Novel MHC class I-related molecule MR1 affects MHC class I expression in 293T cells

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Biochemical and Biophysical Research Communications 366 (2008) 328–334 www.elsevier.com/locate/ybbrc

Novel MHC class I-related molecule MR1 affects MHC class I expression in 293T cells Hatice Aldemir

1

Institut de Pharmacologie Mole´culaire et Cellulaire, CNRS UMR6097, Valbonne 06560, France Received 1 November 2007

Abstract Association with b2-microglobulin and binding a ligand are necessary conditions for cell surface expression of the antigen presenting molecules. MHC class I-related protein, MR1, is suggested to have an antigen presentation function, nevertheless the physiological ligand(s) is (are) still to be determined. In the present study, by characterising the subcellular deportment of human MR1 transfectants, we have shown its differential mobilisation. Our results demonstrated a preferential association of MR1 with b2-microglobulin in MHC class I-deficient B cell lines. Furthermore, we have evidenced diminished expression of classical MHC class I molecules in human MR1transfected 293T cells, showing a possible interaction between MR1 and classical MHC class I molecules.  2007 Elsevier Inc. All rights reserved. Keywords: MR1; MHC class I; b2m

Major histocompatibility complex (MHC) class I molecules play a key role in immune responses by presenting peptides from both self and foreign intracellular proteins. MHC class I complex, which consists of a polymorphic glycosylated heavy chain (HC), a non-polymorphic b2-microglobulin (b2m), and a peptide, travels to the cell surface in most nucleated cells to be recognised by T and NK cells [1]. Novel MHC class I-related protein 1 (MR1) has been shown to associate with b2m [2,3] and it is the first nonMHC encoded class I-like molecule interacting with the peptide-loading complex [3]. A subset of CD4 CD8 double negative T cells with an invariant T cell receptor (TCR) was shown to be selected by MR1 molecule [4]. These MR1-restricted invariant T cells, which was named as Mucosal-associated Invariant T (MAIT) cells, secrete IFN-c, IL-4, IL-5, and IL-10 in response to TCR ligation [5].

E-mail address: [email protected] Present address: Faculte´ de Pharmacie, Universite´ Paris-Sud XI, Chaˆtenay-Malabry 92296, France. 1

0006-291X/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.11.156

MR1 was proposed to present either a microbe-produced or microbe-induced molecule [6]. Evidence was also presented to support that glycolipids can serve as ligands for MAIT cells [7]. Thus the objective of the present study was to ascertain if human (h)MR1 features the necessary conditions for antigen presentation in different cell lines. Subcellular localisation of the hMR1 fusion proteins was evaluated. Association with b2m in MHC class I negative B cell lines and uniform intracellular localisation were demonstrated. Noteworthy, diminished classical MHC class I expression on hMR1-GFP-transfected cell lines was observed. Materials and methods Cell lines and antibodies. Human renal cell line 293T-HEK (293T), HLA-A, -B, -C, and -G negative cell line 721.221 (221), B cell line JY, human cervical adenocarcinoma cell line HeLa and monkey kidney cell line COS-7 were maintained in RPMI 1640 (Cambrex) containing 10% foetal calf serum (HyClone), 100 U/ml P/S, 2 mM L-glutamine and 1.25 mM HEPES (Gibco). The mouse anti-GFP mAb was from Qbiogen and anti-hb2m-HRP and anti-mouse-HRP were from DAKO. Conformational pan anti-human MHC class I antibody W6/32, anti-human b2m ab Bbm.1, HLA-B and

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HLA-C HC-specific ab HC10 and HLA-A HC-specific ab HCA2 were kindly provided by Dr. V. Braud. DNA constructs and fusion proteins. Human MR1 was PCR amplified from the MR24 plasmid (from Dr. S. Bahram, ULP, Strasbourg) by using the following primers: FhMR1GFP: 5 0 -GATATAAGCTTGCCGCCAC CATGGGGGAACTGATG-3 0 ; RhMR1GFP: 5 0 -ATATCGGTACCCCT CGATCTGGTGT-3 0 . Human MR1-GFP inserts were ligated into the neomycin-resistant pEGFP-N1 plasmid (Clontech). Human b2m-expressing hygromycin-resistant plasmid pcDNA3.1-Hyg-hb2m (a kind gift from Dr. G. Lauvau, IPMC) and the ligation products encoding either only GFP or hMR1-GFP sequences were verified by sequencing and were introduced into cells by Ca2PO4 transfection or electroporation. Protein expression was checked by FACS Calibur. Positive cells were FACS sorted and selected in Neomycin (Biowest) or Hygromycin (Invitrogen). FACS data from cell staining were analysed by CellQuestPro (Becton–Dickinson) software. Metabolic labelling, immunoprecipitation (IP), electrophoresis and Western blotting. Cells (107 cells/ml) were labelled with 35S-Met/Cys labelling mix (ICN Biomedicals) at 7.4 MBq/ml, washed in PBS and lysed on ice. Supernatants were collected after centrifugation and cell lysates were precleared by Pansorbin (Calbiochem). Samples were centrifuged and 10 lg antibody per 5 · 106 cells was added into clean tubes. After incubation on ice, Protein A sepharose beads (Amersham) were added. Formed immunocomplexes were collected and boiled in SDS sample buffer and were either treated by endo-b-N-acetylglucosaminidase (EndoH) (Roche) or left untreated. Samples were analysed by SDS–PAGE and gels were either dried and exposed to the films or blotted onto nitrocellulose membranes. Blots were incubated with antibodies and developed with Super Signal (Pierce). Laser scanning confocal microscopy (LSCM). Cells were grown on poly-L-lysine treated coverslips in 12-well plates at a 50–60% confluency. They were fixed with para-formaldehyde for 30 min and then blocked with serum before application of antibodies at suitable concentrations. Cells were permeabilised using 0.5% saponin (Sigma) for intracellular staining. Calnexin, Syntaxin-6 and Lamp-1 mAbs were from Transduction Laboratories. W6/32 antibody or surface biotinylation was used for surface marking by using the membrane non-permeable biotin derivative (Pierce). Anti-mouse-AlexaFluor568 or streptavidin-AlexaFluor568 (Molecular Probes) were used as secondary antibodies.

Results hMR1-GFP is localised in the endoplasmic reticulum (ER) in 293T and HeLa cells while moving into Golgi in class I-deficient 221 B cell line We investigated MR1 intracellular deportment by producing GFP fusion proteins. Subcellular localisation of the hMR1-GFP fusion protein was analysed by IP followed by Endo-H treatment. Endo-H hydrolyses N-glycans of the high mannose type, which are present in the ER and cis-Golgi. Movement from ER/early Golgi renders glycoproteins resistant to Endo-H treatment. In 293T-hMR1GFP cells, following Endo-H digestion, hMR1-GFP was found to migrate faster, indicating an ER/early Golgi localisation (Fig. 1A). Further evidence was sought for an ER localisation of hMR1-GFP by employing colocalisation experiments in hMR1-GFP-transfected 293T, HeLa, and MHC class I-deficient cell line 221. LSCM of transfected cells, costained for the ER marker calnexin, showed that hMR1-GFP was retained in the ER of 293T and HeLa cells, however no colocalisation was observed in 221 cells (Fig. 1B). Calnexin and

Fig. 1. Subcellular localisation of hMR1-GFP in the ER and Golgi. (A) Human MR1-GFP is Endo-H sensitive. IP with anti-GFP followed by Endo-H treatment shows the sensitivity and ER localisation of hMR1. (B) Intra-ER localisation of hMR1-GFP-transfected cells by LSCM. Calnexin colocalises with hMR1-GFP in the ER of 293T and HeLa cells but not in 221 cells. (C) Golgi marker Syntaxin-6 colocalises with hMR1-GFP in 221-hMR1-GFP but not in 293T-hMR1-GFP cells.

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calreticulin are lectin chaperons that interact with and assist in the folding of proteins carrying monoglucosylated Nlinked glycans [8]. Classical MHC class I HCs first associate with calnexin and acquire calreticulin assembly only after b2m is bound [1]. Overall, extended visualisation of MR1GFP molecules with calnexin in 293T and HeLa cells, minimal colocalisation in 221 cells (Fig. 1B) and calreticulin colocalisation at low levels only in 221 cells (data not shown) were observed. These suggested that hMR1-GFP was retained in the ER at a stage before association with b2m in 293T and HeLa cells but association with b2m seemed likely in class I-deficient B cell line 221. Golgi localisation of hMR1-GFP was then checked since b2m association is followed by egress from ER. Colocalisation of hMR1GFP with Golgi marker syntaxin-6 is observed in 221 cells but not in 293T cells displaying its movement into Golgi in class I-deficient B cell line (Fig. 1C). hMR1 associates with b2m in class I-deficient 221 cells Association with b2m was evaluated by IP experiments with anti-hb2m and anti-GFP antibodies. Association of hMR1-GFP HC with b2m was observed in 221 cells only (Fig. 2). To determine whether b2m levels were the limiting factor for HC association, we compared the levels in whole cell extracts from 293T, 221 and HeLa cells (Fig. 2) and also from B cell line JY and monkey kidney cell line COS-7. Less b2m expression in 293T cells was observed. To exclude the possibility that non-specific or altered association with b2m in the absence of class I molecules was occurring in 221 cells because of more free b2m available, we transfected the 293T and HeLa cells with hb2m-expressing pcDNA3.1-Hyg-hb2m plasmid. Increased hb2m expression was observed in these cells. However, IP experiments with anti-GFP did not reveal any association of b2m with hMR1-GFP in either cell line (Fig. 2A and B). To determine whether the expression of hMR1-GFP affects the association of MHC class

I HC with b2m in 293T cells, W6/32 mAb was used in the IP experiments (Fig. 2C). Monoclonal ab W6/32 recognises complexes consisting of class I molecules and b2m [1,9] and the presence of such complexes in the wild-type 293T cells were observed by WB analysis, while this association was impaired in hMR1-GFP-expressing cells (Fig. 2C). Hence, a possible effect of hMR1-GFP expression on the association of class I HC with b2m is suggested. MHC class I expression is impaired in hMR1-GFPtransfected cells MHC class I coexpression was then analysed in hMR1GFP transfectants. Whole cell lysates were loaded on gels and transferred onto a membrane which was then blotted either with HLA-B and HLA-C HC-specific HC10 or HLA-A HC-specific antibody HCA2 (Fig. 3A). Both antibodies revealed lower levels of class I molecules in hMR1GFP-transfected cells. Consistent with the WB results, LSCM visualisation after a surface staining with W6/32 antibody proved lower surface class I intensity in cells expressing hMR1-GFP (Fig. 3B). Additional proof was obtained by evaluating the mean fluorescence intensity (MFI) on FACS following surface staining with W6/32 (Fig. 3C). These results indicate lessened MHC class I surface expression in hMR1-GFP cells, while staining profiles in non-transfected and GFP-transfected cells were not affected. hMR1-GFP recycles to endosomes but does not advance to the cell surface Endosome marker Lamp-1 was used to examine endosomal recycling of hMR1-GFP. Despite the fact that it did not associate with b2m in 293T and HeLa cells, hMR1-GFP was visualised in the endosomal compartments (Fig. 4A). However, no surface expression of

Fig. 2. Human MR1-GFP fusion protein associates with b2m only in class I negative cell line 221. IP of cell lysates from (A) 221 and HeLa cells, (B) 221 and 293T cells (C) 293T cells with different antibodies show b2m association only in MHC class I-deficient 221 cells.

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Fig. 3. hMR1-GFP impairs the normal MHC class I expression in 293T cells. (A) Total MHC class I expression is reduced in MR1-GFP-transfected cells. Equal quantity of total protein from cell lysates were separated and probed either with HC10 (recognises HLA-B and HLA-C HC) or with HCA2 (recognises HLA-A). Relatively lower levels of MHC class I HC were detected in the 293T-hMR1-GPF transfectants. (B) Diminished MHC class I expression was also evidenced by LSCM. MR1-expressing cells (green) show lower surface MHC class I (red) staining. (C) FACS staining with pan antihuman MHC class I antibody W6/32 confirmed impaired MHC class I surface expression in 293T-hMR1-GPF cells. (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)

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Fig. 4. Human MR1-GFP moves through endosomal compartments but does not advance to the cell surface. Human MR1-GPF recycling to endosomal compartments is shown by Lamp-1 colocalisation (A). Human MR1-GFP does not advance to the cells surface as shown by surface biotinylation followed by streptavidin-coupled secondary antibody (B).

hMR1-GFP could be observed in any of the biotinylated cell lines visualised by LSCM in colocalisation experiments (Fig. 4B) or in IP experiments of surface proteins by streptavidin-coupled antibodies. Discussion The function of the MR1 molecule has yet to be identified, and arguments about its antigen presenting role are

intriguing [3,4]. Some MHC class I-related molecules form heterodimers consisting of HC and b2m without an antigen presentation function [10,11]. MR1 protein was interpreted to be atypical in the manner in which it interacts with b2m [3]. The data presented here suggested differential association of hMR1-GFP with b2m. In its heterodimer form hMR1-GFP was observed to enter into ER exit sites in 221 cells and visualised in budding vesicles. Overall, in 221 cells in the absence of MHC class I molecules,

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hMR1-GFP associated with b2m. In turn, whilst awaiting for the supposed ligand, this complex associated with TAP1/2 (data not shown). On the other hand, in 293T cells, in the presence of MHC class I molecules, which readily bind b2m, hMR1-GFP did not show this association. Concurrent to this, relatively weak b2m association of MR1 was evidenced by increased MR1 cell surface expression when a3 domain was swapped with that of a classical class I molecule [3]. Murine MHC class Ib molecule H2-M3 binds a highly restricted pool of peptides, resulting in its intracellular retention under normal conditions [12]. Similar to the current results with MR1-transfected cells, trafficking of MHC class Ia molecules and H2-M3 were shown to differ in distinct human cell lines [13]. In H2M3-transfected HeLa cells exogenous peptides mobilise MHC class Ia molecules and H2-M3 from the ER but only MHC class Ia molecules are mobilised in the heavily mutagenized lymphoblastoid cell line 221 [13]. A possibility of MR1 to bind both TAP-dependent and TAP-independent ligands was already proposed [3] and surface expression of MR1 was shown only in transfected Hom2 cells (human B cell lymphoblast) while in RBL cells (rat basophilic cell line) surface expression was induced sole after PMA-Ionomycin treatment [4], confirming its differential behaviour. MR1 molecule is ubiquitously expressed at the mRNA level in human [14], mouse [15,16] and rat tissues [17] and in many human cell lines [16]. Although endogenous expression at the protein level has not been demonstrated yet [2,3], low levels of epitope-tagged mMR1 surface expression was shown in transfected HeLa cells, still most of the protein remaining intracellular [3]. Even so, in the present study, experiments could not corroborate cell surface expression of neither hMR1-GFP fusion protein nor tagged hMR16His molecules in different transfected cell lines by different methods. This piece of data is in accordance with the mMR1-P388 cells consistently staying intracellular [2]. Complementary experiments were performed to reveal the ligand of MR1 by attempting to induce cell surface expression of hMR1-GFP. Different stress conditions, intracellular and extracellular pathogens and pathogen associated molecules were tested without success to induce surface mobilisation. Not withstanding with this, endogenous protein surface expression could not be shown on ex vivo lymphocytes from different organs even after activation with various mitogens [18]. These data point to a stringent regulation of MR1 expression. A peptide presentation role is suggested for MR1 [3,4,19], however its similarities to non-classical molecules interacting with immune receptors without a ligand are not negligible [10,20–22]. While the soluble Zinc-a2-glycoprotein (ZAG), a lipid mobilising factor, is expressed and secreted by human adipocytes [21], another class I-related molecule, hereditary haemochromatosis protein HFE (HLA-H) is expressed on polarised epithelial cells throughout the gastrointestinal tract [23]. Likewise, cell stress regulated MHC class I-related chain A (MICA) is expressed in

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the gastrointestinal epithelium [22]. A prominent feature of intestinal epithelium is the expression of a wide range of non-classical class I molecules that in addition to the classical ones, enable them to transit lumenal signals to lamina propria (LP) [24]. On the basis of the shown placement of MAIT cells in the gut LP [4], one would expect the selecting molecule MR1 to be expressed on intestinal epithelial cells. However MR1 protein has not been shown yet in these polarised cells, it could participate in mucosal crosstalk between gut-associated lymphocytes and intestinal epithelial cells. In this crosstalk, luminal-basolateral interactions play an important role [24]. Basolateral sorting of some transmembrane proteins depend on a sorting signal within their cytoplasmic domains [25]. Relatedly, MR1 contains a few LV-like sequence in its cytoplasmic domain (GenBank Accession No. AAC50174, http://www.ncbi.nlm. nih.gov/entrez/viewer.fcgi?db=protein&id=940354) and a similar motif was reported to be responsible for MICA localisation to the basolateral side of the polarised cells [22]. However it needs to be proved, one could speculate that MR1 might have a physiological function at the basolateral side similar to iron metabolism implicated HFE that form a high affinity complex with transferrin receptor at the pH of the cell surface [11]. Parallel to shown hMR1-GFP endosome localisation without advancing to the cell surface, HFE-GFP recombinant molecules were shown to follow initially HLA class I intracellular processing but colocalised with transferrin in early endosomes without recycling at the cell surface [23]. In addition, on IP experiments followed by mass spectrometry assays, hMR1-GFP association with sideroflexin-1 (Sfxn1) was also determined. Sfxn1 is a mitochondrial multiple transmembrane protein that is implicated in iron transport [26]. It is postulated that Sfxn1 facilitates the transport of a component required for iron utilisation into or out of mitochondria [26]. Supposedly, if MR1 is involved in this kind of physiological regulation, its surface expression could be a marker of cellular dysfunction/distress that in turn would result in eradication by MAIT cells. T–T hybridomas recognising MR1 directly without a bound ligand [4] could be considered as evidence for this. Pathogenesis of several neurodegenerative diseases including multiple sclerosis is associated with mitochondrial dysfunction [27]. Impaired intra-mitochondrial metabolism with increased free iron levels can recruit reactive T cells. MAIT cells were shown to be accumulated in some of the central nervous system lesions of MS and in the majority of the peripheral nerve samples from Chronic Inflammatory Demyelinating Polyneuropathy (CIDP). This reflects a role for MR1-restricted human invariant T cells in autoimmune inflammatory lesions [28] and corresponds well with rapid and diverse cytokine response by this innate T cell population [5]. Likewise, overrepresentation of this invariant T cell population was also shown to decrease the severity of experimental autoimmune encephalomyelitis (EAE) in mice by reducing the production of proinflammatory cytokines [7]. In conclusion, rigorously controlled expression of evolutionary conserved MR1 protein [15–17], persistent intracel-

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lular localisation of the transfectants and observed differences in MHC class I molecule expression in the presence of MR1 transfectants are in agreement with certainly significant role(s) of MR1 molecule in physiological and/or pathological processes that need to be investigated more profoundly. Acknowledgments This work was supported by a grant from Sidaction and would not be possible without the help of Dr. V. Braud. I would like to thank Dr. H. Tinwell for critical reading of the manuscript and Dr. G. Lauvau for helpful discussions.

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