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Characterisation of RT1-E2, a multigenic family of highly conserved rat non-classical MHC class I molecules initially identified in cells from immunoprivileged sites.

Lau P, Amadou C, Brun H, Rouillon V, McLaren F, Le Rolle AF, Graham M, Butcher GW, Joly E - BMC Immunol. (2003)

Bottom Line: The RT1n MHC haplotype (found in BN rats) carries a single RT1-E2 locus, which lies in the RT1-C/E region of the MHC and displays the typical exon-intron organisation and promoter features seen in other rat MHC class I genes.Compared to other class I molecules, this suggests that RT1-E2 molecules may associate with well defined sets of ligands.Several characteristics point to a certain similarity to the mouse H2-Qa2 and human HLA-G molecules.

View Article: PubMed Central - HTML - PubMed

Affiliation: IFR Claude de Préval, INSERM U563, CHU Purpan, 31300 Toulouse, France. laupoui@pasteur.fr

ABSTRACT

Background: So-called "immunoprivileged sites" are tissues or organs where slow allograft rejection correlates with low levels of expression of MHC class I molecules. Whilst classical class I molecules are recognised by cytotoxic T lymphocytes (CTL), some MHC class I molecules are called "non-classical" because they exhibit low polymorphism and are not widely expressed. These last years, several studies have shown that these can play different, more specialised roles than their classical counterparts. In the course of efforts to characterise MHC class I expression in rat cells obtained from immunoprivileged sites such as the central nervous system or the placenta, a new family of non-classical MHC class I molecules, which we have named RT1-E2, has been uncovered.

Results: Members of the RT1-E2 family are all highly homologous to one another, and the number of RT1-E2 loci varies from one to four per MHC haplotype among the six rat strains studied so far, with some loci predicted to give rise to soluble molecules. The RT1n MHC haplotype (found in BN rats) carries a single RT1-E2 locus, which lies in the RT1-C/E region of the MHC and displays the typical exon-intron organisation and promoter features seen in other rat MHC class I genes. We present evidence that: i) RT1-E2 molecules can be detected at the surface of transfected mouse L cells and simian COS-7 cells, albeit at low levels; ii) their transport to the cell surface is dependent on a functional TAP transporter. In L cells, their transport is also hindered by protease inhibitors, brefeldin A and monensin.

Conclusions: These findings suggest that RT1-E2 molecules probably associate with ligands of peptidic nature. The high homology between the RT1-E2 molecules isolated from divergent rat MHC haplotypes is particularly striking at the level of their extra-cellular portions. Compared to other class I molecules, this suggests that RT1-E2 molecules may associate with well defined sets of ligands. Several characteristics point to a certain similarity to the mouse H2-Qa2 and human HLA-G molecules.

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The transport of RT1-E2u to the surface of transiently transfected COS-7 cells is dependent on a functional TAP transporter. For all panels, graphs are from flow cytometric analyses after staining either for H2-Db with B22-249 (upper four panels), or for RT1-E2u with MRC OX-18 (lower four panels). The fine lines are for cells labelled only with PE-conjugated secondary antibody. For both H2-Db and RT1-E2u, cell surface expression was significantly reduced when an expression-plasmid for the TAP-inhibitor protein ICP47 was co-transfected. For the analyses shown in the right-hand column, the proportion of COS-7 cells expressing the MHC molecule of interest was improved by transfecting bicistronic vectors carrying a Gtx IRES-EGFP sequence after that of the MHC class I molecules (see M&M), and gating only on the EGFP-expressing cells for analysis.
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Figure 8: The transport of RT1-E2u to the surface of transiently transfected COS-7 cells is dependent on a functional TAP transporter. For all panels, graphs are from flow cytometric analyses after staining either for H2-Db with B22-249 (upper four panels), or for RT1-E2u with MRC OX-18 (lower four panels). The fine lines are for cells labelled only with PE-conjugated secondary antibody. For both H2-Db and RT1-E2u, cell surface expression was significantly reduced when an expression-plasmid for the TAP-inhibitor protein ICP47 was co-transfected. For the analyses shown in the right-hand column, the proportion of COS-7 cells expressing the MHC molecule of interest was improved by transfecting bicistronic vectors carrying a Gtx IRES-EGFP sequence after that of the MHC class I molecules (see M&M), and gating only on the EGFP-expressing cells for analysis.

Mentions: In order to investigate the dependence of RT1-E2u expression upon a functional TAP transporter, we made use of the fact that the ICP47 viral glycoprotein derived from HSV-1 (Human Herpes Simplex Virus-1) can block TAP function in the simian COS-7 cell line [45]. As can be seen in Figure 8, co-transfection of either RT1-E2u or H2-Db with ICP47 led to inhibition of cell surface expression for both class I molecules, although this effect was more profound in the case of RT1-E2u. This result was confirmed in parallel experiments in which RT1-E2u and H2-Db were subcloned into bicistronic expression vectors expressing EGFP after the Gtx IRES (see M&M). Gating on the EGFP-expressing cells after transfection of these vectors, a dramatic increase in the proportion of cells expressing the MHC molecules was seen among the cells analysed (Fig. 8, right column). The levels of class I expression attained per cell were, however, slightly lower using this system, and this is particularly noticeable for RT1-E2u (panels C and G). Considered together, these results suggest that RT1-E2 molecules follow intracellular pathways similar to those of classical class I molecules, and the dependence of their surface expression on the TAP transporter and active proteases points to their probable assembly with ligands of a peptidic nature.


Characterisation of RT1-E2, a multigenic family of highly conserved rat non-classical MHC class I molecules initially identified in cells from immunoprivileged sites.

Lau P, Amadou C, Brun H, Rouillon V, McLaren F, Le Rolle AF, Graham M, Butcher GW, Joly E - BMC Immunol. (2003)

The transport of RT1-E2u to the surface of transiently transfected COS-7 cells is dependent on a functional TAP transporter. For all panels, graphs are from flow cytometric analyses after staining either for H2-Db with B22-249 (upper four panels), or for RT1-E2u with MRC OX-18 (lower four panels). The fine lines are for cells labelled only with PE-conjugated secondary antibody. For both H2-Db and RT1-E2u, cell surface expression was significantly reduced when an expression-plasmid for the TAP-inhibitor protein ICP47 was co-transfected. For the analyses shown in the right-hand column, the proportion of COS-7 cells expressing the MHC molecule of interest was improved by transfecting bicistronic vectors carrying a Gtx IRES-EGFP sequence after that of the MHC class I molecules (see M&M), and gating only on the EGFP-expressing cells for analysis.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC183868&req=5

Figure 8: The transport of RT1-E2u to the surface of transiently transfected COS-7 cells is dependent on a functional TAP transporter. For all panels, graphs are from flow cytometric analyses after staining either for H2-Db with B22-249 (upper four panels), or for RT1-E2u with MRC OX-18 (lower four panels). The fine lines are for cells labelled only with PE-conjugated secondary antibody. For both H2-Db and RT1-E2u, cell surface expression was significantly reduced when an expression-plasmid for the TAP-inhibitor protein ICP47 was co-transfected. For the analyses shown in the right-hand column, the proportion of COS-7 cells expressing the MHC molecule of interest was improved by transfecting bicistronic vectors carrying a Gtx IRES-EGFP sequence after that of the MHC class I molecules (see M&M), and gating only on the EGFP-expressing cells for analysis.
Mentions: In order to investigate the dependence of RT1-E2u expression upon a functional TAP transporter, we made use of the fact that the ICP47 viral glycoprotein derived from HSV-1 (Human Herpes Simplex Virus-1) can block TAP function in the simian COS-7 cell line [45]. As can be seen in Figure 8, co-transfection of either RT1-E2u or H2-Db with ICP47 led to inhibition of cell surface expression for both class I molecules, although this effect was more profound in the case of RT1-E2u. This result was confirmed in parallel experiments in which RT1-E2u and H2-Db were subcloned into bicistronic expression vectors expressing EGFP after the Gtx IRES (see M&M). Gating on the EGFP-expressing cells after transfection of these vectors, a dramatic increase in the proportion of cells expressing the MHC molecules was seen among the cells analysed (Fig. 8, right column). The levels of class I expression attained per cell were, however, slightly lower using this system, and this is particularly noticeable for RT1-E2u (panels C and G). Considered together, these results suggest that RT1-E2 molecules follow intracellular pathways similar to those of classical class I molecules, and the dependence of their surface expression on the TAP transporter and active proteases points to their probable assembly with ligands of a peptidic nature.

Bottom Line: The RT1n MHC haplotype (found in BN rats) carries a single RT1-E2 locus, which lies in the RT1-C/E region of the MHC and displays the typical exon-intron organisation and promoter features seen in other rat MHC class I genes.Compared to other class I molecules, this suggests that RT1-E2 molecules may associate with well defined sets of ligands.Several characteristics point to a certain similarity to the mouse H2-Qa2 and human HLA-G molecules.

View Article: PubMed Central - HTML - PubMed

Affiliation: IFR Claude de Préval, INSERM U563, CHU Purpan, 31300 Toulouse, France. laupoui@pasteur.fr

ABSTRACT

Background: So-called "immunoprivileged sites" are tissues or organs where slow allograft rejection correlates with low levels of expression of MHC class I molecules. Whilst classical class I molecules are recognised by cytotoxic T lymphocytes (CTL), some MHC class I molecules are called "non-classical" because they exhibit low polymorphism and are not widely expressed. These last years, several studies have shown that these can play different, more specialised roles than their classical counterparts. In the course of efforts to characterise MHC class I expression in rat cells obtained from immunoprivileged sites such as the central nervous system or the placenta, a new family of non-classical MHC class I molecules, which we have named RT1-E2, has been uncovered.

Results: Members of the RT1-E2 family are all highly homologous to one another, and the number of RT1-E2 loci varies from one to four per MHC haplotype among the six rat strains studied so far, with some loci predicted to give rise to soluble molecules. The RT1n MHC haplotype (found in BN rats) carries a single RT1-E2 locus, which lies in the RT1-C/E region of the MHC and displays the typical exon-intron organisation and promoter features seen in other rat MHC class I genes. We present evidence that: i) RT1-E2 molecules can be detected at the surface of transfected mouse L cells and simian COS-7 cells, albeit at low levels; ii) their transport to the cell surface is dependent on a functional TAP transporter. In L cells, their transport is also hindered by protease inhibitors, brefeldin A and monensin.

Conclusions: These findings suggest that RT1-E2 molecules probably associate with ligands of peptidic nature. The high homology between the RT1-E2 molecules isolated from divergent rat MHC haplotypes is particularly striking at the level of their extra-cellular portions. Compared to other class I molecules, this suggests that RT1-E2 molecules may associate with well defined sets of ligands. Several characteristics point to a certain similarity to the mouse H2-Qa2 and human HLA-G molecules.

Show MeSH