Class I HLA oligomerization at the surface of B cells is controlled by exogenous ß2-microglobulin: implications in activation of cytotoxic T lymphocytes
Andrea Bodnár1,
Zsolt Bacsó2,
Attila Jenei2,4,
Thomas M. Jovin4,
Michael Edidin3,
Sándor Damjanovich1,2 and
János Matkó2,5
1 Cell Biophysical Research Group of the Hungarian Academy of Sciences and 2 Department of Biophysics and Cell Biology, University of Debrecen, Medical Faculty, 4012 Debrecen, Hungary 3 Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, USA 4 Department of Molecular Biology, Max-Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany 5 Department of Immunology, Eötvös Lorand University, 1117 Budapest, Hungary
Correspondence to: J. Matkó, Department of Immunology, Eötvös Lorand University, Pázmány Péter sétány 1/C, 1117 Budapest, Hungary. E-mail: matko{at}cerberus.elte.hu
Transmitting editor: I. Pecht
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Abstract
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Submicroscopic molecular clusters (oligomers) of class I HLA have been detected by physical techniques [e.g. fluorescence resonance energy transfer (FRET) and single particle tracking of molecular diffusion] at the surface of various activated and transformed human cells, including B lymphocytes. Here, the sensitivity of this homotypic association to exogenous ß2-microglobulin (ß2m) and the role of free heavy chains (FHC) in class I HLA oligomerization were investigated on a B lymphoblastoid cell line, JY. Scanning near-field optical microscopy and FRET data both demonstrated that FHC and class I HLA heterodimers are co-clustered at the cell surface. Culturing the cells with excess ß2m resulted in a reduced co-clustering and decreased molecular homotypic association, as assessed by FRET. The decreased HLA clustering on JY target cells (antigen-presenting cells) was accompanied with their reduced susceptibility to specific lysis by allospecific CD8+ cytotoxic T lymphocytes (CTL). JY B cells with reduced HLA clustering also provoked significantly weaker T cell activation signals, such as lower expression of CD69 activation marker and lower magnitude of TCR down-regulation, than did the untreated B cells. These results together suggest that the actual level of ß2m available at the cell surface can control CTL activation and the subsequent cytotoxic effector function through regulation of the homotypic HLA-I association. This might be especially important in some inflammatory and autoimmune diseases where elevated serum ß2m levels are reported.
Keywords: ß2-microglobulin, cell-surface HLA cluster, cytotoxicity, fluorescence resonance energy transfer, free heavy chain, T cell activation
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Introduction
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Class I MHC (MHC-I) glycoproteins, expressed by most nucleated cells, play a key role in the cellular immune response: peptides derived from endogenous (and sometimes from exogenous) antigens are recognized by CD8+ T cells in association with MHC-I molecules (1). It seems that correct folding and proper transport to the cell surface requires the MHC-encoded heavy chain (
chain) to be non-covalently associated with the light chain [ß2-microglobulin (ß2m)] and the antigenic peptide (2). At the surface of cells with defects in peptide transport/loading, a reduced expression and stability of MHC-I can be observed, while in most ß2m-deficient human cells heavy chains do not reach the cell surface at all. Although in the murine system there are some exceptions to this rule, i.e. the H-2Db (3) and H-2Ld alleles, in humans no MHC-I expression was detectable in cells lacking ß2m.
On the other hand, ß2m-free heavy chains (FHC) were detected at the surface of numerous human and murine ß2m+ cells. Although their appearance is thought to be ubiquitous at the surface of all MHC-I-expressing cells (4), abundant expression of FHC can be found mainly in the plasma membrane of activated T and B lymphocytes, some virus-transformed lymphoblasts, fibroblasts, and neuroblastoma cell lines (3,57). The exact functional role of these cell-surface FHC of relatively long (several hours) half-life (4) is still not clear, although some possible functions have been proposed (6,8,9). They may serve, for example, as the major source of soluble HLA (through cleavage by metalloproteases) (10), with an immunoregulatory role (8), and are also assumed to contribute to cell-surface clustering (oligomerization) of class I HLA, based on a good correlation observed between expression of FHC and the appearance of larger HLA clusters (6).
Clustering (self-association) of HLA-I glycoproteins was observed earlier at the surface of several (mostly lymphoid) cell types by fluorescence resonance energy transfer (FRET) and lateral diffusion (single particle tracking) measurements (6,11). A recent detergent-solubility analysis of their self-association properties also confirmed that homotypic association is an inherent property of MHC-I molecules (12), in good accordance with their spontaneous clustering observed earlier upon reconstitution into liposome model systems (13).
Earlier, ß2m was shown to influence MHC clustering in proteoliposome model systems: its addition could prevent or revert the clustering of MHC molecules (13). Since no attempt has been made so far to analyze the effect of HLA-I oligomerization state on the efficiency of cytotoxic activity of effector Tc cells [in antigen-presenting cell (APC)cytotoxic T lymphocyte (CTL) conjugates in situ], in this work we addressed the question whether the expected dispersing effect by exogenous ß2m on the self-assembly of HLA I molecules on APC affects their antigen presentation to T cells and the subsequent T cell activation signals (e.g. CD69 up-regulation and TCR down-regulation) or the cytotoxic effector function of CTL.
A human B lymphoblastoid cell line, JY (HLA-A2, B7+), served as APC throughout these experiments, which characteristically display a reproducibly high degree of MHC I oligomerization (in all stages of life cycle) as well as a substantial FHC expression in their plasma membrane. Exogenously added ß2m diminished HLA-I clusters and also decreased the susceptibility of JY cells to CTL-mediated lysis by allospecific cytotoxic T cells. In addition, TCR down-regulation and expression of the activation marker CD69 were both consistently weaker in Tc lymphocytes conjugated with ß2m-pretreated JY cells than with control, untreated APC. These data suggest that the extent of homotypic HLA-I associationand one of its potential regulators, the exogenous ß2m levelmay control the efficiency of antigen presentation and the subsequent T cell activation in the case of APCTc lymphocyte interactions.
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Methods
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Cells and culture conditions
All chemicals (including ß2m), if otherwise not indicated, were purchased from Sigma (St Louis, MO). JY cells (EpsteinBarr virus-transformed human B lymphoblastoid cell line; HLA-A2, B7, DR4, DQw1,3+) and Raji cells (human Burkitt lymphoma line) were grown in RPMI 1640 medium containing 10% heat-inactivated FCS, 2 mM L-glutamine and 50 µg/ml gentamycin in an air humidified atmosphere containing 5% CO2, at 37°C.
JY cells were treated with ß2m by culturing overnight in medium supplemented with typically 5 µM (or other concentrations) of human ß2m.
mAb
W6/32, KE-2 (anti-HLA-A,B,C), L368 (anti-ß2m) and HC-10 (anti-FHC) mAb were prepared from hybridoma supernatants by Protein A-affinity chromatography. MEM147 (anti-HLA-I) mAb was a kind gift from V. Horejsi (Institute for Molecular Genetics, ASCR, Prague). Conjugation of the antibodies with fluorescein derivatives (FITC and SFX), tetramethylrhodamine isothiocyanate (TRITC; Molecular Probes, Eugene, OR) or with Cy3 and Cy5 (Amersham, Vienna, Austria), and the subsequent purification was carried out as described earlier (14). Further mAb were used against CD8 (2E7; obtained from H. Heyligen, L. Willems Instituut, Belgium), CD69 (FN50; Dako, Glostrup, Denmark) and CD3 (UCHT-1; Sigma).
Cell labeling, and measurement of TCRCD3 and CD69 expression
Cells were washed with PBS twice and then labeled with fluorophore-conjugated antibodies as described elsewhere (14). After labeling, cells were washed with PBS and then fixed in PBS containing 1% formaldehyde (30 min on ice). The expression level of TCRCD3 and CD69 on CTL was measured after labeling with FITC-conjugated mAb, using flow cytometry (FACSCalibur; Becton Dickinson, San Jose, CA).
Generation of effector cytotoxic T cells against JY cells
The CTL specific for JY cells were generated from peripheral blood mononuclear cells (PBMC), and maintained in complete medium supplemented with 10 mM HEPES (pH 7.3) and 50 µM 2-mercaptoethanol (Serva, Heidelberg, Germany). The stimulator JY cells were treated with 50 µg/ml mitomycin C for 40 min and washed 3 times. PBMC were stimulated with mitomycin C-treated JY cells on day 1 and cultured for 2 weeks. From day 4 the medium was supplemented with 20 U/ml human recombinant IL-2 (R & D Systems Europe, Kidlington, UK) and refreshed in every 3 days. On day 14 CTL were re-stimulated with the mitomycin C-treated JY cells and the whole cycle was repeated at least 23 times. The effector Tc cells were used within 5 days following the last stimulation. The homogeneity of CTL population (
95% CD8+) was tested by flow cytometry using FITCanti-CD8 antibody.
Conjugation of JY B cells with CTL
The B and T cells in PBS were mixed in 1:1 ratio (at
106 cells/ml density), and their conjugation (synapse formation) was promoted by centrifugation for 60 s at
200 g (15). Then the cells were gently suspended and the extent of conjugation was determined by fluorescence microscopy. Usually 5070% of B cells were found in conjugates, encountered either with single or multiple T cells (see, e.g. Fig. 4). Cell samples prepared this way were further used in measurements of specific lysis and T cell activation marker expression, alike.
Measurement of CTL activity against JY cells: Eu-release measurements
The measurements were carried out as described earlier (16). Briefly, target cells were labeled with Eu3+-DTPA in the presence of 500 µM dextrane sulfate for 10 min on ice. Then 1 mM Ca2+ and 10 mM glucose was added, and the cells were incubated for another 5 min. Cells were washed 4 times and were used for the cytotoxicity assay. The target cells were mixed with elevating concentrations of effector T cells in the wells of a 96-well plate. Each sample with a given E:T ratio was triplicated. The spontaneous and maximal Eu release were determined from labeled targets incubated in culture medium alone and in 0.5% Triton-X 100 respectively, and expressed as averages of six parallel samples. The mixed cells were centrifuged at 200 g for 1 min (to promote conjugate formation) and incubated for 3 h within culture conditions. After centrifugation (10 min, 200 g) the enhancement solution was added to the supernatant. Time-resolved luminescence intensities were determined with a DELFIA Fluorometer 1232 (LKB-Wallac, Turku, Finland). The specific lysis was calculated as follows:

where Lsample, Lspont and Lmax are the luminescence intensities, measured at each given E:T ratio, corresponding to the actual, spontaneous and maximal Eu release respectively.
Fluorescence resonance energy transfer (FRET) measurements
Flow cytometric energy transfer (FCET) measurements
FCET measurements were performed on a modified Becton Dickinson FACStar flow cytometer. The energy transfer efficiency was determined on a cell-by-cell basis as described earlier (17,18). Briefly, the cells were excited sequentially by the 514 and 488 nm lines of the Ar ion laser adjusted to all-line mode. Fluorescence intensities emitted at 540 ± 20 and above 580 nm were detected using a combination of suitable filters. Three fluorescence intensities I1 (488/540), I2 (488/>580) and I3 (514/>580) were detected from each cell and stored for calculation. (The excitation and emission wavelengths are indicated in parentheses.) From these intensities the energy transfer efficiency can be calculated as follows:

here E is the energy transfer efficiency, and S1, S2, S3 and
are correction parameters obtained from data measured on single labeled cells (17). The necessary parameters were collected in list mode and analyzed by Flowin 2.01 software (SoftFlow, Pecs, Hungary) using gating for forward angle light scatter.
Photobleaching fluorescence resonance energy transfer (pbFRET) measurements
pbFRET measurements (19) were performed in a Zeiss Axiovert 135 TV inverted fluorescent digital imaging microscope equipped with a 75 W Xe arc lamp. The donor (FITCantibody) or donor + acceptor (FITC and TRITCantibody)-labeled cells were illuminated using a 488 (±10 nm) band-pass filter. The emitted fluorescence intensity passing through a 530 (± 15 nm) band-pass filter was detected in time using a CCD camera and an Attofluor 5.44 digital imaging system (Atto Institute, Rockville, MD). Data were then stored and processed by a personal computer. The fluorescence decay (photo bleaching kinetics) data were fitted using the following double exponential function:

where F(t) is the fluorescence intensity, F
is the background fluorescence,
1 and
2 are the time constants, and A1 and A2 are the appropriate amplitudes. The average of
1 and
2 weighted by the amplitudes was used to determine the energy transfer efficiency:

E = 1 Td/Tda(5)
where E is the energy transfer efficiency, and Td and Tda are the average bleaching time constants for the single- and double-labeled samples respectively.
Scanning near-field optical microscopy (SNOM)
The SNOM was an add-on to a Nanoscope-IIIa scanning probe system (Digital Instruments, Santa Barbara, CA) (20). Simultaneous excitation and detection of fluorescence emission were with an uncoated optical fiber tip (20,21). The tips were produced with a commercial pipette puller (P-2000; Sutter Instruments, Novato, CA) from a 9-µm core diameter optical fiber (SMF 1528 CPC6; Siecor, Neustadt, Germany) and mounted in a shear force sensor head (21). Excitation of the Cy5-labeled antibodies was with the 647 nm line of a Performa ArKr laser (Spectra Physics, Mountain View, CA), while excitation of the TRITC-labeled antibodies was with the 543 nm line of a HeNe laser (LHGP 0101; Research Electro Optics, Boulder, CO). Excitation and emitted light was discriminated with a 543/647 nm double dichroic mirror (Chroma, Brattleboro, VT) in the case of samples labeled both with rhodamine- and Cy5-tagged Fab. Emission signal of the two dyes were discriminated with a 620 nm dichroic mirror (Omega Optical, Brattleboro, VT). Rhodamine emission was detected with a 605/55 nm band-pass filter (Chroma), while Cy5 emission was monitored with a 700 nm long-pass filter (Omega Optical). In addition, a 543.5 nm notch filter (Kaiser Optical, Ann Arbor, MI) was also used to block stray light coming from the HeNe laser. Emitted light always passed through a 750 nm short-pass filter (Andover, Salem, NH) to block stray light from the infrared diode laser used for shear force detection.
Determination of protein co-localization
SNOM images of double-labeled cells were processed in Scil-Image (Technical University of Amsterdam, The Netherlands). Non-constant background was removed with either of the following procedures. (i) The original image was low-pass filtered and divided by the filtered one, thereby providing an image lacking most of background intensity variations (22,23). (ii) Top-hat transformation, which gives the difference between the original image and the opening of the original image, was carried out. Visual inspection of resultant images was used to choose the procedure yielding the better result in each case. High-frequency noise was either removed with low-pass filtering or by opening. Images were then segmented using the entropy threshold algorithm of Scil-Image. Segmentation classifies pixels as background or non-background. Background pixels are labeled with digit 0, while non-background ones are labeled with digit 1. Non-background pixels were always found to be clustered in membrane areas with a mean diameter of several hundred nanometers (23). We identify these membrane patches as clusters of membrane proteins. In order to calculate the area of overlap between clusters of different proteins, segmented images recorded in different fluorescence channels were multiplied by each other. Percent overlap was calculated by dividing the area of overlap by the area of one of the membrane protein clusters. In order to obtain color images displaying overlap of membrane protein clusters, gray scale images were first contrast stretched, and then they were used as inputs to the red and green channels of RGB images. Yellow areas correspond to overlap of clusters.
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Results
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ß2m FHC are involved in cell-surface MHC-I clustering
Earlier we have shown that expression of FHC correlated reasonably well with the spontaneous self-association of MHC-I on a number of cell types (6). As no clear quantitative relationship could be drawn from these experiments, here we further analyzed their participation in MHC-I clustering in two ways. (i) We analyzed their co-distribution with intact MHC-I heterodimers with SNOM, based on selective staining with specific antibodies against FHC and MHC-I heterodimers [using Cy3-conjugated HC10 and fluorescein (SFX)- conjugated W6/32 mAb respectively]. As Fig. 1(A) shows, there is a significant overlap [cross-correlation coefficient
0.771, typical error (CV) 6%] between these two molecular species of MHC-I in the microscopically resolvable MHC-I clusters at the surface of human JY B cells. (ii) In addition, FRET data (Table 1) also indicate that they are co-clustered at a molecular (nanometer scale) level, as well. In contrast, no homo-FRET was detectable between antibodies tagging the light chains (ß2m), indicating either their too large distance or unfavorable orientation within the supramolecular MHC clusters.

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Fig. 1. SNOM fluorescence images of the human JY B cell-surface labeled for FHC (with Cy5-HC10 mAb, red) and MHC-I heterodimers (with SFX-W6/32 mAb, green) on control cells (A) and cells treated with 5 µM ß2m (B). The images represent 15 x 15 µm areas; the yellow color indicates membrane regions where the two molecular species are co-localized [cross-correlation coefficients are 0.771 (A) and 0.61 (B) respectively].
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Table 1. FRET between intact HLA-I and FHC determinants at the surface of JY B lymphoblast cells: effect of exogenous ß2m
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A partial (2025%) blocking by HC-10 antibody (against an FHC determinant) of the binding of antibodies W6/32 and MEM-147, reacting with two distinct extracellular domain epitopes on HLA-I heterodimers (data not shown), also reflects an intermixing of FHC and intact HLA-I heterodimers in the MHC-I clusters at the surface of JY cells. No such effect could be observed in human lymphocytes isolated from peripheral blood, with a substantially lower level of MHC-I expression and almost undetectable level of FHC.
MHC-I clusters of B cells are dispersed by excess exogenous ß2m
Expanding the analysis of exogenous ß2m effects on liposome models (13) to live B cells, we investigated here the effect of overnight culturing of B cells with 5 µM ß2m on molecular proximity of (FRET between) the MHC-I species involved in the cell-surface MHC clusters. FRET was determined by two methods: FCET (17,24) and microscopic pbFRET (19), using two different mAb to label heavy chain epitopes of intact MHC-I molecules (W6/32 and KE-2 respectively) and HC10 mAb to label FHC.
As Fig. 2 shows, treatment with ß2m remarkably reduced homotypic association of intact MHC-I molecules (clustering), as revealed by FITC- and TRITC-conjugated KE-2 mAb, using pbFRET. Homo-FRET data obtained between W6/32 mAb-labeled intact MHC-I molecules, as well as hetero-FRET between FITC-HC10-labeled FHC and W6/32-labeled MHC-I molecules further confirmed the view of dispersed MHC clusters upon ß2m-addition to B cells in culture (Table 1). Although on the SNOM images a significant amount of fluorescent patches of Cy-5 HC-10-labeled FHC remained still observable after exogenous ß2m administration, the cross-correlation between FHC and intact MHC-I patches also decreased (Fig. 1B), indicating a moderate dispersing effect of ß2m treatment on the large-scale MHC-I domains, as well.

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Fig. 2. Effect of exogenous ß2m on the HLA-I homoassociation on JY cells as assessed by pbFRET. (A) Black bars represent photobleaching time constants measured in donor (FITCKE2 antibody)-labeled JY cells, while the white bars represent the same time constant measured in double-labeled (donor: FITCKE2 mAb; acceptor: TRITCKE2 mAb) cells, indicating a significant FRET (homoassociation) between HLA-I molecules. (B) The black and white bars represent the same parameters measured in JY cells pretreated with exogenous ß2m. The largely reduced difference in the means of time constant histograms indicates a decreased FRET (homoassociation) upon this treatment.
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JY B cellCTL conjugates: a model system to study functional consequences of ß2m-induced MHC-I reorganization
Mixing of JY B cells with CTL selected against themas described in Methods and previously (25)in a 1:1 ratio and applying a short (60 s) centrifugation (at 200 g) resulted in 6070% conjugation of these cells with different stoichiometries (mostly 1:1, 1:2 or 1:3 B:CTL ratios). Such conjugation (as shown by Fig. 3) represents single or multiple immune synapses between these cells and may serve as a good model system to study functional properties of T cells encountered with B cells (APC) (15).

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Fig. 3. Confocal fluorescence images of JYCTL conjugates. The JY B cells were stained with anti-HLA-I antibody, FITCW6/32, while the CTL were stained with anti-CD8 antibody (TRITC2E7). The images were three-dimensionally reconstructed from 0.4-µm optical slices recorded by a Zeiss LSM4 laser scanning confocal microscope. Images (A) and (B) represent typical multiple and single T cell synapses respectively.
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These conjugates were then the subjects of further studies on how the modulation of the APC surface MHC-I organization by exogenous ß2m affects T cell activation or the susceptibility of B cells to CTL killing.
Effect of exogenous ß2m on the activation of cytotoxic effector T cells by JY B cells
We were also interested in the functional consequences of the ß2m-induced cell-surface MHC-I redistributions in terms of antigen presentation and the activation of allospecific Tc lymphocytes. Antigen-specific activation of CD8+ T lymphocytes by JY B cells resulted in a significant TCR down-regulation in
3 h after conjugation. The TCR down-regulation was significantly weaker when the B cells were pretreated with exogenous ß2m prior to conjugation (Fig. 4). This suggests that ß2m treatment of B cells may cause an upward shift in the threshold of CTL activation, also indicated by a weaker expression of CD69 T cell activation marker on CTL when encountered with ß2m-treated B cells. Approximately one-third level of CD69 expression could be detected by flow cytometry on CTL (4 h after initiation of the conjugation) than in T cells encountered with untreated JY cells (data not shown).

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Fig. 4. TCR down-regulation in CTL upon conjugation with JY B cells, as followed by flow cytometric monitoring of TCRCD3 expression. The TCRCD3 was labeled with FITCanti-CD3 mAb (UCHT-1) in cell samples taken after conjugation of the two cells, at the time points indicated on the abscissa. The expression level on T cells prior to addition of B cells was taken as 100%. The CTL were mixed either with untreated B cells (filled squares) or B cells pretreated with 5 µM ß2m (empty squares).
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Exogenous ß2m decreases the susceptibility of JY B cells to specific killing by CTL
As another important consequence, using Eu-release assay, we demonstrate here that allospecific Tc cells were capable of inducing a significantly weaker specific lysis of B cells pretreated with 5 µM ß2m (for overnight in culture) than that of control (untreated) B cells (Fig. 5). This effect was more profound at lower E:T ratios, possibly because killing is approaching saturation at high E:T ratios in our system (using 3 h incubation). The specificity of the cellular interactions is shown by the lack of specific lysis in the case of Raji cellCTL interaction.

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Fig. 5. Effect of exogenous ß2m on the susceptibility of B cells to cytotoxicity (specific lysis) of CTL. Percentage of specific lysis of target cells determined from Eu-release assay is displayed (with SEM of three measurements) at different E:T cell ratios. Target cells are shown in the insert legend; the human Raji cell line was used as a non-specific negative control.
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Discussion
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Although the functions of MHC I glycoproteins are mainly carried by the transmembrane heavy chain, ß2m is also of essential importance in terms of operation: non-covalent binding of ß2m to the
3 domain of newly synthesized heavy chains in the endoplasmic reticulum is required for acquisition of native
1/
2 conformation and intracellular transport of MHC I molecules (2). In the absence of intracellularly synthesized ß2m, most MHC-I molecules are retained in the endoplasmic reticulum and not transported to the cell surface as demonstrated by experiments with ß2m-deficient human or murine cells (26). In some cases class I heavy chains can reach the cell surface (27) and even can be recognized by CTL, also in ß2m cells (3), e.g. cell-surface expression of H-2Db molecules was observed at the surface of ß2m tumor cells although at a much reduced level compared to cells with restored ß2m production. A small percentage of these molecules are functionally conformed and elicit a cytotoxic immune response (3). Lack of ß2m was presumably overcome by binding of intracellular peptides with sufficient affinity that stabilized the proper conformation of H-2Db molecules in the above- mentioned cells (28). In the human system, so far no example has been found for heavy chains being transported to the cell surface without ß2m. So, despite the exceptions, the role of ß2m in the intracellular transport and structural integrity of MHC-I molecules is indisputable.
Nevertheless, the expression of FHC is not a specific feature of the aforesaid ß2m cells. In the plasma membrane of several ß2m+ human and murine cells MHC I molecules are present in at least two distinct forms: largely as intact ß2m/HC heterodimers and to a much less extent (
20%) as ß2m FHC. Appearance of FHC in these cases requires a previous cell-surface expression of intact ß2m/HC heterodimers, since FHC originate mostly from such ß2m-associated MHC I molecules (9,10,29). The first time FHC were detected on the surface of activated lymphocytes and different virus-transformed cell lines (57). Later, FHC were also reported in lymphocytes of peripheral origin, as well as in splenocytes, although to a much less extent (4). It seems that the appearance of FHC is a ubiquitous attribute of cells bearing the genes of MHC and ß2m; however, their expression level dramatically increases upon activation or viral transformation.
Dissociation of ß2m ends in altered conformation of both the peptide-binding site (loss of
1/
2 epitope) (30) and the cytoplasmic tail of the
chain (31,32), but the conformation of the extracellular
3 domain still resembles the native state. In this form FHC are capable of re-association with ß2m, regaining the native conformation of MHC I molecules. With the lack of excess free ß2m, FHC may undergo irreversible denaturation (loss of the
3 epitope) and/or are internalized or released in soluble form in a metalloprotease-mediated pathway (10,33). Studies with mutant ß2m (34) have shown the importance of certain HC-contacting residues in antigen presentation. These data all suggest that ß2m may play an important role in regulation of heavy chain conformation not only intracellularly, but also at the cell surface.
Our SNOM data have shown a high degree of co-localization of FHC and intact HLA-I molecules within the large HLA-I domains observable on these human B lymphoblast cells. Treatment with exogenous ß2m decreased the magnitude of co-localization, but still a number of FHC- and FHC/HLA-I clusters remained at the cell surface, although with a smaller average size (Fig.1B). These residual domains may represent partially denatured forms of FHC still reacting with HC-10 antibody, but incapable of binding ß2m and/or the net FHC reservoir formed by biosynthesis/endocytosis/recycling pathways and vesicular trafficking (35). The ability of exogenous ß2m treatment to cause HLA-I redistribution at the cell surface was further confirmed by our FRET data indicating that the magnitude of both HLA-IHLA-I and FHCHLA-I molecular interactions (clustering) decreased significantly, depending on the exogenous ß2m dose (Table 1).
According to these results, FHC likely have an important contribution to the HLA-I clustering in the membrane. Although, the molecular structural details are not yet known, FHC may stabilize their conformation by participating in large domains of intact HLA-I. On the other hand, their involvement in these domains seems to stabilize HLA-I patching, vice versa, since no such large HLA-I patches can be observed on lymphocytes of peripheral origin not expressing a significant amount of FHC (J. Matko, unpublished observations).
There are many data showing that FHC are not simply spoiled, functionally inactive versions of MHC-I molecules, but they do have biological role. Due the ability to capture peptides and ß2m, their membrane-bound form can present exogenous antigens to CTL (28,36), while the soluble form can act as a scavenger system for soluble peptides and ß2m (8,10). Presumably they also have a role in protection against NK-mediated lysis, but there are controversial views in this question (37,38). In addition, the inclination of both types of MHC I molecules for aggregation/self-association was also demonstrated in many cases, partly by our recent (13) and present results. Demonstration of FHCHLA-I co-clustering may explain, on the one hand, the surprisingly long cell-surface lifetime of FHC (4). On the other hand, according to our data, these FHC, at least partly, may rebind ß2m. This, in turn, may result in a partial dissolution of cell-surface MHC-I clusters.
At least two subsets of cell-surface FHC can be distinguished: one capable of re-associating with ß2m and the functionally inactive denatured form [visualized as the residual red/yellow spots (clusters) on the SNOM image taken after ß2m treatment, Fig. 1(B)]. Dissociation and association of ß2m was convincingly shown to result in altered conformation of the peptide-binding site and also of the cytoplasmic tail of MHC-I molecules (39,40). Therefore, the concentration of ß2m available at the cell surface may influence both the expression level of FHC and the equilibrium among the two different forms of HLA-I heavy chains (ß2m+ and ß2m respectively) existing at the cell surface.
Several explanations were suggested earlier for the origin of FHC at the cell surface: (i) increased synthesis of ß2m-associated MHC molecules may lead to the accumulation and subsequent dissociation of unstable complexes, (ii) internalization and recycling of MHC molecules may also account for the generation of FHC, and (iii) existence of a limited number of stable heterodimers with different biochemical characteristics (e.g. phosphorylation state) predestining them for dissociation. Thus, the expression level of FHC seems to be regulated by the amount of cell-surface heterodimers, internalization and spontaneous and/or inducible release in soluble form. Summarizing our recent (6,13,22,35,41) and present results, as well as results from other works (9,10,29,33), we propose here a model for a dynamic cell-surface organization of intact HLA-I molecules and FHC on B cells (or perhaps on other APC) influencing the antigen-presentation efficiency (Fig. 6).

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Fig. 6. A possible model proposed for the dynamic cell-surface organization of HLA-I molecules in B cells (APC). Following synthesis, assembly and transport, the HLA-I molecules appear at the surface mostly in a heterotrimeric form (HLp). Consecutive dissociation of a loosely bound peptide and the ß2m may result in formation of FHC (H). FHC may unfold or be clustered in denatured form and subsequently internalized, or may also serve as sources of soluble HLA (sH) through metalloprotease activity. A novel role is proposed for soluble, exogenous ß2m available at the cell surface (depending on the actual serum level): (i) it can shift the equilibrium between HLp and H towards HLp (also providing a chance for HLA to pick up exogenous peptides), and (ii) by partial rebinding to H, it can partially dissolve the large MHC-I clusters shifting another equilibrium backward to HLp. Endocytosis and recycling may also affect the equilibria involving the mixed clusters of HLp and H forms of HLA-I. As shown by the presented evidence, thus exogenous ß2m (through the magnitude of cell-surface HLA-I clustering) may control the efficiency of antigen presentation to CTL and the activation of their subsequent effector function.
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On the basis of recent studies with soluble dimers, trimers or tetramers of peptide-loaded MHC molecules, binding of multimeric ligands in most cases induced a more potent activation/immune response of T cells in comparison with monomers (42,43). Although these experiments were carried out in murine systems, it can be hypothesized that in vivo formation of cell-surface HLA I (and in some cases HLA II) clusters may increase the efficiency of antigen presentation, presumably by promoting formation of a more stable immunological synapse. Our data confirmed this hypothesis and, more importantly, have demonstrated that soluble, extracellular ß2m is one of the potential factors controlling the MHC-I clustering. Moreover, the data obtained with ß2m-pretreated B cells (APC) suggest that the cell-surface redistribution of MHC molecules may have a direct influence on the magnitude of the subsequent cytotoxic effector function of Tc lymphocytes.
In conclusion, our results turn the attention to a possible regulatory role of exogenous ß2m (and FHC) in controlling antigen presentation and the subsequent activation of CTL that may be of physiological relevance, especially in the case of autoimmune disorders with elevated serum ß2m levels [e.g. (44)].
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Acknowledgements
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The skillful technical assistance of A. Harangi and T. Lakatos is gratefully acknowledged. This work was financially supported by research grants FKFP 0518/99 (J. M.), OTKA T034393 (J. M.), T030411 (S. D.) and F034487 (A. J.), and ETT 031/2001 (Zs. B.) and 013/2001 (A. J.). A. J. was the recipient of a long-term fellowship from the European Molecular Biology Organization.
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Abbreviations
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ß2mß2-microglobulin
APCantigen-presenting cell
CTLcytotoxic T lymphocyte
FCETflow cytometric energy transfer
FHCfree heavy chain
FRETfluorescence resonance energy transfer
MHC-IMHC class I
PBMCperipheral blood mononuclear cell
pbFRETphotobleaching FRET
SNOMscanning near field optical microscopy
TRITCtetramethylrhodamine isothiocyanate
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