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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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Transport of RT1-E2u molecules to the cell surface is affected by endoprotease inhibitors, and follows a similar pathway to that of the H2-Kk class Ia molecule. MHC molecules present at the surface of transfected L cells were denatured by a brief acid treatment (see M&M). After being washed, the cells were then returned to 37°C to allow expression of new molecules, either in tissue culture medium only, or in the presence of the indicated drugs. After 6 h, cell surface expression of the transfected RT1-E2u was measured by flow cytometric analysis after staining with MRC OX-18 (striped columns), or of endogenous H2-Kk with 11-4-1 (black columns). Values used are based on means of fluorescence intensity, expressed as a percentage of re-expression compared to the cells recovering in tissue culture medium alone.
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Figure 7: Transport of RT1-E2u molecules to the cell surface is affected by endoprotease inhibitors, and follows a similar pathway to that of the H2-Kk class Ia molecule. MHC molecules present at the surface of transfected L cells were denatured by a brief acid treatment (see M&M). After being washed, the cells were then returned to 37°C to allow expression of new molecules, either in tissue culture medium only, or in the presence of the indicated drugs. After 6 h, cell surface expression of the transfected RT1-E2u was measured by flow cytometric analysis after staining with MRC OX-18 (striped columns), or of endogenous H2-Kk with 11-4-1 (black columns). Values used are based on means of fluorescence intensity, expressed as a percentage of re-expression compared to the cells recovering in tissue culture medium alone.

Mentions: The cellular requirements for RT1-E2 expression were investigated as follows: correctly conformed MHC class I molecules were first removed from the surface of the L cell transfectants by means of a brief acid treatment (pH 3.0). Subsequent recovery of class I expression was measured after six hours of culture in the presence or absence of various inhibitors, using MRC OX-18 to detect RT1-E2 and mAb 11-4-1 to detect H2-Kk (endogenous to L cells). As can be seen in Fig. 7, incubation of the cells with drugs that inhibit either many of the cellular proteases (MG-101) or more specifically the proteasome (MG-132), the secretion pathway upstream of the Golgi apparatus (brefeldin A) or transport through the Golgi apparatus (monensin) resulted in each case in a significant reduction of the re-appearance of both RT1-E2u and H2-Kk at the cell surface. The observation that the treatment with MG101 resulted in a more dramatic inhibition of RT1-E2 expression than that of H2-Kk suggests that, in addition to the proteasome, the generation of peptidic ligands suitable for binding to the RT1-E2 molecules may require the contribution of additional proteases that would be very sensitive to this drug.


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)

Transport of RT1-E2u molecules to the cell surface is affected by endoprotease inhibitors, and follows a similar pathway to that of the H2-Kk class Ia molecule. MHC molecules present at the surface of transfected L cells were denatured by a brief acid treatment (see M&M). After being washed, the cells were then returned to 37°C to allow expression of new molecules, either in tissue culture medium only, or in the presence of the indicated drugs. After 6 h, cell surface expression of the transfected RT1-E2u was measured by flow cytometric analysis after staining with MRC OX-18 (striped columns), or of endogenous H2-Kk with 11-4-1 (black columns). Values used are based on means of fluorescence intensity, expressed as a percentage of re-expression compared to the cells recovering in tissue culture medium alone.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC183868&req=5

Figure 7: Transport of RT1-E2u molecules to the cell surface is affected by endoprotease inhibitors, and follows a similar pathway to that of the H2-Kk class Ia molecule. MHC molecules present at the surface of transfected L cells were denatured by a brief acid treatment (see M&M). After being washed, the cells were then returned to 37°C to allow expression of new molecules, either in tissue culture medium only, or in the presence of the indicated drugs. After 6 h, cell surface expression of the transfected RT1-E2u was measured by flow cytometric analysis after staining with MRC OX-18 (striped columns), or of endogenous H2-Kk with 11-4-1 (black columns). Values used are based on means of fluorescence intensity, expressed as a percentage of re-expression compared to the cells recovering in tissue culture medium alone.
Mentions: The cellular requirements for RT1-E2 expression were investigated as follows: correctly conformed MHC class I molecules were first removed from the surface of the L cell transfectants by means of a brief acid treatment (pH 3.0). Subsequent recovery of class I expression was measured after six hours of culture in the presence or absence of various inhibitors, using MRC OX-18 to detect RT1-E2 and mAb 11-4-1 to detect H2-Kk (endogenous to L cells). As can be seen in Fig. 7, incubation of the cells with drugs that inhibit either many of the cellular proteases (MG-101) or more specifically the proteasome (MG-132), the secretion pathway upstream of the Golgi apparatus (brefeldin A) or transport through the Golgi apparatus (monensin) resulted in each case in a significant reduction of the re-appearance of both RT1-E2u and H2-Kk at the cell surface. The observation that the treatment with MG101 resulted in a more dramatic inhibition of RT1-E2 expression than that of H2-Kk suggests that, in addition to the proteasome, the generation of peptidic ligands suitable for binding to the RT1-E2 molecules may require the contribution of additional proteases that would be very sensitive to this drug.

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