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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 RT1-E2u molecule is expressed at low levels at the surface of transfected L cells. L cells transfected with either pSV2 neo alone, or together with either pCMU-E2u, or pCMU-Au [28], were stained with saturating concentrations of MRC OX-18 hybridoma supernatant, followed by a FITC-conjugated anti-mouse Ig secondary polyclonal reagent.
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Figure 6: The RT1-E2u molecule is expressed at low levels at the surface of transfected L cells. L cells transfected with either pSV2 neo alone, or together with either pCMU-E2u, or pCMU-Au [28], were stained with saturating concentrations of MRC OX-18 hybridoma supernatant, followed by a FITC-conjugated anti-mouse Ig secondary polyclonal reagent.

Mentions: An RT1-E2u cDNA clone derived from PVG-RT1u neurospheres was selected for the stable transfection of the mouse L cell line. In the resulting G418-resistant cells obtained, low levels of expression were detected with MRC OX-18 in approximately 50% of the population. Those cells were selected by flow cytometry, and were later cloned in order to obtain a population that did not lose expression of the RT1-E2u molecule over successive passages. The levels of MRC OX-18 staining attained with these transfectants were better than in transiently transfected COS cells, but much lower than those seen with stable L cell transfectants for many other rat or mouse class I molecules generated using the same procedure [27-29,31,33] (Fig. 6). Treatment of the RT1-E2u L cells transfectants with γ interferon resulted in a three to four fold increase in the levels of cell surface expression detected, to levels that remained very inferior to those detected for class Ia counterparts (not shown). The possibility that this low staining was not due to a low level of cell surface expression but to a low affinity of MRC OX-18 for the RT1-E2u molecule was ruled out by antibody titration experiments showing similar saturating concentrations to those observed for L cells transfected with RT1-A (not shown). Analysis of these transfectants with other RT1-reactive rat monoclonal antibodies has so far failed to identify other antibodies crossreacting with the RT1-E2u molecule (not shown).


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 RT1-E2u molecule is expressed at low levels at the surface of transfected L cells. L cells transfected with either pSV2 neo alone, or together with either pCMU-E2u, or pCMU-Au [28], were stained with saturating concentrations of MRC OX-18 hybridoma supernatant, followed by a FITC-conjugated anti-mouse Ig secondary polyclonal reagent.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC183868&req=5

Figure 6: The RT1-E2u molecule is expressed at low levels at the surface of transfected L cells. L cells transfected with either pSV2 neo alone, or together with either pCMU-E2u, or pCMU-Au [28], were stained with saturating concentrations of MRC OX-18 hybridoma supernatant, followed by a FITC-conjugated anti-mouse Ig secondary polyclonal reagent.
Mentions: An RT1-E2u cDNA clone derived from PVG-RT1u neurospheres was selected for the stable transfection of the mouse L cell line. In the resulting G418-resistant cells obtained, low levels of expression were detected with MRC OX-18 in approximately 50% of the population. Those cells were selected by flow cytometry, and were later cloned in order to obtain a population that did not lose expression of the RT1-E2u molecule over successive passages. The levels of MRC OX-18 staining attained with these transfectants were better than in transiently transfected COS cells, but much lower than those seen with stable L cell transfectants for many other rat or mouse class I molecules generated using the same procedure [27-29,31,33] (Fig. 6). Treatment of the RT1-E2u L cells transfectants with γ interferon resulted in a three to four fold increase in the levels of cell surface expression detected, to levels that remained very inferior to those detected for class Ia counterparts (not shown). The possibility that this low staining was not due to a low level of cell surface expression but to a low affinity of MRC OX-18 for the RT1-E2u molecule was ruled out by antibody titration experiments showing similar saturating concentrations to those observed for L cells transfected with RT1-A (not shown). Analysis of these transfectants with other RT1-reactive rat monoclonal antibodies has so far failed to identify other antibodies crossreacting with the RT1-E2u molecule (not shown).

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