcripts was consistently obtained in six independent TEC lines. Semiquantitative analysis of these PCR products obtained in the linear range of amplification from mRNA of three TEC lines revealed that the HLA-G2 and -G3 transcripts represent 20.5 ± 3.9% and 4.4 ± 3.5% (mean ± SEM) of the HLA-G1 mRNA, respectively. PCR amplification of HLA-G transcripts from mRNA of fresh thymic tissue obtained from three separate donors revealed a similar pattern of frequency of the HLA-G2 and -G3 transcripts relative to the HLA-G1 transcript (e.g., 29.8 ± 4.1% and 0.57 ± 0.2%, respectively). Sequence analysis of HLA-G1 and -G2 cDNAs isolated from two pediatric thymic epithelial cell lines confirmed an intact primary structure and correct splicing of the corresponding transcripts. In particular, HLA-G1 cDNAs revealed a nucleotide sequence consistent with the splicing of exons 1, 2, 3, 4, 5, 6, and 8 of the HLA-G gene (21; Fig. 5 B). In contrast, HLA-G2 cDNAs revealed a sequence consistent with the splicing of exons 1, 2, 4, 5, 6, and 8. Similar analysis of HLA-G1 and -G2 cDNAs obtained from a fetal thymic epithelial cell line revealed an intact primary structure of the HLA-G1 transcript, but an HLA-G2 mRNA containing a 33-nucleotide deletion within exon 4. Fig. 5 B shows the location of the sequence variations identified by our analysis, as compared to the cDNA sequence of the full-length HLA-G reported by Shukla et al. (35). Notably, within the HLA-G1 transcripts, most nucleotide variations resulting in amino acid changes are clustered within exon 3, encoding for the α2 domain, located between the β-pleated sheet and the start of the α2 helix. Interestingly, such amino acid changes fall into the same stretch of sequence (e.g., amino acids 101–138) previously identified as a mutational hot spot by genomic sequence analysis of HLA-G alleles (36). The data indicate that, unlike classical HLA class I molecules in which polymorphic residues restricting peptide binding are present in both the α1 and α2 domains, polymorphism of HLA-G appears to be largely restricted to the α2 domain. The deletion which we report within HLA-G2 cDNA expressed by the fetal cell line includes the loss of cysteine residue 259 located in the α3 domain. It is conceivable that this sequence change results in a gross alteration of protein structure, suggesting that this transcript may either yield a protein with an altered function or behave as a pseudogene.

Bottom Line: 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.

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