The non-classical HLA class I molecule HFE does not influence the NK-like activity contained in fresh human PBMCs and does not interact with NK cells

Steve Pascolo1, Florent Ginhoux2, Nihay Laham3, Steffen Walter1, Oliver Schoor1, Jochen Probst1, Pierre Rohrlich4,5, Florian Obermayr1, Paul Fisch6, Olivier Danos2, Rachel Ehrlich3, Francois A. Lemonnier4 and Hans-Georg Rammensee1

1 Department of Immunology, Auf der Morgenstelle 15, 72076 Tübingen, Germany
2 Genethon CNRS UMR 8115, 1 bis rue de l'Internationale, BP60, Evry Cedex 91002, France
3 Department of Cell Research and Immunology, Tel Aviv University, Tel Aviv, Israel
4 Immunité Cellulaire Antivirale, Institut Pasteur, 28 rue du Dr. Roux, 75715 Paris, France
5 Hospital Robert Debré, 75019 Paris, France
6 Pathologisches Institut, University of Freiburg, Freiburg, Germany

Correspondence to: S. Pascolo; E-mail: steve.pascolo{at}uni-tuebingen.de


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Supplementary data
 References
 
In humans, four ß2-microglobulin-associated non-classical class I molecules are encoded in the MHC: HLA-E, -F, -G and -H. Three of them (HLA-E, -F and -G) were shown to inhibit NK activity. On the contrary, the fourth one, HLA-H, named HFE after it was found to be mutated in patients suffering from inherited hemochromatosis, has been shown to be involved only in the regulation of iron uptake. We tested the capacity of HFE to affect (enhance or reduce) specifically the NK activity contained in non-manipulated fresh human PBMCs. We showed that HFE expression by target cells does not affect their killing by the NK-like activity contained in PBMCs. Moreover, using fluorescent HFE tetramers, we could confirm that blood NK cells as well as blood {gamma}{delta} T cells do not bind HFE. Altogether, our data indicate that HFE does not affect the NK activity contained in the PBMCs.

Keywords: HFE, iron metabolism, NK cells, non-classical MHC class I, tetramer


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Supplementary data
 References
 
With the noticeable exception of H2-M3a, which is a mouse MHC non-classical class I molecule that presents formylated bacteria-derived peptides to T lymphocytes (1) and H2-T22 which is recognized by {gamma}{delta} T cells (2), all the non-classical class I molecules (3) for which an immunological function was found [HLA-E (4), -F (5) and -G (6), the NKG2D-ligand family (7) and H2-Qa-1 (8)] are involved in the control of innate immunity: macrophages, monocytes and in particular NK cells. HLA-H, renamed HFE after it was found to be mutated in most cases of hereditary hemochromatosis (9), is known to interact with the transferrin receptor (TfR), lowering its affinity for the iron-loaded transferrin and thus reducing iron uptake (10). To date, HFE has not been reported to have specific immune functions. Nevertheless, at least two observations highlight a possible link between HFE and immunity: (i) the human cytomegalovirus (HCMV) US2 protein that is involved in the immune escape of HCMV by inducing the degradation of MHC class I molecules is also able to selectively prevent HFE expression by targeting the nascent chain to the proteasome-mediated degradation pathway (11, 12) and (ii) the number of CD8 T lymphocytes is often reduced in HFE-deficient patients (13). In addition, indirect evidences such as hepatic iron overload in TCR delta knockout mice suggested that HFE is recognized by {gamma}{delta} T cells (14, 15). Based on these observations and the known role of most non-classical MHC class I molecules in controlling NK killing, we studied the potential of HFE to affect NK-like activity contained in unmanipulated PBMCs and the capacity of HFE fluorescent tetramers to specifically label some cell populations in the blood. Our results indicate that HFE does not specifically interact with any receptor on PBMCs other than the TfR and that it does not inhibit or activate the spontaneous NK activity contained in PBMCs.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Supplementary data
 References
 
Media and cell culture
All cells were cultivated in RPMI 1640 (Bio-Whittaker, Verviers, Belgium) complemented with 10% of heat-inactivated FCS (PAN Systems, Germany), 2 mM L-glutamine, 100 U ml–1 penicillin and 100 µg ml–1 streptomycin. The cell lines 1257, 1795, 1973 and KNP were kindly provided by Elke Jäger.

Antibody cell staining
Cells were incubated with purified B2.62.2 (anti-hß2m) or 2F5 (anti-HFE) mAbs and, after washes, incubated with an FITC-labeled goat anti-mouse IgG polyclonal serum. Stained cells were analyzed on a FACSCalibur (Becton Dickinson).

Preparation of the HIV-based vectors and infection of HCT cells
For the details of cloning, see Supplementary Data (available at International Immunology Online). Recombinant genes coding HFE monochain, H63D monochain and human ß2-microglobulin (hß2m) under the control of the 500-bp HLA-A*0201 promoter were cloned in the pSIN.PW HIV-1-derived transfer vector (16). Lentiviral transfer vector particles were separately produced as previously described (17). Transduction experiments were carried out by adding serial dilutions of vector particles to 105 HCT cells in a 12-well plate in the presence of polybrane (8 µg ml–1). After 48–72 h, HCT cells were analyzed by FACS for the expression of the transgene. Positively stained cells were purified by cell sorting on a MoFlow (DakoCytomation, Freiburg, Germany) and cloned by limiting dilution. HFE- or hß2m-expressing clones were confirmed by antibody staining and used as targets in cytotoxic assays.

High-density oligonucleotide microarray analysis
For the details of microarray analysis, see Supplementary Data (available at International Immunology Online). Total RNA samples from human tissues were obtained commercially (Ambion, Huntingdon, UK; Clontech, Heidelberg, Germany; Stratagene, Amsterdam, The Netherlands). Tumor (RCC**T) and healthy (RCC**N) kidney samples were obtained from operated renal cell carcinoma patients. The local ethical committee approved this study (272/2000) and informed consent was obtained from the patients. Total RNA from isolated cells, leukocytes, human aortic epithelial cells, human aortic smooth muscle cells, DCs, CD3+ or CD19+ cells or kidney samples, was isolated using Trizol extraction (Invitrogen, Karlsruhe, Germany). RNA quality and quantity was confirmed on the Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 Pico LabChip Kit (Agilent).

Hybridization on Affymetrix U133A GeneChips (Affymetrix, Santa Clara, CA, USA) was done according to the manufacturer's protocols. Significance of detection was judged by the ‘present’ values given by the statistical algorithms implemented in the Microarray Analysis Suite 5.0 software.

Chromium release assay
PBMCs were prepared from fresh heparinized blood using centrifugation on Ficoll. The cells contained in the interface were harvested, washed once with PBS, counted and incubated at different effector/target ratios in triplicate for 6 h with 51Cr-labeled target cells (5000 target cells per well in a 96-well plate). The percentage of killing was determined from the amount of 51Cr released in the medium (A) compared with spontaneous release of target cells (B) and total 51Cr content of 1% Triton X-100-lysed target cells (C) using the formula: % lysis = (A – B)/(C – B) x 100.

Production of soluble HFE monomers
The soluble HFE monomers were based on a previously described soluble HFE–ß2-microglobulin chain (18). For the details of generation, production and purification by HEK293 cells of HFE monomers, see Supplementary Data (available at International Immunology Online).

Tetramer staining
Soluble HFE monochains were biotinylated before fast protein liquid chromatography (FPLC) gel filtration purification by the enzyme BirA ligase (Avidity, Denver, CO, USA) and tetramerized using streptavidin–allophycocyanin (APC) (Molecular Probes).

For flow cytometric analysis, 1 million PBMCs were stained at 4°C with APC-labeled HFE tetramers (at the final concentration of 10 µg ml–1 in PBS containing 50% FCS in a volume of 100 µl) together with CD56 (PE), gdTCR (FITC), CD19 (peridinin–chlorophyll–protein complex) and CD71 (FITC) antibodies (BD PharMingen, Heidelberg, Germany) according to the manufacturer's instructions. After 30 min incubation, the cells were washed twice with PBS, fixed with PBS containing 1% PFA, re-suspended in PBS and analyzed on a FACSCalibur (BD PharMingen).


    Results and discussion
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Supplementary data
 References
 
HFE expression in normal and tumor cells
Using an HFE-specific mouse mAb [2F5 (11)] generated by hyper-immunization of mice with syngeneic cells expressing human HFE, we studied the expression of HFE on the cell surface of tumor cell lines by FACS analysis (Fig. 1) or on solid tumors by immunohistochemistry. As shown in the upper panels of Fig. 1(A), the mAb 2F5 recognizes the hß2m-deficient colon carcinoma cell line HCT when it stably expresses either the wild-type HFE or its H63D mutant. Both recombinant HFE molecules are monochains where the HFE heavy chain is linked to the hß2m by a 15-amino acid linker, thus allowing surface expression of HFE but not restoring endogenous HLA class I expression in ß2m-deficient cells (19). H63D is a natural mutant version of HFE that is found in a lower percentage of hemochromatosis patients than the C282Y mutant (20) but that can egress out of the endoplasmic reticulum. No expression of HFE was detected on most tumor cell lines (Fig. 1A: kidney carcinoma cell lines 1795 and 1973, EBV B cell line KNP or prostate cancer cell line DU145 for example). Staining of these and many other classical MHC class I-positive (expressing a functional hß2m) non-transformed or transformed cells showed the same results even when the labeling was performed on permeabilized cells (using saponin pre-treated cells, data not shown). There was also no specific staining detectable on tumor (kidney and colon) cryosections using 2F5 (data not shown). Only one cell line among those that we studied, the colon carcinoma cell line HT29, showed a detectable natural cell-surface expression of HFE (Fig. 1A). This analysis would indicate that cancer cell lines can express HFE at the cell surface but do so very rarely. Since 2F5 is known to recognize an epitope on HFE that is hidden or destroyed when HFE is associated with TfR (11), the antibody staining may underscore the presence of HFE at the cell surface. Consequently, we decided to investigate HFE expression by genetic tools. Using microarray technology, we studied the presence of the mRNA coding for the full-length HFE in different cells and organs, including healthy and tumor kidney tissues (Fig. 1B). HFE-coding mRNA was detected as present according to the analysis software (the algorithm ‘Microarray Analysis Suite 5.0 software’) in all samples except for the liver. For this reason, no relative expression value can be given for liver: it is not listed in Fig. 1(B). Relative expression was similar in all those samples (the chips are normalized to 100 genes). The overall signal value for HFE is between 20 and 30. Compared with a housekeeping gene expressed in all cells such as glyceraldehyde-3-phosphate dehydrogenase, which has an overall signal value of ~2000–8000, or a transcription factor such as nuclear factor {kappa}B1, that has an overall signal value of 150–300, we can estimate that the HFE gene is weakly transcribed in all cells or expressed in only few cells (in the case of chips hybridized with the total RNA extracted from a tissue). The expression of HFE does not vary significantly between healthy kidney and tumor tissues. This suggests that over-expression of HFE may not be a mechanism of immune suppression. Similar results are obtained with the 12 different splice variants of HFE addressed by the chip which codes for parts of the HFE protein that have no known functions. We conclude from this large expression study that HFE is expressed in a variety of tissues but is not or rarely over-expressed in tumors. Nevertheless, the results obtained with HT29 indicate that cell-surface expression of TfR-free HFE is achieved in some tumor cells. This stimulated our search for a role of HFE in the recognition of target cells by the immune system.



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Fig. 1. Expression of HFE in transfected cells and tumor cell lines. (A) HCT cells expressing HFE or HFE-H63D monochains or hß2m were stained with the HFE-specific antibody 2F5 (upper panels: thick lines; the filled gray histogram corresponds to 2F5-stained HCT cells). The histogram indicates that 2F5 recognizes both the HFE monochain and its H63D mutant. Yet, untransfected renal cell carcinoma lines (1795 or 1973), EBV cell lines (KNP) or the prostate cancer cell line DU145 did not show any staining with 2F5 (lower panels: the thin line corresponds to an isotype control staining and the thick line to the staining with 2F5). During such a screening, the only tumor cell line that showed a positive cell-surface staining with 2F5 was the colon cancer cell line HT29. (B) The relative mRNA expression of HFE in different cells, tissues and pairs of healthy and tumor kidney tissues was analyzed by microarrays. HFE is expressed but the level of expression does not change significantly in-between cells and organs or in-between the tumor tissues and the corresponding (from the same organ and patient) healthy tissues. ‘Small’ stands for small intestine and ‘skeletal’ stands for skeletal muscle.

 
HFE expression and NK activity from fresh PBMCs
The impact of HFE expression on the NK activity contained in fresh PBMCs was tested in a cytotoxic assay. The target cells used in this assay were either the parent hß2m-deficient, HLA class I-negative tumor cell line (HCT) or the transfected versions of this line re-expressing HLA class I (upon expression of a transgenic hß2m gene) or the HFE–ß2-microglobulin monochain. The advantage of such a monochain is that the HFE expression can be obtained at the surface of hß2m-deficient tumor cells without allowing re-expression of classical MHC class I molecule (19). Since HFE by interacting with the TfR decreases iron uptake, the stable expression of HFE for several weeks was lethal in HCT and other hß2m-deficient cell lines that we tested (data not shown). We could not obtain clones that stably express HFE using classical expression vectors and selective media: such clones could not expand and died after a few weeks of culture or lost the transgene expression. Up to now, the only human cell line that was found to be able to grow with stable transgenic expression of the human HFE is HEK293 (12). This cell line expresses a functional hß2m and classical MHC class I molecules; it cannot be used as target to study NK activity. Consequently, for HFE expression in HCT cells we used an expression system based on transduction of cells with lentivirus-derived recombinant vectors encoding the transgene of interest (Fig. 2A). This technology allows high efficiency transduction and does not require long-term culture under selection media for the isolation of transfected clones. As shown in Fig. 2, the genes coding for the hß2m or for the HFE monochain as well as its H63D mutated version were cloned in-between HIV-1 long terminal repeats in a ‘transfer vector’. These plasmid constructs were transfected together with ‘packaging’ and ‘envelope’ plasmids into 293T cells (see Methods) and the recovered infectious particles were used to transduce HCT cells. The transduced cells expressed the transgenic molecules as detected by anti-hß2m antibody staining (Fig. 2B) as well as by HLA class I (W632 in the case of hß2m-expressig HCT) or HFE (2F5 in the case of HFE-expressing cells) specific staining (data not shown). As presented in Fig. 2(C), when such HFE- or hß2m-expressing cells were used as target cells in cytotoxic assays, only the re-expression of HLA class I molecules in HCT/hß2m transfectants and not the expression of HFE or H63D-HFE could protect the cells against NK-like killing activity contained in PBMCs (mediated by NK, NKT and {gamma}{delta} T cells). This experiment shows that HFE surface expression probably does not modulate the NK type of activity contained in fresh PBMCs.



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Fig. 2. HFE expression and NK activity. (A) The three DNA constructs that were generated to be encapsulated in lentiviral-derived particles used to infect HCT cells. The arrow represents the HLA-A*0201 promoter and the thick line between the hß2m and the mature HFE (HFEm) gene segments represents the 15-amino acid linker (19). (B) The analysis of the infected cells by FACS after staining with the hß2m-specific mAb B2.62.2 (thick lines, the filled gray histograms correspond to staining with an isotype control) shows that all cells express the expected transgenic molecules at similar levels (C) When used as target cells in a cytotoxicity assay with PBMCs as effector cells, the non-transduced (MHC class I deficient) HCT cells (circles) are killed by the NK activity contained in fresh PBMCs. On the contrary and as expected, the HCT cells transduced with the particles that deliver the hß2m gene (squares) are resistant to such killing. Both HCT cells expressing the wild type or the H63D mutant of the HFE monochain (diamonds and triangles, respectively) are killed by fresh PBMCs similar to non-infected HCT cells. This experiment shows that HFE surface expression does not inhibit or activate the NK activity contained in fresh PBMCs.

 
Since a receptor that would recognize HFE may be expressed only by few cells capable of NK-type killing (recognition of HLA class I-negative cells), the recognition of HFE-expressing cells by NK clones should be studied in order to conclude that no HFER is expressed on cells capable of NK activity. Such studies would require the derivation of many NK clones from several different donors. Moreover, NK clones are kept in vitro through continuous activation by high amounts of IL-2 and may consequently not reflect their in vivo status. Besides, such in vitro expansion may bias the analysis since some NK cells may not be compatible with the in vitro culture conditions and consequently not be represented in the panel of studied clones. Consequently, in order to test for the expression of an HFER at the cell level and using non-manipulated cell populations, we decided to use the MHC tetramer technology.

HFE tetramers recognize only CD71-positive cells
Initially, we made several attempts to refold HFE monochains produced in bacteria but it appeared that we could not detect soluble HFE molecules under several conditions tested. Using a refolding buffer that contained an artificial chaperone [System CTAB/methyl-ß-cyclodextrin (21)] we could obtain small amounts of HFE proteins, but these molecules aggregated during biotinylation. The glycosylation of HFE may be necessary to stabilize the tertiary structure of such peptide-free MHC class I molecules. Consequently, we produced HFE monomers by stable transfection of HEK293 with a plasmid encoding a soluble HFE monochain protein associated to a histidine and a biotinylation tag. Soluble HFE monochains produced with this method were previously shown to be functional (22). The supernatants of some selected clones were shown to contain well-folded HFE as demonstrated by immunoprecipitation using conformation-dependent antibodies (data not shown). The monomers were isolated from the supernatant of large-scale cultures through binding to a Ni column and were biotinylated before purification on a size-exclusion FPLC column. Several hundred micrograms were obtained and tetramerized using streptavidin–APC. The tetramers were tested for specific binding to an HFE-restricted mouse hybridoma (P. Rohrlich, submitted for publication). As shown in Fig. 3(A), the hybridoma that expresses a TCR which recognizes HFE as a major histocompatibility antigen was specifically stained with the HFE tetramer and not with an HLA-A*0201 tetramer folded around the cytomegalovirus (CMV) pp65-dominant epitope. When incubated with fresh PBMCs this HFE tetramer specifically stained a small population of cells (0.05%). The background staining is ~0.01% as shown in Fig. 3(B) with the HLA-A*0201 tetramer folded around the CMV pp65-dominant epitope for a donor who does not have a detectable CTL population recognized by this tetramer. As shown earlier (23), CD19-positive B cells are the main cell type to be responsible for the artifactual staining observed with MHC class I tetramers. The HFE tetramer is also binding on a small number of B cells: 0.1% of the PBMCs, i.e. <1% of the B cells. Such background staining is mostly recognizable by the distribution of the cells in the quadrant: cells are dispersed instead of being grouped in a compact, homogeneous population. Aside from the few probably unspecifically stained CD19 cells, the HFE tetramer-positive cells are not B cells, NK cells (CD56 positive) or {gamma}{delta} T cells (Fig. 3B). The cells stained with the HFE tetramer are also not CD3- or CD14-expressing cells (data not shown). A double staining with HFE tetramer and CD71 (specific for the TfR) showed that the cells that are specifically labeled by the HFE tetramer (the cell population that appears as a focused homogeneous cluster in the upper quadrants) express high levels of CD71. Consequently, some uncharacterized CD71-expressing cells are stained with the HFE tetramer but no CD71-negative cells such as NK or {gamma}{delta} T cells are specifically labeled by the HFE tetramer. This indicates that in the PBMCs there are no detectable cells capable of NK activity (NK, NKT and {gamma}{delta} T cells) that express a specific receptor for HFE.



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Fig. 3. Analysis of blood cells with fluorescent HFE tetramers. A mouse hybridoma that expresses a TCR able to recognize the HFE molecule was incubated with titrated amounts of APC-labeled HFE tetramers: no tetramer, 2.5, 5 and 10 µg ml–1 in PBS with 50% FCS represented by the filled histogram, thick line, dotted line and hatched line, respectively (Panel A). As a control, the same amounts of an APC-labeled HLA-A*0201 tetramer were used and resulted in no staining of the hybridoma. (B) The dot plots show the results of a FACS analysis of human fresh PBMCs incubated with the HFE (upper panel) or an HLA-A*0201 (lower panel) tetramer labeled with APC and different fluorescent antibodies specific for B cells (CD19), NK and NKT cells (CD56), {gamma}{delta} T cells ({gamma}{delta}) and TfR-expressing cells (CD71). In order to see the background staining obtained with tetramer analysis, the results shown here were obtained with the blood of a healthy donor that does not have detectable T cells specific for the HLA-A*0201 epitope derived from the human CMV pp65 protein. The data indicate that B, NK, NKT and {gamma}{delta} T cells are not able to bind HFE but that all cells expressing high levels of CD71 are stained with the HFE tetramer.

 
Several organs contain specific immune cell types that may not be found in PBMCs. We cannot exclude that some of these specialized cells cannot recognize HFE. For example, intra-epithelial {gamma}{delta} T cells (IELs) present in the gut were found to influence iron uptake (TCR delta knockout mice have hepatic iron overload) and hypothesized to recognize HFE (15). An HFE tetramer analysis of human IELs may document this hypothesis. Meanwhile, our results with PBMCs show that HFE does not detectably interact with any blood cell type.

From our HFE expression studies, in vitro NK activity test and HFE tetramer staining, we conclude that as opposed to most non-classical MHC class I molecules in humans, HFE does not have an influence on NK activity contained in blood. Although viral manipulation of HFE has been observed (11, 12) and suggested as a means of improved pathogen survival due to regulation of iron metabolism (24), it is unlikely that targeting HFE is a tool for tumors or viruses to escape from the detection by NK cells.


    Supplementary data
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Supplementary data
 References
 
Supplementary data are available at International Immunology Online.


    Acknowledgements
 
This work was supported by the EU grant: QLQ1-CT-1999-00665, ‘Regulation of expression and function of HFE (HFER)’.


    Abbreviations
 
APC   allophycocyanin
CMV   cytomegalovirus
FPLC   fast protein liquid chromatography
HCMV   human cytomegalovirus
HFER   HFE receptor
hß2m   human ß2-microglobulin
IELs   intra-epithelial {gamma}{delta} T cells
TfR   transferrin receptor

    Notes
 
Transmitting editor: E. Vivier

Received 29 September 2004, accepted 27 October 2004.


    References
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Supplementary data
 References
 

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