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The Journal of Immunology, 2001, 167: 3800-3808.
Copyright © 2001 by The American Association of Immunologists

Induction of TCR V{beta}-Specific CD8+ CTLs by TCR V{beta}-Derived Peptides Bound to HLA-E1

Jianfeng Li2, Itamar Goldstein2, Eva Glickman-Nir, Hong Jiang and Leonard Chess3

Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY 10032


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous studies have identified murine and human regulatory CD8+ T cells specific for TCR-V{beta} families expressed on autologous activated CD4+ T cells. In the mouse, these regulatory CD8+ T cells were shown to be restricted by the MHC class Ib molecule, Qa-1. In the present study, we asked whether HLA-E, the human functional equivalent of Qa-1, binds V{beta} peptides and whether the HLA-E/V{beta}-peptide complex induces and restricts human CD8+ CTLs. We first created stable HLA-E gene transfectants of the C1R cell line (C1R-E). Two putative HLA-E binding nonapeptides identified in human TCR V{beta}1 and V{beta}2 chains (SLELGDSAL and LLLGPGSGL, respectively) were shown to bind to HLA-E. CD8+ T cells could be primed in vitro by C1R-E cells loaded with the V{beta}1 (C1R-E/V1) or V{beta}2 (C1R-E/V2) peptide to preferentially kill C1R-E cells loaded with the respective inducing V{beta} peptide, compared with targets loaded with the other peptides. Priming CD8+ T cells with untreated C1R-E cells did not induce V{beta}-specific CTLs. Of perhaps more physiological relevance was the finding that the CD8+ CTLs primed by C1R-E/V1 also preferentially killed activated autologous TCR V{beta}1+. Similar results were observed in reciprocal experiments using C1R-E/V2 for priming. Furthermore, anti-CD8 and anti-MHC class I mAbs inhibited this V{beta}-specific killing of C1R-E and CD4+ T cell targets. Taken together, the data provide evidence that certain TCR-V{beta} peptides can be presented by HLA-E to further induce V{beta}-specific CD8+ CTLs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
To regulate the immune response and dampen the potential for autoimmunity, the immune system has evolved several mechanisms to down-regulate and control the outgrowth and differentiation of activated CD4+ T cells. One level of control, mediated during the initial interaction of the CD4+ T cell with MHC/peptide complexes on the surface of APCs, determines whether T cell activation, anergy, or apoptosis will ensue (1, 2, 3). A second level of control, mediated by cytokines, regulates the growth and differentiation of activated CD4+ T cells. Different cytokines secreted by CD4+ or CD8+ T cells either stimulate or inhibit CD4+ T cell proliferation and determine whether a naive TH precursor cell differentiates as an IFN-{gamma}-producing TH1 cell or as an IL-4- and IL-10-producing TH2 cell (4, 5). A third level of control resides in the regulatory T cells including both CD4+ (6) and CD8+ T cell populations. Ample data demonstrate the ability of CD8+ T cells to regulate CD4+ T cell responses (7, 8, 9, 10, 11). For example, in murine systems during Ag-driven CD4+ T cell responses, in vivo, CD8+ T cells emerge that specifically regulate activated CD4+ T cells in a TCR V{beta}-specific manner. These CD8+ T cells preferentially recognize Ag-activated CD4+ T cell clones expressing certain TCR V{beta} molecules and, interestingly, are restricted by the class Ib MHC molecule, Qa-1 (12, 13). Unlike conventional MHC class Ia molecules, Qa-1 molecules are only expressed at low levels on resting T cells but are increased following Ag activation (14, 15). These data are consistent with a model of specific immunoregulation in which, following Ag activation, CD4+ T cells express membrane Qa-1/TCR V{beta} motifs that are recognized by the {alpha}{beta} TCR expressed by precursor regulatory CD8+ T cells (RTC).4 These CD8+ T cells are induced to differentiate and down-regulate CD4+ T cells expressing the particular Qa-1/TCR V{beta} motifs.

In addition, V{beta}-specific CD8+ T cells exist in human peripheral blood, which can be induced to kill autologous CD4+ T cells based, at least in part, on the recognition of the TCR V{beta}s expressed (16, 17). It is currently unknown whether these CD8+ T cells are restricted by the HLA-E molecule known to be the human functional equivalent of the murine Qa-1 molecule. HLA-E, like Qa-1, is relatively nonpolymorphic (18, 19) and is expressed on the surface of activated T cells (20).

There are a number of biologic features of the Qa-1 molecule that are of interest with respect to its potential function as a restricting element to present self-TCR peptides to CD8+ T cells. First, although Qa-1 is expressed on a variety of hemopoietic tissues, it is only minimally or not expressed at all on resting T cells, and expression is significantly increased following peripheral T cell activation by Ag (14, 15). Moreover, peripheral expression is short lived and persists for only a few days on activated CD4+ T cells (13); this may exclude resting T cells from down-regulation by Qa-1-restricted RTC. It is also of interest that many features of the Qa-1 binding cleft are conserved as well in the human HLA-E, and these two proteins bind homologous peptides derived from leader sequences of MHC class Ia molecules. For example, Qa-1 binds with high affinity to a nine-amino-acid peptide (AMAPRTLLL) derived from the leader sequence of H-2D (21, 22, 23, 24). HLA-E binds leader sequences of human MHC class I molecules, like the nonapeptide (VMAPRTVLL) derived from the HLA-B7 leader sequence (25, 26). Functionally, many studies have shown that HLA-E (27, 28) and Qa-1 (29, 30) complexed with these peptides are the dominant ligands for the CD94/NKG2 heterodimer. This cell receptor is comprised of an invariant C-type lectin chain, CD94, that is disulfide-bonded with the NKG2 glycoproteins (31) and known to regulate cytotoxic activity of both NK cells and CD8+ CTLs (32, 33, 34).

Furthermore, several studies suggest that Qa-1 (35, 36, 37, 38) and HLA-E, when introduced into the mouse genome (39), can present self- and foreign Ags to CD8+ CTLs. These observations raise the possibility that Qa-1 and HLA-E may have the capacity to bind and present a diverse repertoire of peptides to the TCR complex. Moreover, it has also been shown that HLA-E binds to CD94/NKG2 receptors expressed on CD8+ T cells and can regulate conventional CD8+ CTL activity (33, 40). In this report, we present evidence that HLA-E can physically bind certain human TCR V{beta}-derived peptides, and that HLA-E/V{beta} peptide complexes can induce the differentiation of CD8+ CTL, which preferentially lyse targets in a TCR V{beta}-specific manner.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Peptides and Abs

TCR V{beta}1 (V1) peptide (SLELGDSAL), TCR V{beta}2 (V2) peptide (LLLGPGSGL), HLA-B7 (B7) signal sequence-derived peptide (VMAPRTVLL), and the control (N) peptide (GILGFVFTL) were all synthesized on a SMPS-350 automated peptide synthesizer (Alpha Diagnostics, San Antonio, TX). The peptides were precipitated with ether, lyophilized, and subjected to HPLC to ascertain purity (>95%) and concentration. Abs to human CD4 and CD8 were purchased from BD PharMingen (San Diego, CA), and mAbs to TCR V{beta}2 and TCR V{beta}1 were purchased from Immunotech (Marseille, France). The anti-green fluorescent protein (GFP) mAb was purchased from Clontech Laboratories (Palo Alto, CA). Hybridomas producing mAbs W6/32, OKT3, and OKT8 were purchased from American Type Culture Collection (Manassas, VA). Ascites containing these Abs were produced and purified as described (41). Fluorescein-conjugated F(ab')2 goat anti-mouse IgG+IgM were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).

Transfection

Transfection of the C1R cell line with either the human HLA-E 0101 gene in the pGEFP-N3 vector (provided by Dr. Robert Winchester, Columbia University, New York, NY) or with the empty pGEFP-N3 vector (Clontech Laboratories) was performed using the FuGEN 6 Transfection Reagent, as recommended by the manufacturer (Roche Diagnostics, Indianapolis, IN). Transfection solution was prepared by mixing 1 µg of plasmid DNA and 3 µl of FuGEN 6 Transfection Reagent in 100 µl of serum-free medium. After incubation at room temperature for 15 min, the mixture was added to tissue culture wells containing 1 x 106 cells in 2 ml of complete culture medium. For stable transfection, G418 selection (Life Technologies, Grand Island, NY) was started on day 3 after transfection.

Peptide binding assays

We used flow cytometry to analyze the capacity of the V{beta} peptides to bind and thermostabilize the empty HLA-E/{beta}2-microglobulin ({beta}2 µ) complex on the cell surface of cold-treated cells as described (26). Briefly, cells were precultured at 26°C for 24 h and than pulsed overnight with 100 µM of peptide, and thereafter incubated at 37°C for an additional 4 h to disassociate the empty HLA-E/{beta}2 µ complexes. At that point, cells were harvested for immunostaining with mAb W6/32 followed by fluorescein-conjugated goat anti-mouse Ig-specific Abs.

The assembly assay has been adopted with some modifications from Braud et al. (42). Briefly, C1R transfected with HLA-E (C1R-E) cells were metabolically labeled with 100 µCi [35S]methionine and [35S]cysteine (Amersham, Little Chalfont, U.K.) for 60 min after being starved in methionine- and cysteine-free medium for 1 h. Cells were then washed with ice-cold PBS and incubated for 30 min at 4°C in lysis buffer (40 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 0.5% Mega 9, 5 mM iodoacetamide, and 2 mM PMSF) in the presence or absence of 100 µM of the relevant peptide. Supernatants were precleared by the addition of 10% protein A-Sepharose (Sigma, St. Louis, MO) and overnight incubation at 4°C. To disassociate the empty HLA-E/{beta}2 µ complexes, the lysate was incubated at 45°C for 2 min, after which immunoprecipitation was performed using anti-GFP mAb adsorbed to protein A-Sepharose. Samples were analyzed using 10% SDS-PAGE, and gels were dried and exposed to x-ray film.

V{beta}1+ or V{beta}2+ CD4+ T cell clones and CD8+ CTL lines

PBL were isolated from heparinized blood of healthy donors by Histopaque (Sigma) gradient centrifugation. Subsequently, CD4+ or CD8+ T cells were further isolated from PBL by MACS, in accordance with manufacturer recommendations (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were further maintained in culture medium consisting of RPMI 1640, 10% FCS (HyClone Laboratories, Logan, UT), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin in a humidified 37°C, 5% CO2 incubator.

To generate V{beta}1+ or V{beta}2+ CD4+ T cell lines, purified CD4+ cells were plated in 96-well U-bottom plates at a density of 3, 1, or 0.3 cells/well containing 1 x 105 irradiated autologous PBL (2500 rad) or EBV-transformed B lymphoblastoid cell line (5000 rad). Cells were stimulated with either 0.1 µg/ml toxic shock syndrome toxin-1 (Sigma) or 5 µg/ml OKT3. Independently of the method of cloning, the medium was supplemented with 50 U/ml rIL-2 (Hoffman-LaRoche, Nutley, NJ) every 2–3 days. Proliferating wells were screened for exclusive TCR V{beta}1 or V{beta}2 usage. Selected V{beta}1 and V{beta}2 lines were expanded and maintained by stimulation using 20 ng/ml phorbol 12,13-dibutyrate and 0.8 µM ionomycin (both obtained from Sigma) for 6 h at 37°C every 10–14 days.

To generate CD8+ CTL lines, the purified CD8+ cells were cocultured with either irradiated V{beta} peptide-loaded C1R-E or untreated C1R-E, both at a ratio of 10:1 (C1R-E/CD8+). After 7 days, CD8+ T cells were collected by Histopaque gradient centrifugation and recultured with the irradiated original stimulators at the same ratio. Three days later, CD8+ T cells were collected again by Histopaque gradient centrifugation and further expanded in a medium supplemented with 50 U/ml rIL-2 for an additional 4 days. Cells were then harvested for further study. V{beta} peptide-loaded C1R-E stimulators were prepared by preincubation at 26°C for 8 h and than pulsed with 100 µM of the respective peptide overnight at 26°C. Unprimed naive CD8+ T cells were cultured for the same duration and under similar conditions.

51Cr-release assay

Cytotoxic activity of CD8+ CTLs against peptide-loaded C1R-E or CD4+ T cell clones was tested in a 4-h (C1R-E) or 8-h (CD4+ T cells) standard 51Cr-release assay. C1R-E targets were prepared by overnight incubation at 26°C, with or without 100 µM indicated peptide and 100 µCi/ml 51Cr (NEN, Boston, MA) and placed at 1 x 104 cells/well in 96-well U-bottom plates in triplicate. CD4+ T cell targets were harvested 3–5 days after the last stimulation and labeled with 100 µCi of 51Cr for 1 h at 37°C and placed at 2 x 104 cells/well in 96-well U-bottom plates in triplicate. Supernatants were harvested and counted in an LKB gamma counter (Pharmacia, Gaithersburg, MD). The percentage of specific lysis was calculated as (sample - spontaneous release)/(total - spontaneous release) x 100. For the Ab blocking assays, either CD8+ CTLs or targets were preincubated with the specified Ab at the indicated concentration for 30 min at room temperature.

FACS analysis

Cells were first treated with aggregated human Ig (Enzyme International, Fallbrook, CA) to block nonspecific Ig binding, and were subsequently incubated with saturating concentrations of the indicated mAb for 15 min at 4°C. Cells were then rinsed and incubated for 15 min at 4°C with fluorescein-conjugated F(ab')2 goat anti-mouse IgG+IgM. Fluorescence intensity was measured on a FACScan cytofluorograph (BD Biosciences, San Jose, CA) in the presence of 10 µg/ml propidium iodide to eliminate dead cells from analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
V{beta} peptides can bind and stabilize the HLA-E complex

We first established an HLA-E-expressing cell line by stable transfection of the MHC class I defective human B cell lymphoblastoid line C1R with the human HLA-E 0101 gene in the pEGFP-N vector that expresses HLA-E fused at its C terminus with the GFP tag. The C1R cell line is known to be defective in the expression of HLA-A and B molecules, but expresses intermediate levels of Cw4 (43). Stable transfection of C1R with HLA-E/GFP was established, and expression was confirmed by RT-PCR (data not shown) and immunoprecipitation designed to detect the chimeric gene products using anti-GFP mAbs (see Fig. 2Go).



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  		FIGURE 2. TCR V{beta}-derived peptides are able to stabilize HLA-E complex. HLA-E-transfected C1R cells were lysed in the presence or absence of each peptide, and the HLA-E complex was immunoprecipitated by anti-GFP mAb. The graph shows the relative mean density of {beta}2 µ bands normalized to the HLA-E/GFP band density, obtained from the analysis of three independent experiments. The relative signal intensity of the C1R-E cells without loaded peptides was arbitrarily assigned a value of 1. Inset, Results of a representative immunoprecipitation experiment.

 
Leu at P9 and Leu or Met at P2 were previously identified as dominant anchor residues for HLA-E and Qa-1 (42). Using a computer-based data search, we identified nonapeptides with these dominant anchor residues in several human TCR V{beta} chains. Of this pool V1 peptide (SLELGDSAL) and V2 peptide (LLLGPGSGL) derived from the human TCR V{beta}1 and TCR V{beta}2 chain, respectively, were chosen for further study.

To investigate surface expression of HLA-E in the C1R-E cells, we asked whether the canonical HLA-E binding nonapeptide, VMAPRTVLL, derived from HLA-B7 signal sequence would induce surface HLA-E. In these studies we took advantage of a well-established strategy to up-regulate HLA-E surface expression in MHC class I-deficient cells, such as LCL 721.22, by incubating them at 26°C to permit empty HLA-E/{beta}2 µ dimers to accumulate on the cell surface (26, 28). In contrast, empty HLA-E/{beta}2 µ dimers are poorly maintained on the cell surface at 37°C. Thus, we first incubated C1R-E in the presence of the B7 peptide at 26°C followed by an incubation at 37°C to disassociate the empty HLA-E/{beta}2 µ. We used flow cytometry to detect "supershift" of green fluorescence attributable to W6/32-FITC staining above the GFP background of transfection. As shown in Fig. 1Go, the B7 peptide induced a small, but reproducible increase in mean fluorescence intensity (MFI) compared with the medium control (62.25 vs 19.48).



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  		FIGURE 1. HLA-E surface expression was induced by TCR V{beta}-derived peptides. C1R cells transfected by HLA-E gene (left panel) or empty pEGFP vector (right panel) were incubated in the presence of indicated peptide (solid thick lines) at 26°C for 18 h after incubation at 26°C for 24 h. Cells were transferred to 37°C and incubated for 4 h. The surface expression of W6/32-recognized molecules in these cells was determined by flow cytometry, as compared with the same transfected cells cultured under the same conditions but in the absence of peptide (solid thin lines). Control mouse IgG is represented by the broken line histograms. The data shown are representative of at least three independent experiments.

 
Using this assay, we next analyzed the capacity of the V1 peptide and the V2 peptide to bind and thermostabilize the HLA-E complex on the C1R-E cell surface. As noted above, these V{beta} peptides were selected because they contain anchor residues at P2 and P9. Both the V1 and V2 peptides stabilized the HLA-E complex on the surface of C1R-E (MFI = 57.95 and MFI = 64.32, respectively). A control nonapeptide, which does not contain at position 2 an anchor residue capable of binding HLA-E, did not increase surface immunostaining (MFI = 17.18). To confirm that the increase in staining intensity was indeed HLA-E dependent, we used the empty-vector-transfected C1R cells in parallel. We were unable to detect any significant change in cell surface immunostaining profiles with any peptide when the empty-vector-transfectants were used (Fig. 1Go, right panel). In addition, staining the samples with HLA-C-specific alloantisera revealed no detectable increase in surface expression regardless of the peptide used (data not shown).

Further evidence that the V{beta} peptides physically bind HLA-E molecules was obtained using a modified version of the assembly assay developed by Braud and coworkers (42). This assay is based on the observation that MHC class I signal sequence-derived peptides stabilize the HLA-E/{beta}2 µ complex in MHC-deficient cell line lysates. Thus at 45°C empty HLA-E/{beta}2 µ complexes dissociate, whereas peptide-loaded complexes are stabilized and can be immunoprecipitated as a heterodimer containing both HLA-E and{beta}2 µ. In our experiments we assessed the capacity of V1, V2, B7, andthe control N peptides to stabilize to HLA-E/{beta}2 µ complex at 45°C by specifically immunoprecipitating the chimeric protein HLA-E/GFP with anti-GFP mAb. As shown in Fig. 2Go, the B7 peptide, as predicted, stabilized the HLA-E complex and enhanced the coprecipitation of {beta}2 µ along with HLA-E/GFP. Moreover, both V1 and V2 peptides also stabilized the HLA-E complex, whereas the control N peptide did not. Taken together, these studies demonstrate that the V{beta}1- and V{beta}2-derived nonapeptides bind HLA-E and stabilize the HLA-E/{beta}2 µ complex.

Induction of V{beta}-specific and HLA-E-restricted CD8+ CTLs

In previous studies we showed that V{beta}-specific CD8+ T cells exist in human peripheral blood, which can be induced to kill autologous CD4+ T cells in a TCR V{beta}-specific fashion (16). The MHC restricting element(s) for these CD8+ CTL are unknown. However, similar TCR V{beta}-specific murine CD8+ CTL are restricted by Qa-1 (12, 13), a MHC molecule known to be functionally equivalent to HLA-E. Therefore, to address the potential immunologic significance of V{beta} peptide binding to HLA-E, we asked whether V{beta} peptide-loaded C1R-E cells can induce CD8+ CTL capable of recognizing and lysing targets in a V{beta}-specific manner. PBL-derived CD8+ T cells were stimulated twice by either untreated C1R-E or by V{beta} peptide-loaded C1R-E cells and then further expanded in IL-2 containing medium. Unprimed control CD8+ cells cultured under similar conditions served as an additional control.

To characterize the TCR V{beta} specificity of the various CD8+ lines generated, we tested their CTL activity toward C1R-E cells loaded with V1 peptides (C1R-E/V1), V2 peptides (C1R-E/V2), or control N peptides (C1R-E/N) in a 51Cr-release assay. As shown in Fig. 3GoA, unprimed CD8+ T cells displayed a low level of non-TCR V{beta}-specific CTL activity toward all targets (9.1–10.9% specific lysis). Similarly, CD8+ T cells primed by the C1R-E cells also showed a low level of nonspecific cytotoxicity (18–19%) toward all targets. In contrast, the C1R-E/V1- or C1R-E/V2-primed CD8+ T cells generated CTLs with specific activity toward their respective C1R-E/V1 or C1R-E/V2 targets (36.5 ± 4.4 and 32.4 ± 4.1, respectively; p < 0.01 for both, by Student’s t test), compared with the background NK-like activity (9.1–19%) seen toward the other targets. In complementary experiments, we tested the CTL activity of these various CD8+ lines toward the C1R mock transfectants loaded with the different peptides. Indeed, no significant specificity was detected toward any of these targets (Fig. 3GoB), further supporting the likelihood that HLA-E is the restricting element.



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  		FIGURE 3. V{beta} peptide-specific CD8+ CTLs can be induced by C1R-E/V{beta} priming. CD8+ CTLs were generated by priming purified CD8+ T cells with V{beta} peptide-loaded C1R-E. Control CD8+ T cells were stimulated with untreated C1R-E or C1R, or grown in culture medium alone. The targets consisted of C1R-E or C1R loaded with either no peptide or 100 µM of the various peptides. Killing was measured in a 4-h 51Cr-release assay, and the data are representative of at least three independent experiments all performed in triplicate and at an E:T ratio of 20:1. A, Lysis of the various C1R-E targets by either unprimed CD8+ T cells or by CD8+ CTLs primed as described above. B, Lysis of the different C1R targets by either unprimed CD8+ T cells or by the various CD8+ CTLs. For Ab blocking experiments, either C1R-E/V1- (C) or C1R-E/V2 (D)-primed CD8+ CTLs were preincubated with 50 µg/ml OKT8, or target cells were preincubated with 50 µg/ml W6/32 before the cytotoxic assay was performed.

 
To determine whether the V{beta}-specific cytotoxicity mediated by C1R-E/V{beta}1- or C1R-E/V{beta}2-primed CD8+ CTL lines was dependent on CD8 molecules and involved recognition of HLA-E, we tested the capacity of anti-CD8 (OKT8) and anti-MHC class I (W6/32) mAbs to block cytotoxicity directed toward the various targets. Both anti-CD8 and W6/32 mAbs blocked the V{beta}1- (Fig. 3GoC) and V{beta}2 (Fig. 3GoD)-specific killing. In contrast, the background killing of either C1R-E/N or the noninducing C1R-E/V{beta} targets was not significantly blocked. In these experiments the background killing of 14–22% was probably mediated by NK-like receptors, which are known not to be blocked by these mAbs. Furthermore, as shown in Fig. 4Go, A and B, both the V{beta}1- and V{beta}2-specific CTL activity was highly dependent on the E:T ratio (Fig. 4Go, A and B), and it was evident even at lower concentrations than 100 µM peptide, but reached a plateau at the 100–150 µM range (Fig. 4Go, C and D).



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  		FIGURE 4. Plots of either E:T ratio or peptide concentration vs specific lysis. The lysis of C1R-E target cells loaded with 100 µM of indicated peptide by CD8+ CTLs primed with either C1R-E/V1 (A) or C1R-E/V2 (B) measured at varying E:T ratios. The lysis of target cells loaded with varying concentrations of each peptide by CTLs primed with either C1R-E/V1 (C) or C1R-E/V2 (D) measured at an E:T ratio of 20:1. Killing was measured in a 4-h 51Cr-release assay, and the data are representative of at least three independent experiments all performed in triplicate.

 
CD8+ CTLs induced by C1R-E/V1 or V2 peptide-loaded cells specifically kill TCR V{beta}1+ or V{beta}2+ CD4+ T cells, respectively

To further address the potential immunologic significance of V{beta} peptide binding to HLA-E, we asked whether CD8+ T cell lines induced by C1R-E/V{beta} peptide are capable of lysing normal CD4+ T cell targets in a V{beta}-specific manner. For these studies, peripheral CD8+ T cells were stimulated twice by either C1R-E/V1 or C1R-E/V2 cells, as above, and tested for CTL activity toward autologous CD4+ T cell clones expressing either V{beta}1 or V{beta}2. CD8+ T cells either unprimed or stimulated by untreated C1R or C1R-E were also tested as controls. As seen in Fig. 5GoA, the CD8+ CTL line primed by either V1 or V2 peptide-loaded C1R-E exhibited specific cytotoxic activity against their respective V{beta}1+ or V{beta}2+ CD4+ T cell line, while showing low activity toward the other V{beta} line. In addition, the V{beta}-specific lysis of the CD4+ T cells was inhibited by the mAbs OKT8 and W6/32 (Fig. 5Go, B and C). Taken together, these data suggest that the CD8-mediated TCR V{beta}-specific CTL killing of CD4+ T cells is restricted by HLA-E/V{beta}-peptide complexes.



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  		FIGURE 5. CD8+ CTLs can be induced by C1R-E/V{beta} peptide priming to kill autologous V{beta}1 or V{beta}2 CD4+ T cell lines. CD8+ CTLs were generated by priming purified CD8+ T cells with peptide-loaded C1R-E, as described in Materials and Methods. The targets consisted of either autologous V{beta}1 or V{beta}2 CD4+ T cell lines. The lysis of target cells was detected in an 8-h 51Cr-release assay at an E:T ration of 20:1, and the data are representative of at least three independent experiments all performed in triplicate. A, Lysis of autologous V{beta}1 and V{beta}2 targets by CD8+ T cells either unprimed or primed by C1R, C1R-E, C1R-E/V1, or C1R-E/V2. For Ab blocking studies, either the C1R-E/V1-induced (B) or C1R-E/V2-induced CD8+ CTL (C) were preincubated with 50 µg/ml OKT8 or, alternatively, the targets were preincubated with 50 µg/ml W6/32 before the cytotoxic assay was performed.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is known that the human HLA-E molecule preferentially binds peptides derived from the signal sequences of many MHC class I molecules (25, 26) and that these complexes function as ligands for CD94/NKG2 receptors. In this way it is thought that HLA-E molecules function, in part, to regulate NK cells (27, 28, 44). However, it is also known that the murine Qa-1 binds other peptides and functions, like MHC class Ia molecules, to restrict {alpha}{beta} CD8+ CTLs and trigger lysis of targets. For example, both HLA-E and Qa-1 bind peptides derived from other sources including heat shock, bacterial, or viral proteins and can interact with {alpha}{beta} TCRs expressed on CD8+ T cells to trigger conventional CTL function (20, 35, 36, 37, 38). The peptides that preferentially bind to HLA-E or Qa-1 are nonamers, which have as dominant anchor residues a Leu at P9 and a Leu or Met at P2 (42). Such peptide motifs, first identified in MHC leader sequence peptide, are present in a variety of other self-peptides. In the present report, we used these putative biochemical determinants of HLA-E binding to identify TCR V{beta}1 and V{beta}2 peptides, which bind to HLA-E. Furthermore, we showed that these HLA-E/V{beta}-peptide complexes expressed on the cell surface can induce V{beta}-specific human CD8+ CTL function.

We used the C1R-E cells to provide direct evidence that TCR V{beta}1 and TCR V{beta}2 nonapeptides bind to HLA-E molecules. First, the binding of these peptides was documented by changes in the surface immunofluorescent staining profiles of cold-treated C1R-E cells using mAb W6/32. Following loading with the two TCR V{beta} peptides or the B7 peptide, we observed an increase in immunostaining (Fig. 1Go) that was not observed on mock transfectants or after staining with HLA-C-specific alloantisera. Second, binding of these TCR V{beta} peptides was confirmed using an assembly assay in which an anti-GFP mAb was used to specifically immunoprecipitate the chimeric HLA-E/GFP molecule from C1R-E lysates. In this assay, preincubating with peptides known to physically bind HLA-E (like B7) increases the thermal stability of HLA-E/{beta}2 µ complexes. A similar increase in the thermal stability of the complex was also observed following loading with V1 and V2 peptides, but not with the control N peptide (Fig. 2Go).

The potential immunological significance of this V{beta} peptide binding to HLA-E was investigated by experiments demonstrating that CD8+ T cells isolated from PBL of normal donors could be induced in vitro by C1R-E cells loaded with the V{beta}1 or V{beta}2 peptide to specifically lyse targets loaded with the respective inducing V{beta} peptide, but not the other peptides. The data are consistent with the idea that HLA-E-restricted CD8+ T cell precursors exist in human peripheral blood and can be further induced to expand by HLA-E/V{beta}-peptide complexes. Of perhaps more significance were the findings (Figs. 3Go and 4Go, A and B) that CTLs induced by C1R-E/V1 also specifically lysed activated autologous V{beta}1+ CD4+ T cell targets. Furthermore, in reciprocal experiments using C1R-E/V2 for priming, similar results were obtained. Taken together, these results demonstrate that both V{beta} peptides can be presented by the HLA-E molecule and induce V{beta}-specific CD8+ CTLs.

Evidence that the CD8+ T cell-mediated killing was, in fact, a consequence of {alpha}{beta} TCR triggering came from Ab blocking data demonstrating the ability of both anti-CD8 and W6/32 to inhibit this specific killing (Figs. 3Go, C and D, and 5, B and C). Although there was a background cytotoxic activity observed against all targets, independent of peptide loading or TCR V{beta} expression, no inhibition of this nonspecific NK-like killing was seen with either mAb. In this regard, it is theoretically possible that HLA-E/V{beta}-peptide complexes could be recognized by CD94/NKG2 receptors to either inhibit CD8+ T cell-mediated killing or activate killing, if CD94/NKG2C or CD94/NKG2E receptors were expressed on the CTLs. However, the fact that the V{beta}-specific killing was inhibited by anti-CD8 mAbs argues strongly that it was not secondary to such paradoxical NK activation.

The conceptual origins of the current study emerged from various experimental data (7, 8, 9, 10, 11, 12, 13), as recently reviewed by us (45), suggesting that CD8+ T cells regulate normal immune responses and play a critical role in the regulation of autoimmune responses. In particular, evidence has emerged that such cells mediate resistance to myelin basic protein (MBP)-induced experimental autoimmune encephalomyelitis (EAE). In this model, Qa-1-restricted TCR V{beta}-specific CD8+ T cells emerge during the course of EAE and mediate this protection. In vivo depletion of CD8+ T cells abrogates this protection. Moreover, CD8+ T cell hybridomas isolated from EAE-recovered mice are specifically activated by MBP-reactive CD4+ cells in a TCR V{beta}-specific and Qa-1-restricted manner. These hybridomas were inhibited by anti-CD8, anti-Qa-1, and anti-TCR mAbs. Of potential therapeutic interest is the recent finding that such RTC can also be induced by T cell vaccination with MBP-reactive CD4+ V{beta}8+ T cell clones (13). Furthermore, this T cell vaccination-induced protection from EAE was abolished by depleting mice of CD8+ T cells (46).

In addition, a variety of studies in the human immune system provide evidence that recognition of TCR-derived elements may be integral to the specificity of immune regulation between T cell subsets. For example, it was shown that CD8+ T cells raised to V{beta}2+ CD4+ T cell clones, in vitro, differentiated into CTL, which specifically lyse independently isolated autologous CD4+ T cell targets expressing V{beta}2+ but not other V{beta} chains. Moreover, in reciprocal experiments, CD8+ T cells raised against autologous CD4+ T cell clones with different TCR V{beta} usage were not cytotoxic to V{beta}2+ cells. Taken together, these experiments were interpreted to suggest that some RTC can recognize and kill autologous CD4+ T cells based, at least in part, on the recognition of the V{beta} chain expressed (16). Interestingly, in these early studies the V{beta}2-specific cytotoxicity of one CTL clone TC12/7 was not blocked by the mAb W6/32. However, our current findings suggest that this mAb can block the V{beta}-specific CTL killing. Therefore, this prior observation could have reflected a unique MHC restriction, other than class Ia or HLA-E, of a particular CTL clone. Other investigators have confirmed that TCR-specific human CD8+ CTLs that recognize and kill autoreactive CD4+ T cell clones could be identified (17, 47). In these studies some populations of CD8+ CTLs were anti-idiotypic, whereas others killed autologous CD4+ T cells in a TCR V{beta}-specific manner or lysed all activated clones independent of TCR V{beta} expression (anti-ergotypic; Ref. 48). In both studies the killing of the autologous CD4+ T cells was blocked by W6/32 mAb. The study reported here represents the first demonstration that HLA-E can serve as an MHC restricting element for TCR V{beta}-specific CD8+ CTLs, but does not rule out the possibility that other MHC class I molecules may also restrict RTCs that recognize TCR-derived peptides.

Although the predominant self-peptides presented by HLA-E and Qa-1 on resting T cells are leader sequence of certain MHC class I molecules (23, 25), it is known that a variety of other self-peptides that are up-regulated on certain cells can also bind to these molecules (35, 36, 37, 38). We hypothesize that resting human CD4+ T cells may preferentially express HLA-E/MHC class Ia leader peptide complexes at rest, and following activation other peptides (including V{beta} peptides) may bind. In this regard, it is known that the Qa-1 molecule, and presumably HLA-E, display little charge heterogeneity on resting lymphocytes, but that the level of charge heterogeneity is increased after activation, due, in part, to altered sialylation of their linked oligosaccharides (15). However, the biological significance of these glycosylation variants, and whether they may facilitate subtle conformational changes at the sites involved in ligand binding, is currently unknown.

We envision that these putative HLA-E or Qa-1/TCR V{beta} complexes will be recognized by {alpha}{beta} TCRs on the surface of the RTC that we have described. Moreover, recent data have shown that CD94/NKG2 receptors are also expressed on a large fraction of CD8+ T cells where their function is not completely understood. The CD94/NKG2 receptors expressed on CD8+ T cells may function either as killer inhibitory or activating receptors, and perhaps maintain CTL homeostasis by regulating signaling from the {alpha}{beta} TCR (49, 50). In this regard, in preliminary studies we have directly tested whether these HLA-E/V{beta} peptides could inhibit NK cell function. We found that the cytotoxicity of the CD94/NKG2A-expressing NK92 cell line toward C1R-E was not significantly inhibited by either V1 or V2 peptide loading (data not shown). These observations are consistent with studies showing that not all peptides confer protection against lysis by CD94/NKG2A+ NK cells (51, 52). Thus, this heterodimer can functionally discriminate between HLA-E/ligand complexes containing peptides with various substitutions at nonanchor positions. Therefore, hypothetically the functional consequence might be that resting CD4+ T cells, which preferentially express HLA-E/class I leader peptide, may for the most part trigger inhibitory signals via CD94/NKG2A ligation, whereas upon activation, the prevalence of HLA-E/V{beta} peptide complexes expressed may reach a threshold sufficient to activate CTLs through TCR ligation without triggering inhibitory signals via CD94/NKG2A.

In summary, the data presented here provide direct evidence in humans that TCR V{beta} peptide presentation by HLA-E can induce TCR V{beta}-specific CTLs. Similar Qa-1-restricted RTC have been described in murine systems where they have been shown to be involved in the regulation of autoimmune disease. Further studies of the immunoregulatory pathway, involving the induction of similar V{beta}-specific HLA-E-restricted human RTC, may provide insight into the immunopathophysiology of human autoimmune disease and may suggest therapeutic strategies to treat these disorders.


   Acknowledgments
 
We thank Drs. Xiaoqin Qu and Jianshe Fan for their technical assistance, and Dr. Robert Winchester for kindly providing the HLA-E cDNA construct.


   Footnotes
 
1 This work was funded in part by National Institutes of Health Grants U19 AI46132, R29 AI39630, and RO1 AI4492, and by National Multiple Sclerosis Society Grant RG2938. Back

2 J.L. and I.G. contributed equally to this manuscript. Back

3 Address correspondence and reprint requests to Dr. Leonard Chess, Department of Medicine, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, PH8E, Suite 101, New York, NY 10032. E-mail address: lc19{at}columbia.edu Back

4 Abbreviations used in this paper: RTC, regulatory CD8+ T cell; C1R-E, C1R transfected with HLA-E; MFI, mean fluorescence intensity; GFP, green fluorescent protein; {beta}2 µ, {beta}2-microglobulin; MBP, myelin basic protein; EAE, experimental autoimmune encephalomyelitis. Back

Received for publication April 26, 2001. Accepted for publication August 7, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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