Chimeric ß2 microglobulin/CD3{zeta} polypeptides expressed in T cells convert MHC class I peptide ligands into T cell activation receptors: a potential tool for specific targeting of pathogenic CD8+ T cells

Alon Margalit1,2, Sigal Fishman1, Dikla Berko1, Jan Engberg3 and Gideon Gross1,2

1 MIGAL, Galilee Technology Center, South Industrial Zone, Kiryat Shmona, 11016, Israel 2 Department of Biotechnology, Tel Hai Academic College, Upper Galilee, 12210, Israel 3 Department of Pharmacology, The Royal Danish School of Pharmacy, Jagtvejen 160,DK 2100 Copenhagen, Denmark

Correspondence to: G. Gross, MIGAL, Galilee Technology Center, South Industrial Zone, PO Box 831 Kiryat Shmona 11016, Israel. E-mail: gidi{at}migal.org.il
Transmitting editor: I. Pecht


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CD8+ T cells are key mediators of transplant rejection and graft-versus-host disease and contribute to the pathogenesis of autoimmune diseases. We tested whether TCR ligands can be converted into T cell activation receptors, redirecting genetically modified T cells at pathogenic CD8+ T cells. For this purpose we exploited the ability of the non-polymorphic ß2 microglobulin light chain to pair with all MHC class I heavy chains. In this report we describe the design and expression in a T cell hybridoma of two modalities of ß2 microglobulin polypeptides, fused with the transmembrane and intracellular portion of CD3{zeta} chain. In the absence of a particular antigenic peptide, the chimeric product associates with different endogenous MHC class I heavy chains and triggers T cell activation upon heavy chain cross-linking. When an antigenic peptide is covalently attached to the N-terminus of the chimeric polypeptide, transfectants express high level of surface peptide–class I complexes and respond to antibodies and target T cells in a peptide-specific manner. Our results provide the basis for a universal genetic approach aimed at antigen-specific immunotargeting of pathogenic CD8+ T cells.

Keywords: adoptive immunotherapy, chimeric MHC protein, cytotoxic T lymphocyte, T cellre-programming, T cell specificity


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CD8+ T cell specificity is dictated by the clonotypic TCR, which recognizes short antigenic peptides presented on MHC class I molecules. These proteins are non-covalent heterodimers, comprising a membrane-integral, highly polymorphic {alpha} heavy chain, structurally divided into three extracellular domains ({alpha}1–{alpha}3), and a non-membrane attached, non-polymorphic ß2 microglobulin 2m) light chain. The membrane-distal {alpha}1 and {alpha}2 domains form the peptide-binding groove, whereas the membrane-proximal {alpha}3 domain contributes most of the interactions with ß2m. On the T cell membrane, the TCR associates non-covalently with a group of invariant proteins, collectively termed CD3, which transduce T cell activation signals via immunoreceptor tyrosine-based activation motifs (ITAMs) found in their cytoplasmic regions. The CD3{zeta} chain, which appears primarily as a disulfide-linked homodimer, harbors three ITAMs and plays a pivotal role in TCR signaling. When fused with heterologous extracellular recognition units and expressed in T cells, CD3{zeta} ITAMs transduce T cell activation signals upon engagement with non-MHC ligands [see (13) for review]. Such chimeric receptors have been used to redirect T cell specificity at will in a wide range of CD8+ and CD4+ T cells, both in vitro and in vivo.

MHC class I alloreactive CD8+ cytotoxic T lymphocytes (CTLs) play an essential role in acute and chronic allograft rejection. Early studies have suggested that alloreactive CTLs can recognize certain class I epitopes induced by bound peptides irrespective of their precise amino acid composition (4,5). The notion that CTL alloreactivity can, in part, be directed at peptide-independent epitopes on the foreign MHC molecule has recently gained further support (6). However, a certain degree of peptide selectivity is exhibited by at least some alloreactive CTL clones (79). Autoreactive CD8+ CTLs are involved in the pathogenesis of autoimmune diseases such as insulin-dependent diabetes mellitus (IDDM) and multiple sclerosis (MS). Insight to the role of these cells mostly comes from studies of rodent models, which have been useful in recent years in identifying autoantigen-derived peptides recognized by autoreactive CTL clones: peptides from insulin B chain (10) and GAD65 (11) were implicated in initiation of IDDM in NOD mice while CD8+ T cell clones specific to peptides from myelin basic protein (12) and myelin oligodendrocyte glycoprotein (13) were encephalitogenic in mouse models for MS.

We have reasoned that endowing MHC class I molecules expressed in T cells with T cell activation capacity, through engraftment of CD3{zeta} ITAMs, could convert these molecules into activation receptors directed at specific TCRs expressed on class I restricted pathogenic CD8+ CTLs. Most recently, transgenic T cells expressing chimeric MHC class II/CD3{zeta} molecules with a genetically linked autoantigenic peptide were shown to specifically target autoreactive CD4+ T cells and suppress autoimmune encephalomyelitis in model mice (14). Here we report the assembly and expression of genetic constructs encoding single chimeric ß2m/CD3{zeta} polypeptides and double chimeric peptide/ß2m/CD3{zeta} polypeptides, the latter harboring pre-selected antigenic peptides covalently linked to the N-terminus of ß2m/CD3{zeta}. A T cell hybridoma-based system was designed to evaluate the capacity of these chimeras to confer on transfected cells the ability to respond to antigenic stimuli, which are analogous to those exerted by alloreactive and autoreactive CD8+ T cells. We show that these polypeptides functionally associate with MHC class I heavy chains on the surface of transfected T cells and are capable of redirecting T cell recognition in a peptide-specific manner.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
DNA vectors and plasmids
The eukaryotic expression vector pBJ1-Neo was previously described (15). The nuclear factor of activated T cells (NFAT)-lacZ reporter construct contains the lacZ gene under the control of the NFAT-binding element of the human IL-2 enhancer (16), and was a kind gift from Dr N. Shastri, University of California Berkeley.

Assembly of genetic constructs
ß2m/CD3{zeta}. Full length human ß2m (hß2m) was cloned from Jurkat cells by RT–PCR with the forward primer 30287: 5' GGG TCT AGA GCC GAG ATG TCT CGC TCC GTG 3' and the reverse primer 4337: 5' G CTG GCT CGA GGG CTC CCA TCT CAG CAT GTC TCG ATC CCA CTT 3'. The transmembrane and cytoplasmic portion of mouse CD3{zeta} were cloned by RT–PCR from mRNA of MD45 cells with the forward primer 4840: 5' GAG CCC TCG AGC CAG CCC ACC ATC CCC ATC CTC TGC TAC TTG CTA GAT 3' and the reverse primer 27246: 5' GCG GAA TTC TTA GCG AGG GGC CAG GGT 3'. Both products were cloned in a single step into pBJ1-Neo, producing plasmid 21-2.

Peptide/ß2m/CD3{zeta}. A gene segment encoding the leader peptide of hß2m was cloned by PCR with the forward primer 30287 and either the reverse primer 6086: 5' CGC GGA TCC GCC ACC TCC GAT CAA CCG CCC TTC ATA ATC ACT AGC CTC AAG GCC AGA AAG 3' or 6087: 5'CGC GGA TCC GCC ACC TCC AAT TAG ATT ACC AGT ACT CTC AAA AGC CTC AAG GCC AGA AAG 3'. The mature hß2m protein was cloned by PCR with the forward primer 5187: 5'CGC GGA TCC GCC ACC TCC AAT TAG ATT ACC AGT ACT CTC AAA AGC CTC AAG GCC AGA AAG 3' and the reverse primer 4337. These two products, together with the fragment encoding CD3{zeta}, were cloned into pBJ1-Neo, producing plasmids 406-20 (with primer 6086) and 407-24 (with primer 6087).

Antibodies
mAbs to mouse H-2Kk (clone AF3-12.1) and H-2Kd (clone SF1-1.1) were from PharMingen (San Diego, CA). 2C11 is a hamster mAb specific to mouse CD3{epsilon}. Fab13.4.1 is specific to the influenza virus peptide HA255–262 in the context of Kk (17). mAb against hß2m (clone BM-63) was from Sigma (St Louis, MO). FITC-labeled hamster anti-mouse Fas ligand (clone FLIM58) was purchased from MBL (Nagoya, Japan).

Cell lines
MD45 is an H-2Db-allospecific, H-2k/d mouse CTL hybridoma of BALB/c origin (18). HK9.5-24 and HK8.3-5H3 are MHC class I-restricted T cell hybridomas of BALB/k origin (19) and were a generous gift from Dr A. Stryhn, University of Copenhagen.

Flow cytometry
Cells (106) were washed with PBS and incubated for 30 min on ice with 100 µl first (or control) antibody at 10 µg/ml. Cells were then washed and incubated on ice with 100 µl 1:100 dilution of goat anti-mouse IgG (Fab-specific) FITC conjugated polyclonal antibodies (Sigma) for 30 min, washed, resuspended in PBS and analyzed with FACSCalibur (BD Biosciences, Mountain View, CA). Quantitative analysis of cell surface antigens was performed with QIFIKIT (DAKO, Carpinteria, CA) according to the manufacturer’s instructions. For double staining analysis, cells were washed three times in PBS, resuspended in PBS containing 1 µM Cell-Tracker Orange (CTO) (Molecular Probes, Eugene, OR) and incubated for 1 h at 37°C. Cells were then washed three times and resuspended in DMEM, 10% FCS and supplements at 1 x 106 cells/ml.

Co-stimulation experiments
Cells were washed twice with PBS and resuspended with their growth medium (depleted of selective drugs) at 5 x 105 cells/ml. A 0.5 ml aliquot of each interacting cell (for 1:1 ratio) was added to 24-well plates in triplicates and incubated overnight at 37°C.

ß-Galactosidase enzymatic assay
Cells at 5 x 105/ml were incubated overnight in 24-well plates pre-coated with antibodies (usually at 5 µg/ml) or with target cells at 5 x 105 cells/ml. Twenty to twenty-four hours post-stimulation, cells were harvested in 1.5 ml microfuge tubes and centrifuged at 7000 r.p.m. (4,500 g) for 2 min at room temperature. Supernatant was collected for an IL-2 production assay. Pellet was washed three times with fresh PBS and assayed with ß-Galactosidase (ß-Gal) Enzyme Assay System Kit (Promega, Madison, WI) according to the manufacturer’s instructions. The assay was developed in 96-well plates and was read with SLT Spectra ELISA Reader (SLT-Labinstruments, Salzburg, Austria) at 415 nm, against a standard ß-Gal curve.

In-cell X-Gal staining
Cells in 96-well plates were washed twice with PBS and fixed with 0.25% glutaraldehyde for 15 min, washed three times in PBS, incubated for 4 h with 100 µl of X-Gal solution [0.2% X-Gal, 2 mM MgCl2, 5 mM K4Fe(CN)6.3H2O, 5 mM K3Fe(CN)6 in PBS] and scored under the microscope for blue staining.

IL-2 production assay
CTL-L indicator cells were washed three times and resuspended at 2 x 105 cells/ml. An aliquot of 50 µl of cells was placed in a microtiter plate well in the presence of 50 µl supernatant to be assayed. Following 20 h incubation, 10 µl of 5 mg/ml MTT (Sigma) was added and plates were incubated for an additional 4 h. Precipitate was dissolved with 100 µl of isopropanol containing 0.04 M HCl. Plates were read at 570 nm with 630 nm as reference in a SLT Spectra ELISA Reader.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Design and construction of chimeric ß2m/CD3{zeta}
Expression plasmid 21-2 encodes full hß2m, linked to mouse CD3{zeta} chain transmembrane and cytoplasmic region via a short peptide bridge. This bridge comprises the 13 membrane-proximal amino acids of the extracellular portion of HLA-A2, LRWEPSSNPTIPI, which encompass the proline-rich connecting peptide. Based on HLA-A2 crystallography data (20), this sequence was predicted to bridge hß2m to the cell membrane with minimal structural constraint. Human ß2m efficiently associates with the majority of mouse MHC class I heavy chains tested (21,22) and it serves as a useful marker for cell surface expression on transfected mouse cells. Figure 1 shows schemes of plasmid 21-2 and its expected chimeric product.



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Fig. 1. Schemes of the genetic constructs (A) and the expected single and double chimeric ß2m polypeptides in the context of an MHC class I heavy chain on the cell membrane (B). A partial restriction map of the genetic constructs, including major restriction sites used for cloning is depicted. Abbreviations: pr, promoter; lead, leader peptide; li, linker peptide; p, antigenic peptide; br, bridge; tm+cyt, transmembrane and cytoplasmic region. The antigenic peptides are either HA255–262 or NP50–57 of the influenza virus. Ellipses represent ITAMs in the cytoplasmic portion of the {zeta} chain.

 
Function of chimeric ß2m/CD3{zeta}
If the chimeric ß2m/CD3{zeta} encoded by plasmid 21-2 associates on the surface of transfected T cells with endogenous heavy chains, it is expected to form a functional MHC class I receptor complex, capable of T cell activation. This modality allows the evaluation of the response of chimeric class I molecules to heavy chain cross-linking by immobilized allele-specific antibodies, a stimulus corresponding to that induced by allo reactive T cells. To test this prediction, MD45 cells were transfected with plasmid 21-2 DNA and one subclone of a stable transfectant, designated 412-19-1.6, was further analyzed. 412-19-1.6 cells were incubated in the presence of two immobilized class I heavy chain-specific mAb, anti-H-2Kk (AF3-12.1) and anti-H-2Kd (SF1-1.1). T cell activation was monitored by IL-2 production. As shown in Fig. 2, 412-19-1.6 cells were activated by both anti-heavy chain antibodies and an anti-CD3{epsilon} mAb, whereas MD45 cells were only stimulated by the latter. These results clearly demonstrate that the chimeric ß2m/CD3{zeta} functionally pairs on the cell surface with different class I heavy chains, and transduce an activation signal comparable in intensity to that triggered by the TCR.



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Fig. 2. Activation of 412-19-1.6 cells expressing the single chimeric ß2m/CD3{zeta} genetic construct and the parental MD45 T hybridoma cells by anti-MHC class I heavy chain antibodies. Both transfectants and parental cells were incubated with solid-phase antibodies: anti-H-2Kk (AF3-12.1), anti-H-2Kd (SF1-1.1), anti-CD3{epsilon} (2C11) and an irrelevant antibody. Supernatants were subjected to a standard IL-2 bioassay. One unit of IL-2 is defined as the inverse of the dilution that supported 50% growth of the CTL-L indicator cells.

 
Design, construction and surface expression of double chimeric peptide/ß2m/CD3{zeta}
The above data indicate that chimeric ß2m/CD3{zeta} assembles with different MHC class I heavy chains and converts class I molecules into T cell activation receptors. A critical parameter concerning the potential use of chimeric MHC class I molecules in adoptive immunotherapy, which is particularly relevant to autoimmune diseases, is the ability of antigenic peptides presented by these molecules to confer reactivity against the distinct set of peptide-specific, class I-restricted TCRs expressed by pathogenic T cells. To this end we genetically linked two different antigenic peptides to the N-terminus of the chimeric ß2m/CD3{zeta}. This was done through the assembly of plasmids 406-20 and 407-4, which respectively encode two H-2Kk-restricted peptides, derived from influenza virus: nucleoprotein 50–57 (NP50–57, SDYEGRLI) and hemagglutinin 255–262 (HA255–262, FESTGNLI). These peptides are recognized in the context of Kk by the T cell hybridomas HK9.5-24 (NP50–57) and HK8.3-5H3 (HA255–262) (19). The sequences encoding each of the two peptides were cloned downstream to the leader peptide segment. A DNA stretch encoding the peptide linker G4S(G3S)2 (23) was inserted between the antigenic peptides and the mature ß2m sequences. Maps of plasmids 406-20 and 407-4 and the expected double chimeric protein products are shown in Fig. 1.

The NFAT-lacZ reporter gene (16) allows the unambiguous monitoring of activation of the IL-2 gene in transfectants by antigen-specific T cells, which are also of mouse origin. DNA of expression plasmids 406-20 and 407-4, together with the NFAT-lacZ reporter gene, were introduced sequentially into MD45 cells, to yield transfectant series 425 and 427, respectively. Figure 3 shows the flow cytometry analysis of cell surface expression of the chimeric polypeptides on two clones, 425-44 and 427-24. As expected, hß2m is highly expressed on both transfectants, but not on MD45 parental cells. However, this staining is not in itself indicative of pairing of the chimeric polypeptides with class I heavy chains. The unique binding properties of Fab13.4.1, which only recognizes H-2Kk in complex with HA255–262 (17), render it a specific marker for correct pairing. Indeed, this Fab strongly stains 427-24 cells, which express the HA255–262 peptide, but not 425-44 cells, which express NP50–57. These findings indicate that, at least for 427-24 cells, the antigenic HA255–262 peptide is properly presented by the restricting H-2Kk molecule.



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Fig. 3. Flow cytometry analysis of MD45 parental cells and two of the transfectants expressing double chimeric peptide/ß2m/CD3{zeta} genetic constructs: 427-24, expressing peptide HA255–262 and 425-44, expressing NP50–57. Cells were analyzed with antibodies against H-2Kk (AF3-12.1), hß2m (BM-63) and HA255–262–Kk complex (Fab13.4.1) and detected with goat anti-mouse IgG (Fab-specific)–FITC-conjugated polyclonal antibodies. Staining with secondary antibody only is presented by the open histograms.

 
Expression of specific CD8+ T cell ligands at a minimal level, which can trigger conventional T cell activation, may not suffice upon encountering target T cells, when these ligands are to serve as T cell activation receptors. We therefore went on to evaluate the actual density of specific complexes on clone 427-24, using a commercial kit for quantification of cell surface antigens and both Fab13.4.1 and the anti-H-2Kk mAb. This analysis (Table 1) reveals an average number of more than 5700 complexes per cell, constituting ~20% of total surface H-2Kk molecules expressed by these cells.


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Table 1. Quantitative analysis of surface antigens of transfectants 425-44 and 427-24 and parental MD45 cells
 
Functional analysis of double chimeric peptide/ß2m/CD3{zeta}
In order to assess the ability of the double chimeric peptide/ß2m/CD3{zeta} to activate transfectants in a peptide-specific manner, we first used Fab13.4.1. As shown in Fig. 4, immobilized Fab only activated 427-24 cells, which express the HA255–262 peptide, but not 425-44 cells, expressing NP50–57. In this assay, both transfectants responded equally well to the anti-H-2Kk mAb. Interestingly, the percentage of 427-24 cells activated by the anti-Kk mAb was comparable to that achieved by Fab13.4.1.



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Fig. 4. Activation of transfectants expressing double chimeric HA255–2622m/CD3{zeta} and NP50–572m/CD3{zeta} by the HA255–262–Kk complex-specific Fab13.4.1. Transfectants 425-44 (425), expressing the NP50–57 peptide and 427-24 (427), expressing the HA255–262 peptide, were incubated overnight in triplicate wells in the presence of the immobilized Fab, or anti-H-2Kk mAb AF3-12.1 (positive control for activation), or with no antibody and were then subjected to an in-cell X-Gal staining. Results are presented as percentage of activated cells, which were stained blue.

 
The ability of transfectants to be activated by peptide-specific, class I-restricted T cells is of critical importance, especially in the context of autoimmune diseases. To evaluate this ability we co-incubated transfectants 425-44 and 427-24 with the HK9.5-24 and HK8.3-5H3 T cell hybridomas and monitored cellular activation by the ß-Gal enzymatic assay. The results are presented in Fig. 5 as ß-Gal units. Repeating experiments (n = 15) showed with high significance (P < 0.01 for both clones) that only incubation of a transfectant with a target cell displaying matching peptide specificity triggered cellular response. The level of response following incubation with the non-matching target did not exceed with statistical significance the basal level of activation, which was observed under non-stimulatory conditions. These results demonstrate that peptide/ß2m/CD3{zeta} polypeptides confer on transfected T cells the ability to respond to peptide-specific, class I-restricted T cells.



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Fig. 5. Activation of T cells expressing double chimeric peptide/ß2m/CD3{zeta} by target T cells. Transfectants 425-44, expressing the NP50–57 peptide and 427-24, expressing the HA255–262 peptide were each incubated at a 1:1 ratio with the T hybridoma target cells HK8.3-5H3 (5H3, specific for HA255–262-Kk) and HK9.5–24 (24, specific for NP50–57-Kk) and with no target cells (–). Specific activation of transfectants was monitored by a ß-Gal enzymatic assay, based on the NFAT-lacZ reporter gene. The figure compiles the results of 15 independent experiments.

 
The ability of genetically modified T cells to exert their function against target cells before they are disabled by the targets’ counter response is of crucial importance considering therapeutic application. We were therefore interested to evaluate the magnitude of response manifested by transfectants and target cells separately, following their co-culture. For these experiments we used two other MD45 transfectants, 797-10 (transfected with plasmid 406-20, encoding NP50–57) and 798-2 (expressing HA255–262 through plasmid 407-4), which do not express the NFAT-lacZ reporter construct. Both cells express a high level of hß2m, but only 798-2 is stained with Fab13.4.1 (Fig. 6A). Figure 6(B) shows that co-incubation of these two clones with the two target T cell hybridomas results in IL-2 secretion only in the appropriate combinations. In order to discriminate between the two cell populations, target cells were pre-stained with CTO and the cell mixture was reacted with an anti-Fas ligand antibody following termination of the co-incubation to evaluate the magnitude of response. A representative dot-blot FACS analysis of clone 797-10 is presented in Fig. 7(A) and Fig. 7(B) summarizes three independent experiments. It is evident from these data that the relative increase in the fraction of activated cells in both populations following co-incubation is comparable, varying between ~10- and 20-fold. These results suggest that engagement of genetically modified T cells with their targets bears similar functional consequences for both interacting cells.



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Fig. 6. Expression of double chimeric peptide/ß2m/CD3{zeta} by clones 797-10 and 798-2. (A) Flow cytometry analysis of 797-10 cells, expressing NP50–57 and 798-2, expressing HA255–262. Cells were analyzed with antibodies against hß2m (BM-63) and HA255–262–Kk complex (Fab13.4.1) and detected with goat anti-mouse IgG (Fab-specific)–FITC-conjugated polyclonal antibodies. Staining with secondary antibody only is presented by the open histograms. (B) IL-2 secreted into the assay medium following co-incubation of 797-10 and 798-2 with either HK8.3-5H3 (5H3, specific for HA255–262-Kk) or HK9.5-24 (24, specific for NP50–57-Kk) target T cell hybridomas. Results are presented as OD570 (with OD630 as a reference) of an MTT-based bio-assay using CTL-L indicator cells.

 


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Fig. 7. Direct evaluation of the response of transfectants and target cells following their co-incubation. (A) Representative double staining dot-blot analysis. (B) Summary of three indpendent co-stimulation experiments. The four different histogram patterns represent the four transfectant/target combinations. Response of the indicated (underlined) clones is presented in the left-hand panel and that of the underlined targets in the righ-hand panel. Results are given as percent of activated cells of each cell population in a given combination, as determined by dot-blot FACSCalibur statistical analysis. 797-10 (797) and 798-2 (798) cells were co-incubated overnight at a 1:1 ratio with CTO-pre-stained target cells HK8.3-5H3 (5H3) and HK9.5-24 (24). Activation was monitored using FITC-conjugated hamster anti-mouse Fas ligand mAb.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study we evaluated the capacity of MHC class I molecules supplemented with a T cell activation domain to redirect genetically modified T cells at class I-binding molecular and cellular targets. Such class I molecules can serve in adoptive immunotherapy to selectively target damage-inflicting alloreactive or autoreactive CD8+ T cell clones. For this purpose we have chosen to link the signaling domain from the CD3{zeta} chain to the ß2m light chain of the class I molecule. This design offers several functional and practical advantages over the use of the class I heavy chain, as was recently reported in the same context by Nguyen and Geiger (24). The major advantage stems from the fact that ß2m is non-polymorphic in humans and can pair with all allelic forms of the different class I heavy chain gene products. This is particularly important with regard to immunotargeting of class I alloreactive CTL clones in transplantation, as a single species of a ß2m-based construct can pair with all class I heavy chains on the expressing T cell, thus covering the entire class I makeup of the allograft or of the recipient (in treatment of graft versus host disease). When autoreactive CTL clones specific to a particular peptide–class I ligand are to be targeted, direct joining of the peptide to the restricting class I heavy chain, as was accomplished in antigen presenting cells for CTL induction (25,26), can also be envisaged. However, wide application of this configuration requires a large panel of expression plasmids, each encoding a distinct allelic product of a class I heavy chain gene. In contrast, a single ß2m expression cassette provides a universal scaffold for expression of any antigenic peptide and thus suffices for all specificities in all individuals.

The ß2m light chain is not an integral membrane protein, and fusion with an intracellular signaling component first requires its attachment to the cell membrane through an appropriate bridge. This bridge should retain proper positioning of the chimeric ß2m with regard to the class I heavy chain, so that the signal mediated by engagement of the class I molecule can be effectively transduced across the cell membrane. The first 3D structure of a class I heterodimer, obtained by Bjorkman et al. for HLA-A2 (20), is instructive in this regard. It appears that the non-crystallized, 13-amino-acid stretch, which includes the proline-rich connecting peptide at the C-terminus of the HLA-A2 {alpha}3 domain, spans an equivalent distance to the cell membrane as that required for ß2m. Our results from flow cytometry and T cell activation experiments indicate that this stretch indeed allows both correct pairing of chimeric ß2m with class I heavy chains and functional coupling of the class I molecule to the cellular activation pathway.

As the activation moiety we chose the entire CD3{zeta} intracellular domain, harboring three ITAMs. This T cell activation element has been utilized in numerous chimeric receptor studies and has been shown to be sufficient for conferring full T cell responsiveness. As evident from our data (Fig. 2), both anti-H-2Kk and anti-Kd mAb failed to elicit detectable secretion of IL-2 by the parental MD45 hybridoma cells. In contrast, transfectant 412-19-1.6 was highly activated by both antibodies in the apparent absence of any co-stimulation. These findings confirm that the CD3{zeta} activation domain, which is provided by the chimeric ß2m, converts class I molecules into T cell activation receptors. Previous studies demonstrated that ligation of natural MHC class I molecules on the surface of T cells by immobilized antibodies induce new phenotypes and functions [for review see (27)]. Of a special interest is the reported ability of class I ligation to induce IL-2 production in Jurkat cells, which requires PMA co-stimulation (2830). Our results show that expression of the chimeric ß2m/CD3{zeta} polypeptide is both necessary and sufficient for cellular activation through class I cross-linking as judged by IL-2 production.

In order to demonstrate peptide specificity of response, we linked two Kk-restricted peptides derived from influenza virus proteins to the N-terminus of the chimeric ß2m/CD3{zeta}, via a short flexible peptide linker. This strategy for epitope linking allows insertion of antigenic peptides into the peptide binding groove of class I molecules, both when added exogenously as a soluble peptide/ß2m protein produced in bacteria (23,3133), or as a cell-expressed protein following gene delivery (31,33). Flow cytometry analysis of transfectants 425-44, 427-24, 797-10 and 798-2, using anti-hß2m mAb, indicated that the peptide/ß2m/CD3{zeta} constructs were highly expressed on the cell surface. FACS analysis with Fab13.4.1 also revealed that HA255–262-Kk complexes were present on clones 427-24 and 798-2 but not on 425-44 or 797-10 (Figs 3 and 6A), confirming that, at least in the case of HA255–262, the antigenic peptide was properly associated with the class I heavy chain. Furthermore, using Fab13.4.1 we could determine that the absolute number of specific HA255–262-Kk complexes per cell is more than 5700, constituting 20% of all H-2Kk molecules expressed by these cells (Table 1). Indeed, when 427-24 cells were incubated with immobilized Fab13.4.1, the percentage of activated cells was comparable to that observed following their activation by the anti-H2-Kk mAb (Fig. 4). Activation of transfectants by their target T cells (Figs 5 and 7), and their ability to stimulate their targets (Fig. 7) and trigger IL-2 secretion following co-incubation (Fig. 6B), all provide direct evidence that the bound peptides are properly encased in the Kk binding groove and can interact with the TCR.

The results presented in Fig. 5 are highly significant (P < 0.01 for both transfectants). However, the magnitude of the response was relatively low, compared with that achieved under similar experimental conditions with the immobilized anti-H-2Kk antibody (data not shown). We tend to attribute this relatively low reactivity to the lack of the CD8 co-receptor on the target hybridoma cells, as reported by Stryhn et al. (19) and confirmed by us (data not shown). It is well established that CD8 markedly increases the avidity of TCR interaction with class I ligands (3436) and its expression by the target T cells in our setting is therefore expected to govern the magnitude of response. In this study we were unable to demonstrate peptide-specific killing of either of the two target T cells hybridomas. However, in the same experiments we could not detect any cytotoxic activity of our MD45 transfectants against their original EL4 target cells, although they do express fully functional TCR (data not shown). We conclude that both transfectants have lost their lytic ability, as frequently observed with CTL hybridomas, especially after lengthy tissue culture propagation. In fact, this is also the case with both target hybridomas (A. Stryhn, personal communication). Thus, the outcome of the predicted two-way cytolysis, which is an important issue concerning therapeutic application of this approach, could not be directly assessed and awaits in vivo studies, which are underway. Nonetheless, the evidence presented in Fig. 7 suggests that both effectors and targets respond at a comparable level to their encounter at a 1:1 ratio, both with respect to the relative increase in the fraction of cells featuring an activated phenotype and the average magnitude of activation. These results are supported by the observation that retrovirally transduced T cell lines expressing a chimeric MHC class I heavy chain and loaded with exogenous peptide could efficiently lyse antigen-specific CTL precursors (24).

A particularly high level of presentation of the pre-selected epitope is mandatory when the epitope is to serve as a key component of a T cell activation receptor. Indeed, non-membrane-attached chimeric peptide/ß2m expressed by antigen presenting cells was reported to sensitize specific CTL clones (31,33). However, whereas hundreds to tens of specific peptide–class I complexes on the antigen presenting cell (37,38), or even one (39), are usually sufficient for CTL activation, from the T cell side activation appears to require signaling through an exceedingly larger number of TCR molecules. This has been proposed to entail serial engagement of as many as 100–200 TCR molecules by a single peptide–MHC ligand (4042). Expression of several thousands of particular class I complexes with the pre-selected peptide on a single transfected T cell, as demonstrated in this setting (Table 1), assures an exceeding amount of signaling events, which can be transduced by the modified class I molecules upon encountering target T cells. The T cell hybridoma we used throughout this study is not known to bear any defect in its class I processing and presentation pathway, implicating that this high level of specific complexes is formed in the presence of competing peptides derived from cellular proteins processed in the cytosol. This is most likely achieved through both sustained persistence of the peptide on the cell membrane and advantage over conventional complexes during class I assembly in the endoplasmic reticulum. This high peptide density also implies that the chimeric ß2m successfully competes with endogenous ß2m on pairing with the corresponding heavy chain. Additional biochemical and functional properties of antigen presentation through membrane-bound ß2m are currently being investigated.

Adoptive T cell immunotherapy mediated by genetically modified T cells, which express chimeric activation receptors, emerges as a promising avenue for targeted treatment of human diseases. The ability to re-program T cell specificity at will, by coupling T cell activation pathway to a heterologous T cell recognition unit, offers a broad spectrum of applications. Recently (14), CD4+ T cell ligands, engrafted as recognition units in the form of chimeric peptide/MHC class II/CD3{zeta} proteins, have been shown to redirect modified transgenic T cells at autoreactive CD4+ T cells of pre-selected specificity in vivo, leading to suppression of an autoimmune disease. The use of ß2m-based chimeras described herein is the first demonstration that this non-polymorphic polypeptide can be harnessed as a universal component of chimeric MHC class I molecules, designed for adoptive immunotherapy of CD8+ T cell-mediated disorders.


    Acknowledgements
 
The authors thank Dr Anette Stryhn for the HK9.5-24 and HK8.3-5H3 cells and Dr Nilabh Shastri for the NFAT-lacZ construct. This work was supported by the Chief Scientist of the Ministry of Industry and Trade (Israel).


    Abbreviations
 
ß2m—ß2 microglobulin

ß-Gal—ß-galactosidase

CTL—cytotoxic T lymphocyte

2m—human ß2m

IDDM—insulin-dependent diabetes mellitus

ITAM—immunoreceptor tyrosine-based activation motif

MS—multiple sclerosis

NFAT—nuclear factor of activated T cells


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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