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The Journal of Immunology, 2002, 168: 3266-3274.
Copyright © 2002 by The American Association of Immunologists

Genetically Modified HLA Class I Molecules Able to Inhibit Human NK Cells Without Provoking Alloreactive CD8+ CTLs1

Alexandra Sharland*, Amy Patel*, Josie Han Lee*, Aimee E. Cestra*, Susan Saidman{dagger} and Gerald L. Waneck2,*

* Laboratory of Molecular and Cellular Immunology, Transplantation Biology Research Center, and {dagger} Histocompatability Laboratory, Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02129


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human NK cells are likely to be important effectors of xenograft rejection. Expression of HLA class I molecules by transfected porcine cells can protect them from human NK cell-mediated lysis; however, this strategy has the potential to augment the anti-graft response by recipient CD8+ T cells recognizing foreign pig peptides presented by HLA. In this study we show that the introduction of a mutation (D227K) in the {alpha}3 domain of HLA-Cw3 abrogates its recognition by CD8-dependent T cells but leaves intact its ability to function as an inhibitory ligand for NK cells. Such genetically modified molecules may have potential therapeutic applications in the prevention of delayed xenograft rejection and in the facilitation of allogeneic and xenogeneic bone marrow engraftment.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Xenotransplantation is being actively pursued as a means of alleviating the current critical shortage of donor organs (1). For ethical and practical reasons, the pig (miniature swine) is the most likely candidate donor species (2). Human NK cells mediate a vigorous anti-pig cytotoxic response in vitro (3, 4, 5), suggesting that they might be important effectors in the rejection of porcine xenografts. Moreover, NK cells express low-affinity IgGRs (Fc{gamma}RIIIA, CD16) and may kill xenoreactive natural Ab-coated pig cells through Ab-dependent cell-mediated cytotoxicity (3, 5, 6). In a number of xenogeneic combinations, including pig-to-primate, delayed xenograft rejection of solid organs is characterized by a cellular infiltrate comprising mainly NK cells and macrophages, with surprisingly few T cells (4, 7, 8). NK cells strongly resist the engraftment of xenogeneic bone marrow (9) and also mediate the rejection of allogeneic bone marrow (10, 11). Thus, it seems likely that strategies to inhibit NK cell-mediated cytotoxicity will need to be developed to facilitate successful clinical transplantation between species (12).

Whereas CTLs kill through recognition of foreign MHC class I (MHC-I),3 NK cells are naturally cytolytic unless they are inhibited through recognition of self MHC-I (13). In this manner, NK cells are able to kill tumor cells and virus-infected cells that have down-regulated self MHC-I ("missing self") (13, 14, 15). Inhibition of killing is mediated by inhibitory NK receptors (iNKR) (16), which in humans recognize certain groups of HLA class I (HLA-I) allotypes rather than individual HLA-I/peptide complexes (17, 18, 19, 20, 21, 22, 23, 24, 25). HLA-I-specific human iNKRs include CD158 killer Ig-like receptors (KIRs), CD85 leukocyte Ig-like receptors (LILRs), and CD94/NKG2 lectin-like receptors. The LILR family members have also been called Ig-like transcripts (ILTs), leukocyte Ig-like receptors (LIRs), and monocyte/macrophage Ig-related receptors (MIRs). CD158a (KIR2DL1) binds to HLA-C group 1 alleles (e.g., Cw4); CD158b1 (KIR2DL2) and CD158b2 (KIR2DL3) bind to HLA-C group 2 alleles (e.g., Cw3); CD158d (KIR2DL4) binds toHLA-G; CD158e1 (KIR3DL1) binds to Bw4 serotypes; CD158k (KIR3DL2) binds to HLA-A3 and -A11; CD85j (LILRB1/ILT2/LIR-1/MIR-7) binds to the {alpha}3 domain of various HLA-I molecules; CD85d (LILRB2/ILT4/LIR-2/MIR-10) binds to HLA-F; and CD94/NKG2a binds to HLA-E (26). Swine leukocyte Ag class I (SLA-I) molecules do not appreciably protect porcine cells from killing by human NK cells (3), and the nucleotide sequence of a number of SLA class I cDNAs (27) has revealed that critical residues for recognition by human CD158 are different from those of HLA-C. Expression in porcine cells of single human iNKR ligands, such as HLA-C, -G, or -E, can completely protect these cells from killing by NK clones that have the appropriate iNKR (28, 29, 30, 31). However, because iNKRs are heterogeneously expressed on overlapping subpopulations of NK cells (32), this strategy only partially protects cells from killing by polyclonal NK populations (28, 29, 30, 31). Thus, expression of the correct combination of human iNKR ligands in porcine tissues may help prevent delayed xenograft rejection by polyclonal NK populations and may facilitate the development of mixed xenogeneic bone marrow chimerism for the induction of central tolerance (33, 34).

Although human CD8+ T cells can recognize SLA-I Ags directly (35, 36, 37, 38, 39, 40), the frequency of SLA-I-reactive CD8+ CTL precursors (CTLp) appears to be considerably lower than that of CTLp reacting directly against allogeneic HLA-I (41, 42), and CD8+ T cells do not appear to play a major role in the human anti-pig cytotoxic response in vitro (3, 37, 43, 44). The low frequency of xenoreactive human anti-pig CTLp might be due to evolutionary differences between HLA-I and SLA-I in {alpha}1/{alpha}2 framework residues bound by the human TCR and/or residues important for interaction with human CD8 that influence positive and negative selection of the CD8+ T cell repertoire (27, 45, 46, 47, 48, 49, 50). Unfortunately, the expression of HLA-I ligands to inhibit human anti-pig NK cytotoxicity might restore a strong CD8+ CTL response by providing the correct structures for allorecognition. Even HLA-I ligands identical with the recipient’s could present many pig peptides that could be recognized as multiple minor histocompatibility Ags and generate a strong CTL response leading to rejection (51, 52, 53). To avoid this consequence, HLA-I molecules that fail to stimulate CTLs, while retaining the ability to inhibit NK cells, would be required.

CD8 plays a critical role both in generation of the MHC-I-restricted immune response and in effector CTL lysis of target cells (54, 55, 56, 57, 58). Disruption of the CD8/MHC-I interaction by Ab blockade, exon exchange, or mutagenesis abrogates the CTL response (55, 59, 60, 61, 62, 63, 64, 65). Four regions of the MHC-I molecule are important for CD8 binding: residues 115, 122, and 128 in the {alpha}2 domain and residues 222–229, 233–235, and 245–247 in the {alpha}3 domain (62, 63, 64, 65, 66). Conversely, HLA-C residues important for inhibition of NK cell killing by CD158 are located close to the peptide binding groove at positions 73, 76, 80, and 90 in the {alpha}1 domain (67).

Given the spatial separation of the CD8/{alpha}3 and CD158/{alpha}1 binding sites (68, 69), we reasoned that it should be possible to introduce a mutation in the HLA-C {alpha}3 domain to prevent CD8 binding without affecting the HLA-C/CD158 interaction. Therefore, we introduced a well-characterized (62, 63, 64, 70) aspartic acid (D) to lysine (K) mutation at position 227 into HLA-Cw3, a group 2 ligand for CD158b. The wild-type (Cw3-wt) and mutant (Cw3-D227K) constructs were expressed in the HLA-A,-B,-C, and -G-negative human NK target cell line LCL 721.221 (221 cells) (71). By selecting responders who share all HLA class II DR and DQ alleles expressed by 221 cells, we were able to examine the response to a single transfected HLA-C molecule in isolation. In the following experiments, we demonstrate that, as predicted, generation of CTLs is impaired and lysis of the Cw3-D227K transfectant by CD8-dependent CTLs is abolished, whereas inhibition of clonal and polyclonal NK cells via binding of Cw3-D227K to CD158b is preserved.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Constructs

The expression vector used in the construction of both Cw3-wt and Cw3-D227K was pcDNA3.1- (Invitrogen, Carlsbad, CA), which encodes resistance to G418. For the Cw3-wt construct, a 4-kb XbaI fragment from pCw3 (28, 72), containing the entire HLA-Cw3 gene minus the promoter region, was subcloned into the dephosphorylated XbaI site of pcDNA3.1-, behind the CMV promoter. For the Cw3-D227K construct, the D227K mutation was introduced by PCR mutagenesis using Pfu I (Stratagene, La Jolla, CA). Two gene fragments were prepared that could be joined at an EcoRI restriction site created by the mutagenic primers. Introduction of the EcoRI site required a semiconservative change of leucine to phenylalanine at position 230. To amplify the 5' gene fragment (750 bp), the forward primer 5'-TCAGTTTAGGCCAAAATCCC-3' was predicted from the HLA-Cw3 sequence (72, 73) from intron 2 positions 798–817, and the mutagenic reverse primer 5'-CCTGCTGGCCTGGTCTCCACgAatTCgGTcTtCTGAGTTTGGTCCTCCCC-3' (mutant bases in lowercase letters; EcoRI site and D227K codon underlined) was predicted from positions 1855–1904 of exon 4. To amplify the 3' gene fragment (1.1 kb), the forward primer 5'-GGGGAGGACCAAACTCAGaAgAccGaatTcGTGGAGACCAGGCCAGCAG-3' was deduced from the reverse complement of the mutagenic primer above, and the reverse primer 5'-TAGAAGGCACAGTCGAGG-3' was predicted from the antisense strand of the pcDNA3.1- bovine growth hormone polyadenylation signal. The primers were prepared by the Massachusetts General Hospital Molecular Biology/Endocrine Unit core facility. After PCR amplification using Cw3-wt as a template, the two fragments were electrophoresed through 1% genetic technology grade agarose (FMC BioProducts, Rockland, ME), electroeluted into dialysis bags (Life Technologies, Grand Island, NY), extracted with phenol chloroform (pH 8) (Fisher Biotech, Fair Lawn, NJ), and desalted by precipitation with ethanol (Aaper, Shelbyville, KY). To create ligatable ends, the 5' Cw3-D227K fragment was digested with Acc65 I (New England Biolabs, Beverly, MA) and EcoRI, and the 3' fragment was digested with EcoRI and BamHI (New England Biolabs). The digested fragments were repurified and quantitated. A four-way ligation was performed using 0.6 pmols of each fragment and 0.1 pmol each of two "vector arms" prepared from separate digestions of a truncated Cw3-wt construct containing a 2.9-kb XbaI-BamHI Cw3 gene fragment. This BamHI site interrupts exon 8 of the Cw3 gene. The left arm was a 1.1-kb XbaI-Acc65 I fragment representing the 5' end of Cw3, and the right arm was the 5.4-kb XbaI-BamHI cut pcDNA3.1- vector. This strategy was required to avoid complications from unwanted additional Acc65 I and BamHI sites. After ligation and transformation of electrocompetent DH10B bacteria (Life Technologies), recombinant Cw3-D227K plasmid DNAs were screened by restriction digestion to confirm the introduction of the EcoRI site and the ability to recut the ligated Acc65 I and BamHI sites. The exon and splice junction sequences of Cw3-D227K between the Acc65 I and BamHI sites were confirmed by DNA sequencing at the Massachusetts General Hospital Molecular Biology/Endocrine Unit core facility (Boston, MA). To reconstruct the full-length Cw3-D227K gene, the mutant 1.8-kb Acc65 I-BamHI fragment was joined in a three-way ligation to the left and right vector arms above. The construct was completed by the addition of a 1.1-kb BamHI-BamHI 3' gene fragment from full-length Cw3-wt, which extends from the BamHI site before exon 8, past the XbaI site, to a BamHI site in the polylinker of pcDNA3.1-.

Cell lines

The human lymphoblastoid NK target cell line 221 (ATCC no. CRL 1855; American Type Culture Collection, Manassas, VA) was provided by Dr. J. Strominger (Harvard University, Cambridge, MA). The 221 cell line has a partial deletion of chromosome 6 and lacks HLA-A, -B, -C, and -G (71). The 221 cells previously transfected with HLA-A2 (221/A2) (74) were a gift of Dr. G. Cohen (Massachusetts General Hospital, Charlestown, MA). LAZ-388 human B lymphoblastoid cells (75) used to prepare leukocyte-conditioned medium and NK cell restimulation mix were a gift of Dr. J. Ritz (Dana-Farber Cancer Institute, Boston, MA). The HLA-Cw3-positive cell line GRC-212 (IHW9364) was obtained from the European Collection of Cell Cultures (Wiltshire, U.K.) (76). HLA typing of cell lines was performed by the Massachusetts General Hospital Histocompatability Laboratory (Table IGo). All cell lines were cultured in growth medium (RPMI 1640 (Mediatech, Herndon, VA) supplemented to contain 10% bovine calf serum (JRH Biosciences, Lenexa, KS), 2 mM L-glutamine (Mediatech), and 5 x 10-5 M 2-ME (Bio-Rad, Richmond, CA)).


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  		Table I. HLA typing of cell lines and donor PBMC

 
Transfection and cloning of 221 cells

Untransfected 221 cells (221/ut; 1 x 107) were electroporated with 10 µg of plasmid DNA linearized by ScaI (New England Biolabs) in Dulbecco’s PBS without Ca2+ or Mg2+, using a Bio-Rad gene pulser set at 340 V and a capacitance extender set at 960 microfarads. The transfected cell population was subcloned by limiting dilution (1 x 104 cells/well) in culture medium containing 1.5 mg/ml G418 (Mediatech). Ten to 14 days after plating, drug-resistant colonies were expanded in cultures containing 0.75 mg/ml G418. Shortly thereafter, cells were analyzed for expression of HLA-C by immunofluorescent staining and flow cytometry. Clones 221/Cw3XX.5 (transfected with Cw3-wt), 221/Cw3-D227K.58 (transfected with mutant Cw3), and 221/neo.1 (transfected with empty vector) were selected for functional analysis.

Flow cytometry

Expression of HLA-C on the surface of transfected 221 cells was determined by indirect immunofluorescent staining with the mAbs W6/32 (ATCC no. HB-95; American Type Culture Collection) (77) and F4/326 (obtained from Dr. J. Strominger) (78). Cells (1 x 106) were incubated with a saturating amount of mAb (in hybridoma supernatants) for 45 min on ice, washed, and then incubated for a further 45 min with FITC-conjugated goat anti-mouse Ig (Cappel, Durham, NC). Unfractionated and fractionated PBMCs, effector cells generated in MLR, and NK cell populations were characterized using the following mAbs: anti-CD3-FITC (UCHT1), anti-CD4-PE (RPA-T4), anti-CD8-PE (RPA-T8), anti-CD16-biotin (3G8), anti-CD19-PE (HIB19), anti-CD56-PE (B159), and CD94-FITC (HP-3D9) (BD PharMingen, San Diego, CA); and anti-CD158a (EB6), anti-CD158b-PE (GL183), and anti-NKG2a (Z199) (Immunotech, Marseille, France). Irrelevant mouse IgG1 (W3/25) conjugated with either FITC or PE (Serotec, Oxford, U.K.), IgG2a (HOPC1; BD PharMingen), and IgG2b (TEN-0; Serotec) mAbs were used as negative controls. Secondary reagents included anti-mouse IgG2b-FITC (LO-MG2b), anti-mouse IgG1-biotin (LO-MG1), goat anti-mouse IgG-allophycocyanin, and streptavidin-allophycocyanin (all from Caltag Laboratories, Burlingame, CA). Normal human serum (Sigma-Aldrich, St. Louis, MO) was included in the buffer with anti-mouse Abs to adsorb any anti-human reactivity. Three-color staining for iNKR required the use of fluorochrome-conjugated or biotinylated anti-mouse Abs as second-step reagents, followed by further staining with directly conjugated mouse mAb. In these instances, cell suspensions were incubated with normal mouse serum (Accurate Chemical and Scientific, Westbury, NY) after staining with anti-mouse Abs to block any free combining sites. Stained cells were fixed for 5 min in freshly prepared 2% paraformaldehyde (Sigma-Aldrich) in PBS. Samples were analyzed on a FACSCalibur flow cytometer using CellQuest software (BD Biosciences, Mountain View, CA).

Limiting dilution assay for precursor frequency

This assay was adapted from the methods of Skinner and Marbrook (79), Lefkovitz and Waldmann (80), and Kaminski et al. (81). Responder cells were obtained from a Cw3-negative donor (SA), who was matched with 221 cells at HLA-DR and -DQ (Table IGo). PBMCs were prepared from whole blood by density gradient centrifugation over lymphocyte separation medium (ICN Biomedicals, Aurora, OH). Responder cells were suspended in cell-mediated lympholysis (CML) medium (RPMI 1640 with 2 mM L-glutamine, 25 mM HEPES (Mediatech), 5 x 10-5 M 2-ME, nonessential amino acids (Life Technologies), 1 mM Na pyruvate (Mediatech), 100 U/ml penicillin (Life Technologies), 100 µg/ml streptomycin (Life Technologies), and 10% human AB serum (Sigma-Aldrich) screened by us for its ability to support T cell proliferation). Cells at concentrations ranging from 103 to 105/well were plated at 50 µl each into 24 wells of 96-well V-bottom plates (Costar, Corning, NY) for each responder-stimulator combination. The 221/ut, 221/Cw3-wt, and 221/Cw3-D227K were used as stimulators. Stimulator cells were washed in CML medium and irradiated at 10,000 rad before use. The indicated numbers of responders/well were cocultured with 104 irradiated stimulators for 5 days in a total volume of 100 µl/well. Cultures were fed on day 3 with human rIL-2 to a final concentration of 20 U/ml. Human rIL-2 (82) was obtained from Dr. M. Gately (Hoffmann-LaRoche, Nutley, NJ) through the AIDS Research and Reference Reagent Program (Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD). Cw3-positive target cells (PHA blasts) lacking other HLA Ags in common with 221 or the transfectants were generated concurrently from PBMCs of donor SL (Table IGo). On day 5, 51Cr-labeled targets (4,000/well) were added to each responder well and incubated for 4 h. The 51Cr released from lysed cells was quantitated in a gamma counter (Isomedic 4/600 HE, ICN Biomedicals). Wells showing >10% specific lysis were scored as positive (79). Regression analysis of a semilog plot of the percent negative wells vs the number of responders/well showed single hit kinetics, and the CTLp frequency was calculated from the responder dilution that yielded 37% negative cultures.

CML assays using 221 transfectants as stimulators

Effector cells were also generated under bulk culture conditions using the stimulator-responder combinations above. Stimulators and responders were each prepared at a concentration of 2 x 106/ml and mixed in equal volumes. Two milliliters of this mixture were added to each of 10 wells of a 24-well culture plate (Costar) and incubated for 7 days. Cultures were fed with human rIL-2 as above on days 3 and 5. On day 7, aliquots of cells were removed for staining and flow cytometric analysis, and the remaining cells were assayed for their ability to kill Cw3+ PHA blasts from donor SL in a 4-h 51Cr release assay.

CML assays using 221 transfectants as targets

CTLs were generated in bulk cultures as described above, using Cw3-positive GRC-212 cells as stimulators. Responders were obtained from a donor (AK) who was Cw3-negative and matched with 221 at DR and DQ (Table IGo). Lysis of 221 transfectants by these CTLs was evaluated by a 4-h 51Cr release assay as above. In some assays, plated effector cells were incubated with an excess of anti-CD8 blocking mAb B9-11 (Immunotech) at a minimum concentration of 5 µg of mAb/4 x 104 effectors for 30 min before addition of the labeled targets and throughout the assay period.

Generation of NK cell populations

NK cells were purified from donor GLW PBMCs (Table IGo) by negative selection using a MACS NK cell isolation kit (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instructions. Polyclonal NK cell cultures were derived according to standard methods (83). Briefly, 104 purified NK cells were mixed with a restimulation mix composed of 107 irradiated (5,000 rad) allogeneic PBMCs and 106 irradiated (10,000 rad) LAZ-388 cells in a total volume of 20 ml of NK cell medium (RPMI 1640 supplemented with 100 µg/ml L-glutamine, 5 x 10-5 M 2-ME, 1 mM sodium pyruvate, nonessential amino acids, 5% human AB serum (not heat inactivated), 100 U/ml penicillin, 100 µg/ml streptomycin, and 500 U/ml human rIL-2). Next, 1 µl/ml PHA-M (Difco, Detroit, MI) was added to the cell suspension, which was then plated (200 µl/well) in U-bottom 96-well plates (Costar). After 7 days of culture, cells were transferred to six-well plates (Costar) and further expanded. Polyclonal NK cells were phenotyped before use in functional assays (see above). NK clones were generated using the method of Seebach et al. (28), but with a higher concentration (500 U/ml) of human rIL-2. Clones that were CD158b+ and CD94dim were chosen for use in additional experiments.

NK cell functional assays

Lysis of 221/ut, 221/Cw3-wt, and 221/Cw3-D227K by polyclonal NK cells and of 221/neo, 221/Cw3-wt, 221/Cw3-D227K, and autologous PHA blasts by NK cell clones was evaluated in 4-h 51Cr releases assays. Some experiments were performed in the presence of Ab blockade with the anti-NKG2a mAb Z199 or with a combination of Z199 and the anti-CD158b mAb GL183. In these assays, 3 µl of each Ab (yielding a minimum concentration of 0.64 µg/1.6 x 105 cells) was added to each well of NK effectors and incubated for 30 min before the addition of labeled targets and for the duration of the assay.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cw3-wt and Cw3-D227K molecules are expressed at similar high levels on transfected 221 cells

HLA-I surface expression by 221 transfectants was assessed by staining with mAb W6/32, which recognizes a monomorphic determinant on the class I molecule (77). The 221/ut (data not shown) and 221 cells transfected with the empty vector (221/neo) were stained by W6/32 to the same degree (Fig. 1Go) and were moderately positive compared with cells stained with the isotype-matched negative control mAb (Fig. 1Go). The 221/Cw3-wt and 221/Cw3-D227K transfectants expressed similar high levels of HLA-I on their surface compared with 221/neo cells (Fig. 1Go, a and b). To distinguish expression of HLA-C from HLA-E on the surface of 221/Cw3-wt and 221/Cw3-D227K, these cells were also stained with mAb F4/326, which reacts strongly with HLA-C, weakly with HLA-B27, and negligibly with other HLA molecules tested (28, 78). The 221 cells transfected with HLA-A2, a molecule that does not react with F4/326 but is able to provide the peptide necessary for HLA-E surface expression (25, 84), were used as a control. The 221/ut (data not shown) and 221/neo (Fig. 1Go) were stained by F4/326 to the same degree and were weakly positive compared with cells stained with the isotype-matched negative control mAb. Bright staining by W6/32 of 221/A2 cells (Fig. 1Goc) was similar to that of 221/Cw3-wt (Fig. 1Goa) and 221/Cw3-D227K (Fig. 1Gob). In contrast, staining by F4/326 of 221/A2 was not greater than background levels (Fig. 1Gof), whereas both 221/Cw3-wt (Fig. 1God) and 221/Cw3-D227K (Fig. 1Goe) were stained strongly. These results suggest that F4/326 does not appreciably recognize HLA-E and provide further evidence that Cw3-wt and Cw3-D227K molecules are expressed at similar high levels on the 221 transfectants.



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  		FIGURE 1. Cw3-wt and Cw3-D227K are expressed at similarly high levels on transfected 221 cells. The 221 cells electroporated with the constructs Cw3-wt or Cw3-D227K or the neoR expression vector pcDNA 3.1- were cloned by limiting dilution under G418 selection to generate the cell lines 221/Cw3-wt, 221/Cw3-D227K, and 221/neo, respectively. Transfectants were screened for HLA-I expression by staining with mAb W6/32 (a–c) and for HLA-C expression by staining with mAb F4/326 (d–f). HLA-I is expressed at similarly high levels on 221/Cw3-wt (a), 221/Cw3-D227K (b), and the positive control line 221/A2 (c) compared with an isotype-matched control mAb. The 221/neo (a–c) and 221/ut cells (data not shown) were stained moderately by W6/32. F4/326 stains HLA-C at similarly high levels on 221/Cw3-wt (d) and 221/Cw3-D227K (e), but it does not stain 221/A2 (f) above background levels present on 221/neo.

 
221/Cw3-D227K cells prime alloreactive CTL less effectively than 221/Cw3-wt

The ability of 221/ut, 221/Cw3-D227K, and 221/Cw3-wt cells to prime CTL was compared using limiting dilution and bulk CML assays. Cw3-negative responder cells from donor SA shared all DR and DQ alleles expressed by 221 cells (Table IGo), and no response against 221/ut was detected (Fig. 2Goa). The precursor frequency of CTLs generated against Cw3-D227K (1/240,000 ± 15,000) was significantly less (p = 0.039) than that generated against Cw3-wt (1/55,000 ± 850) (Fig. 2Goa). CTLs were also generated against 221 transfectants under bulk culture conditions (Fig. 2Gob). There were no obvious differences in the compositions of effector cell populations between those generated against Cw3-wt or Cw3-D227K. The proportion of CD8+ T cells was very similar in both cultures (33.7% for Cw3-wt and 36.1% for Cw3-D227K), as were the proportions of CD4+ T cells (48.9 and 46.9%, respectively), NK cells (7.4 and 8.1%), and B cells (5.2 and 3.8%). Nonetheless, CTLs generated against Cw3-wt were significantly better able (p = 0.0003) to lyse Cw3-bearing third party targets (percentage of specific lysis, 22.9 ± 2.8%) than CTLs generated against Cw3-E227K (percentage of specific lysis, 2.3 ± 1.3%) at an E:T ratio of 100:1 (Fig. 2Gob).



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  		FIGURE 2. The 221/Cw3-D227K primes alloreactive CTLs less effectively than 221/Cw3-wt. a, Limiting dilution assays were used to measure the frequency of CTLp responding to 221/ut cells and 221/Cw3-wt and Cw3-D227K stimulators. Responder cells were prepared from a Cw3- donor (SA), who was HLA matched with 221 cells for DR1 or DQ5, and were assayed against PHA blasts generated from a Cw3+ individual (SL), who did not share DR and DQ with the stimulator or responder (Table IGo). Thus, the only HLA Ag shared by stimulator and target cells was Cw3. CTLp frequency was calculated from the responder dilution that yielded 37% negative cultures. The CTLp frequency for 221/Cw3-D227K (1/240,000 ± 15,000) was significantly less (p = 0.04) than that for 221/Cw3-wt (1/55,000 ± 850). b, Using the same stimulator-responder-target combination described above, CTLs were generated in 7-day bulk cultures against 221/Cw3-wt or 221/Cw3-D227K and were assayed for killing of 51Cr-labeled Cw3+ PHA blasts at various E:T ratios. At an E:T ratio of 100:1, specific lysis by CTLs primed against Cw3-D227K (2.3 ± 1.3%) was significantly less (p = 0.0003) than that against Cw3-wt (22.9 ± 2.8%).

 
221/Cw3-D227K cells cannot be killed by CD8-dependent anti-Cw3 CTLs

To determine the effect of the D227K mutation on target cell recognition, CTLs from donor AK were primed by Cw3-positive GRC-212 cells and used in CML assays against 221/Cw3-wt and 221/Cw3-D227K. Although AK responder cells differ from GRC-212 stimulator cells at HLA-DQ and -DR, they share both alleles of the 221 targets at these loci (Table IGo), thus minimizing the impact of responses directed against HLA class II Ags on the CML assay readout. As shown in Fig. 3Go, 221/Cw3-D227K target cells were relatively resistant to killing by AK CTLs compared with 221/Cw3-wt. When the interaction of CD8 with Cw3 was blocked by addition of anti-CD8 mAb B9-11, killing of 221/Cw3-wt was reduced to the same level as that of 221/Cw3-D227K (Fig. 3Go). These results confirm that the reduced lysis of 221/Cw3D227K by anti-Cw3 CTLs is due to disruption of the CD8/{alpha}3 interaction.



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  		FIGURE 3. The D227K mutation of Cw3 abolishes CD8-dependent killing of 221 transfectants by anti-Cw3 CTLs. CTLs were generated by in vitro stimulation of DR1+DQ5+Cw3- PBMCs (donor AK) with irradiated DR1-DQ5-Cw3+ B lymphoblastoid cells (GRC-212) for 7 days and then assayed against 51Cr-labeled 221 transfectants in the presence or absence of anti-CD8 blocking mAb B9-11. The 221/Cw3-wt cells were killed at high levels compared with 221/Cw3-D227K. Inclusion of saturating amounts of anti-CD8 mAb during the assay had no effect on 221/Cw3-D227K but reduced the killing of 221/Cw3-wt to the same level as 221/Cw3-D227K with or without mAb.

 
Phenotypic analysis of clonal and polyclonal NK effector cell populations

All NK cell populations were phenotyped before use in functional assays. High purity of NK cells (97% CD56+CD3-) with <1% T cell contamination was obtained by MACS bead purication of NK cells from PBMCs (data not shown). These were used as the starting population for generating polyclonal and clonal NK effectors. The polyclonal NK cell population used in these experiments was free of contaminating T cells (Fig. 4Goa). A total of 83.9% of the cells were CD16+ (Fig. 4Gob); 23.9% were positive for CD158a alone, 10% were positive for CD158b alone, 14.5% carried both CD158a and CD158b (Fig. 4Goc), and 51.6% of cells had neither CD158a nor CD158b (Fig. 4Goc). A total of 87% of cells in the polyclonal NK cell population were CD94bright (Fig. 4God). NK clone GLW-NK.69 was also free from contamination by T cells (Fig. 4Goe). These cells were positive for both CD56 and CD16 (Fig. 4Gof) and expressed both CD158a and CD158b (Fig. 4Gog). This clone showed low-level expression of CD94 (Fig. 4Goh) compared with the polyclonal population in Fig. 4God.



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  		FIGURE 4. Phenotypic analysis of NK effector cell populations. a, The polyclonal NK cell cultures used for experiments shown in Fig. 5Go were 97% CD56+CD3-, with <1% contaminating T cells. b, A total of 83.9% of the polyclonal cells were CD16+. c, A total of 23.9% of the polyclonal cells were CD158a single positive, 10% were CD158b single positive, and 14.5% were CD158a+CD158b+. A total of 51.6% of the cells were negative for CD158. d, More than 87% of the polyclonal cells were CD94bright. The phenotype of NK clone GLW-NK.69 used for experiments shown in Figs. 6Go and 7Go was CD56+CD3- (e), CD16+ (f), CD158a+CD158b+ (g), and CD94dim (h).

 
Cw3-D227K is indistinguishable from Cw3-wt in providing partial protection against killing by polyclonal NK cells

The ability of Cw3-D227K and Cw3-wt to protect transfected 221 cells from NK-mediated killing was assesssed using 4-h 51Cr release assays at E:T ratios ranging from 100:1 to 6.3:1. At all E:T ratios tested, the expression of Cw3-D227K in 221 cells provided significant protection against killing by the polyclonal NK cell population, which was indistinguishable from the effect mediated by expression of Cw3-wt. Killing of both transfectants increased similarly in proportion to the E:T ratio, consistent with partial inhibition (Fig. 5Go). This increase in killing was parallel to that of 221/ut, which was not protected.



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  		FIGURE 5. Cw3-D227K is indistinguishable from Cw3-wt in providing significant partial protection against killing by polyclonal NK cells. The ability of transfected Cw3 isoforms to protect 221 cells from NK-mediated killing was assesssed using 4-h 51Cr release assays. The partial protection provided by Cw3-wt and Cw3-D227K remained constant through all E:T ratios tested and paralleled the killing of 221/ut, which was not protected.

 
Cw3-D227K and Cw3-wt completely inhibit CD158b+ NK clones

Expression of Cw3 in 221 cells has the potential to up-regulate expression of HLA-E, a ligand for CD94/NKG2a (25, 84). To isolate the inhibitory effects of Cw3 binding to CD158b from that mediated by HLA-E binding to CD94/NKG2a, we generated CD94dim NK cell clones expressing CD158b and tested these in 4-h 51Cr release assays at E:T ratios ranging from 40:1 to 1.3:1. Killing of 221 cells by GLW-NK.69 was completely inhibited by expression of either Cw3-D227K or Cw3-wt (Fig. 6Go). To exclude the possibility of inhibition mediated by the low level of CD94/NKG2a on NK clone GLW-NK.69, we blocked NKG2a using saturating amounts of mAb Z199. As shown in Fig. 7Go, complete inhibition of killing of both transfectants was maintained in the presence of Z199, similar to cultures with no added mAb. However, addition of mAb GL183 to block inhibition through CD158b restored lysis of both transfectants to the same degree. Restored killing was similar to that observed for 221/neo (Fig. 6Go), which represents the level of killing obtained in the absence of all known inhibitory ligands. These results confirm that Cw3-D227K mediates inhibition through CD158b, which is indistinguishable from that mediated by Cw3-wt.



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  		FIGURE 6. Cw3-D227K completely protects against killing by CD158b+ NK clones. To isolate the effects of Cw3 binding to CD158b from those mediated by other receptor-ligand combinations, we generated CD94dim NK cell clones expressing CD158b and tested these in 4-h 51Cr release assays at E:T ratios ranging from 40:1 to 1.3:1. At the highest E:T ratio, expression of either Cw3-D227K or Cw3-wt provided complete protection from killing by clone GLW-NK.69, compared with killing of 221/neo.

 


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  		FIGURE 7. Cw3-D227K inhibits NK cell killing via binding to CD158b. To exclude the possibility of inhibition mediated by the low level of CD94/NKG2a on NK clone GLW-NK.69 binding to HLA-E on Cw3-transfected 221 cells, we blocked NKG2a using mAb Z199. Complete inhibition of killing of both transfectants was maintained in the presence of Z199, similar to cultures with no added mAb. The addition of mAb GL183 to block inhibition through CD158b restored killing of both Cw3-wt (a) and Cw3-D227K (b) to the same degree. The level of restored killing was similar to that determined for 221/neo in Fig. 6Go.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In vitro studies have shown that NK cells form an important component of the human anti-pig cellular immune response (3, 43, 44). The strong cytotoxic activity of unfractionated human PBMCs against porcine target cells is not restricted by the SLA haplotype of the stimulator cells, nor can cytolysis by responding PBMCs be blocked by anti-T cell Abs (37). Although CD8+ CTLs can directly recognize specific SLA-I alleles (37, 38, 40), the precursor frequency of these cells appears to be low (41, 42), and the predominant T cell response to SLA-I results from indirect presentation of processed Ags to CD4+ cells (85, 86, 87). Taken together, these findings suggest a combined failure of SLA-I molecules to serve as stimulatory ligands for human CD8 and/or TCRs and as inhibitory ligands for human NK receptors. Several studies have shown that the expression of HLA-I molecules in pig cells can protect them from killing by human NK cells (28, 29, 30, 31), suggesting a possible therapeutic strategy for xenotransplantation. However, we believe that this strategy has the potential to restore an unwanted strong CD8+ CTL response by providing the recognition structures missing on SLA-I. Based on this premise, we engineered an HLA-Cw3 molecule (Cw3-D227K) that is defective in CD8 interaction and demonstrated that it still retained the full ability to inhibit NK cells through the iNKR CD158b.

To isolate the effect of the D227K mutation on CTL and NK cell recognition of HLA-Cw3 and to demonstrate a molecular proof-of-principle toward a therapeutic strategy for xenotransplantation, we transfected human HLA-I-negative 221 cells, a well-characterized human NK cell target. We were surprised to find detectable levels of W6/32 staining on 221/ut cells. This observation has also been made by others (88), although the nature of the determinant is not currently known. HLA-E genes are transcribed in 221 cells, but surface expression of HLA-E on these cells is limited by availability of the necessary binding peptide, which can be derived from the leader signal sequence of HLA-A, -B, -C, or -G (19, 22, 25, 84). It is possible that small amounts of HLA-E may reach the cell surface by binding non-HLA peptides with similar anchor motifs. It has also been postulated that HLA-F expression may account for this staining (88), but reports of HLA-F expression at the cell surface have been contradictory (88, 89, 90, 91). Alternatively, W6/32 may cross-react with other as yet uncharacterized non-HLA molecules. Nonetheless, any HLA-I molecules present on the surface of 221 cells are apparently insufficient to inhibit human NK cells. The F4/326 mAb binds to HLA-C but does not appreciably recognize HLA-E expressed on 221/A2 transfectants. This confirms that Cw3-wt and Cw3-D227K are expressed at similar high levels on transfected 221 cells and that differences in the cytotoxic response to these transfectants are not a result of inequalities in Cw3 expression levels.

As anticipated (62, 63, 64, 70), introduction of the D227K mutation into HLA-Cw3 significantly compromised priming of alloreactive CTLs and abrogated CD8-dependent killing of 221 transfectants by anti-Cw3-specific CTLs. Some background killing of 221/Cw3-D227K still occurred, which was similar to that of 221/Cw3-wt in the presence of anti-CD8 mAb blockade. There are two possible explanations for this background. Although the effector cells were derived from PBMCs under culture conditions designed to limit the generation of lymphokine-activated killer cells, we did not deplete NK cells. A subpopulation of CD158b-negative NK cells in these bulk cultures, therefore, could mediate cytotoxicity against 221/Cw3 transfectants. In support of this possibility, 221/ut cells were lysed by this effector population (data not shown). Alternatively, but not exclusively, the background killing could be due to recognition of Cw3 by CD8-independent CTLs. These two possibilities could be resolved in future experiments with NK-depleted effector cells and by blocking with anti-HLA mAbs as well as anti-CD8.

Based on the known footprints of CD8 and CD158b on HLA-I molecules (68, 69), we reasoned that mutation of Cw3 {alpha}3 domain residues responsible for CD8 binding should not interfere with the ability of CD158b to bind Cw3 {alpha}1/{alpha}2 residues. In our experiments using polyclonal NK cells, Cw3-D227K partially protected 221 transfectants to the same degree as Cw3-wt. Partial protection by Cw3 is consistent with the composition of the NK population we studied, in which only ~25% of the cells expressed CD158b. However, the majority of these polyclonal NK cells were CD94bright, and HLA-E expressed on the Cw3 transfectants interacting with CD94/NKG2a on NK cells might have accounted for the observed partial inhibition. In experiments using CD158b+CD94dim NK clones, Cw3-D227K completely protected 221 transfectants as effectively as Cw3-wt. Complete protection was maintained in the presence of anti-NKG2a blocking mAb but was reversed by blockade of CD158b. These experiments establish that the identical protective effect of the two Cw3 isoforms is mediated through CD158b.

HLA-C is the least polymorphic of the classical HLA loci. Nevertheless, mismatch at HLA-C can elicit an alloreactive response leading to organ graft rejection (92, 93), bone marrow transplantation failure (94), or graft-vs-host disease (95). Clinically significant donor anti-recipient reactivity against HLA-C Ags has been defined as a precursor frequency >1/105 (95). Our precursor frequency estimate of 0.42/105 suggests that introduction of the D227K mutation has converted HLA-Cw3 from a clinically significant transplantation Ag to a clinically silent one. The Cw3-D227K molecule nevertheless fully inhibits human NK cells through ligation of CD158b. Therefore, these experiments provide a proof-of-principle for the rational design of "next generation" NK cell ligands, which may have therapeutic value in overcoming NK cell-mediated cytotoxicity in transplantation without amplifying the CD8+ CTL response against the graft (96).

An individual’s NK cells express overlapping subsets of inhibitory receptors (32), and no single molecule thus far tested has been able to consistently confer complete protection against killing of porcine targets by polyclonal NK populations (28, 29, 30, 31). To achieve this, ligands capable of inhibiting NK cells through more than one type of iNKR will be needed. Based on current knowledge (17, 18, 19, 20, 21, 22, 23, 24, 25), complete protection might be achieved by the combined expression of HLA-A3, HLA-B27, HLA-Cw3, HLA-Cw4, HLA-E, and HLA-G. Each of these NK ligands could be genetically modified to make them less alloreactive to CD8+ CTLs, as we have demonstrated in this study. Moreover, it should be possible to engineer mutant HLA-C molecules expressing a "superinhibitor" motif found in both Cw3 and Cw4 (67), thereby reducing the number of ligands that must be expressed. As more information becomes available regarding molecular interactions of iNKR with their HLA-I ligands, other genetic modifications may be able to reduce this number further. The iNKR ligands could be expressed as transgenes in porcine tissues, complementing existing strategies to prevent the Ab-mediated hyperacute rejection and vascular coagulopathy that occur in pig-to-primate transplantation (97, 98, 99). Alternatively, expression of these molecules in porcine bone marrow cells through stem cell transduction may facilitate the development of central tolerance through mixed xenogeneic chimerism (33, 34), making it possible to perform subsequent pig-to-human organ transplantation. Although protection of pig cells from both human NK cell and CTL lysis was the rationale for this study, a similar stem cell transduction strategy could be applied to human allogeneic bone marrow transplantation.


   Acknowledgments
 
We thank Joerg Seebach, Megan Sykes, Henry Winn, and Wayne Yokoyama for critical review of the manuscript, Jon Boyson and George Cohen for helpful discussions, Harout DerSimonian for advice on NK cell cloning, David Dombkowski for assistance with flow cytometry, and Basya Rybalov for help in obtaining cell lines.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant AI43440. A.S. received a C.J. Martin Fellowship from the National Health and Medical Research Council of Australia and a Don and Lorraine Jacquot Traveling Fellowship from the Royal Australasian College of Physicians. A.P. conducted this work in partial fulfillment of her undergraduate senior honors thesis from Tufts University (Medford, MA). Back

2 Address correspondence and reprint requests to Dr. Gerald L. Waneck, Department of Surgery, Massachusetts General Hospital, Transplantation Biology Research Center, 149 13th Street, Charlestown, MA 02129. E-mail address: waneck{at}helix.mgh.harvard.edu Back

3 Abbreviations used in this paper: MHC-I, MHC class I; iNKR, inhibitory NK receptor; HLA-I, HLA class I; SLA-I, swine leukocyte Ag class I; CTLp, CTL precursor; wt, wild type; 221, LCL 721.221; 221/A2, 221 cells transfected with HLA-A2; ut, untransfected; KIR, killer cell Ig-like receptor; LIR/LILR, leukocyte Ig-like receptor; ILT, Ig-like transcript; MIR, monocyte/macrophage Ig-related receptor; CML, cell-mediated lympholysis. Back

Received for publication July 16, 2001. Accepted for publication January 25, 2002.


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