Design and use of conditional MHC class I ligands

© 2006 Nature Publishing Group http://www.nature.com/naturemedicine TECHNICAL REPORTS Design and use of conditional MHC class I ligands Mireille Toebes1, Miriam Coccoris1,4, Adriaan Bins1,4, Boris Rodenko2, Raquel Gomez1, Nella J Nieuwkoop3, Willeke van de Kasteele1, Guus F Rimmelzwaan3, John B A G Haanen1, Huib Ovaa2 & Ton N M Schumacher1 Major histocompatibility complex (MHC) class I molecules associate with a variety of peptide ligands during biosynthesis and present these ligands on the cell surface for recognition by cytotoxic T cells. We have designed conditional MHC ligands that form stable complexes with MHC molecules but degrade on command, by exposure to a defined photostimulus. ‘Empty MHC molecules’ generated in this manner can be loaded with arrays of peptide ligands to determine MHC binding properties and to monitor antigen-specific T-cell responses in a highthroughput manner. We document the value of this approach by identifying cytotoxic T-cell epitopes within the H5N1 influenza A/Vietnam/1194/04 genome. MHC class I molecules are heterotrimers that consist of an invariant light chain, a polymorphic heavy chain and an 8–10-amino-acid peptide ligand. The peptide forms an essential subunit of the MHC class I complex, as MHC class I molecules that do not associate with peptide ligand are unstable1,2. Association of peptides with MHC class I molecules is in large part based on shape and electrostatic complementarity between two amino-acid side chains at the anchor positions of the peptide and MHC allele–specific pockets3,4. In addition, binding of peptide ligands depends on the interaction of the MHC molecule with the terminal a-amino and carboxyl groups of the peptide5,6. Because the sequence requirements for binding to MHC are largely restricted to two dominant anchor residues7, the average protein contains several dozen potential T-cell epitopes and, as an example, the approximately 100 open reading frames of a member of the herpesviridae family contain an estimated 4,000 potential T-cell epitopes. Definition of tumor-and pathogen-encoded MHC ligands and detection of T-cell responses specific for such ligands remains a major challenge. The visualization of antigen-specific T-cell responses was first made possible with the development of tetrameric MHC reagents8. In this strategy, soluble MHC monomers complexed with a peptide of interest are biotinylated and converted to tetravalent structures by binding to fluorochrome-conjugated streptavidin or avidin. The resulting MHC tetramers have become essential reagents for the detection of antigen-specific CD4+ and CD8+ T cells by flow cytometry. More recent work indicates that characterization of antigenspecific T-cell responses by MHC microarray–based strategies is also feasible9,10. Furthermore, MHC-based selection of antigen-specific T cells has been proposed as a strategy to boost melanoma-specific T-cell responses in individuals with melanoma11 and to provide defined minor histocompatibility antigen–specific and virus-specific T cells to recipients of allogeneic stem cell transplants and other immunocompromised individuals12–17. At present, the major limitation of MHC tetramer–based technologies is the involved and lengthy nature of the refolding and purification steps required to generate every individual peptide-MHC complex. Consequently, it has not been feasible to apply MHC tetramer–based technology for highthroughput applications. To address this issue, we have explored the possibility of identifying conditional MHC ligands that can be used to generate peptide-receptive MHC class I molecules at will. RESULTS Design of conditional MHC class I ligands We set out to create MHC class I ligands that would disintegrate on command while bound to the MHC complex. As a building block for such conditional ligands, we synthesized a 9-fluorenylmethyloxycarbonyl (Fmoc)-derivative of 3-amino-3-(2-nitro)phenyl-propionic acid (abbreviated ‘J’ in the amino acid sequence). Subsequently, we used this building block to generate variants of the human leukocyte antigen (HLA)-A2.1–restricted influenza A matrix 1 (M1)(58–66) epitope (sequence, GILGFVFTL). An example of such a variant, in which Thr8 is replaced by 3-amino-3-(2-nitro)phenyl-propionic acid, is compound I (sequence, GILGFVFJL; Fig. 1a). Consistent with published literature18,19, this compound disintegrates upon exposure to ultraviolet (UV) light (Fig. 1a), and after exposure to UV-light, a molecule with a mass-to-charge ratio of 751.4 becomes apparent, corresponding to the mass of the heptameric peptide acetamide fragment (compound II; Fig. 1). We did not study further the small, C-terminal peptide fragment that is also generated during this cleavage reaction. To establish whether UV light–sensitive ligands can be used to generate peptide-MHC complexes, we used two influenza A M1(58– 66) variants with 3-amino-3-(2-nitro)phenyl-propionic acid incorporated at position 4 (compound III; GILJFVFTL; Supplementary Fig. 1 online) or position 8 (compound I; Fig. 1a) in MHC class I refolding reactions20. MHC class I refolding reactions with either GILJFVFTL or GILGFVFJL produced high yields of HLA-A2.1–peptide complexes. As 1Division of Immunology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands. 2Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands. 3Department of Virology and WHO National Influenza Center, Erasmus Medical Center, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. 4These authors contributed equally to this work. Correspondence should be addressed to T.N.M.S. ([email protected]) or H.O. ([email protected]). Received 11 November 2005; accepted 13 December 2005; published online 5 February 2006; doi:10.1038/nm1360 246 VOLUME 12 [ NUMBER 2 [ FEBRUARY 2006 NATURE MEDICINE TECHNICAL REPORTS a H N O N H O O H N N H O H N O N H* O OH N H I O O H2N N H H N O O H N N H O O O H N N H *2 NH II O b 1,057.6 [I-H+] Percent 100 TOF MS ES+ 1.84e4 1,058.6 0 100 m/z 751.5 [II-H+] TOF MS ES+ 1.02e4 Percent © 2006 Nature Publishing Group http://www.nature.com/naturemedicine UV light 752.5 0 750 800 850 900 950 m/z 1,000 1,050 1,100 a first crude test of the sensitivity of the non-natural MHC-bound ligands to UV light–mediated cleavage, we exposed HLA-A2.1–peptide complexes containing either the unmodified influenza A M1(58–66) epitope, or the GILJFVFTL or GILGFVFJL variant to UV light and analyzed the reaction products by gel-filtration chromatography. Whereas the unmodified HLA-A2.1–peptide complex was not affected by exposure to 366-nm light, exposure of either HLA-A2.1– GILJFVFTL (data not shown) or HLA-A2.1–GILGFVFJL (Supplementary Fig. 2 online) resulted in a substantial reduction in recovery of MHC molecules. Furthermore, when we prepared HLA-A2.1– peptide complexes with an influenza A M1(58–66) derivative Figure 2 UV light–mediated peptide exchange. Mass spectrometric analysis of HLA-A2.1–associated peptide of HLA-A2.1–GILGFVFJL complexes before (top) and after (bottom) exposure to UV light in the presence of CMV pp65(495–503) peptide. Observed mass-to-charge ratios and assignments are indicated. Expected masses: Na+ ion of compound I: 1,079.6; Na+ ion of CMV pp65(495–503): 965.5. There was a lack of detectable compound I–Na+ after UV light–induced peptide exchange (arrow). We exposed 25 mM HLA-A2.1–GILGFVFJL in 20 mM Tris-HCl (pH 7.0), 150 mM NaCl and 0.5 mM DTT to 366-nm light for 1 h on ice in the presence of 500 mM CMV pp65(495–503) peptide. Subsequently, HLA-A2.1-peptide complexes were purified by gel-filtration chromatography on a Biosep SEC S-3000 column in 25 mM NH4OAc (pH 7.0) and directly used for analysis on a Waters LCT ESI mass spectrometer by direct infusion under optimized conditions (160 V cone voltage, 160 1C desolvation temperature). The use of high cone voltage and high temperature resulted in the detection of predominantly sodiated species. Numbers in the upper right corner reflect the total ion counts in time-of-flight positive electrospray ionization mode. NATURE MEDICINE VOLUME 12 [ NUMBER 2 [ FEBRUARY 2006 containing the UV light–resistant building block 3-amino-3-phenylpropionic acid21, the resulting MHC complexes were insensitive to exposure to UV-light, showing that the UV light–induced decay of HLA-A2.1–GILJFVFTL and HLA-A2.1–GILGFVFJL requires the presence of the nitrophenyl moiety. Notably, the observed decrease in the amount of folded MHC upon exposure to UV light of the HLA-A2.1– GILGFVFJL complex can be prevented by inclusion of HLA-A2.1– binding peptides during the cleavage reaction, but not by inclusion of a control HLA-A3–binding peptide (Supplementary Fig. 2 online), suggesting efficient peptide exchange. Although these data indicate the sensitivity to UV light of HLA complexes containing 3-amino-3-(2-nitro)phenyl-propionic acid– based peptide ligands, it is difficult to establish the efficiency of this cleavage reaction by gel-filtration chromatography (Supplementary Fig. 2 online). As a more stringent test for replacement of the conditional ligand by the newly added peptide, we exposed HLAA2.1–GILGFVFJL complexes to UV light in the presence of the cytomegalovirus (CMV) pp65(495–503) epitope, and after this reaction, we purified the peptide–MHC complexes and analyzed them by mass spectrometry. The major peptide mass that is visible before exposure to UV light corresponds to the Na+ ion of GILGFVFJL, providing formal proof that this ligand forms a stable complex with HLA-A2.1 (Fig. 2). After UV light–mediated cleavage, no detectable amount of GILGFVFJL remains associated with HLA-A2.1. Furthermore, the amount of compound II complexed with HLA-A2.1 is less than background, suggesting that dissociation of this heptameric peptide fragment is essentially complete. Instead, upon UV light– mediated cleavage the sole detectable peptide mass associated with a + 100 1,079.6 (Compound I–Na ) TOF MS ES+ 73.3 Percent N H Figure 1 Photocleavage strategy. (a) Structure of compound I (top), the photocleavable analog of the influenza A M1(58–66) epitope obtained as an approximate one-to-one mixture of diastereoisomers. Amino acid sequence, GILGFVFJL. Structure of compound II (bottom), the heptameric peptide fragment generated upon UV light–induced degradation of compound I. Asterisks in compounds I and II indicate the preferred cleavage site. (b) Liquid chromatography–mass spectrometry of compound I before (top) and after (bottom) exposure to 366-nm light for 60 min, in the presence of 0.5 mM DTT. Expected masses: single-protonated compound I: 1,057.6; single-protonated compound II: 751.4. Numbers in the upper right corner reflect the total ion counts in time-of-flight positive electrospray ionization mode. 1,080.6 1,057.6 0 m/z b 100 + 965.6 (CMV pp65(455–503)–Na ) TOF MS ES+ 89.8 Percent O H2N NO2 O 966.6 943.6 0 700 750 800 850 900 950 1,000 1,050 1,100 m/z 1,150 247 TECHNICAL REPORTS A2.1–MART-1(26–35)(A2L)-PE A2.1-(I) UV MART-1(26–35)(A2L)-PE A2.1-(I) UV HY(311–319)-PE anti–CD8-FITC * 0.25% Peptide + UV A2.1-pp65(495–503)-PE 0.02% 0.97% A2.1-M1(58–66)-PE 0.26% + A2.1-(I) UV HY(311–319)-PE c A2.1-(I) UV MART-1(26–35)(WT)-PE 0.94% b A2.1-(I) UV pp65(495–503)-PE strep-pe A2.1-(I) UV M1(58–66)-PE 0.0% Control anti–CD8-APC © 2006 Nature Publishing Group http://www.nature.com/naturemedicine anti–CD8-FITC a Figure 3 T-cell staining with MHC exchange tetramers. (a) Flow cytometric analysis of MHC tetramer staining of a MART-1(26–35)-specific CTL clone with classical MHC tetramers containing the MART-1(26–35)(A2L) epitope (top left), or MHC exchange tetramers containing the MART-1(26–35)(A2L) epitope (top right), a control peptide HY(311–319) (bottom left), or the naturally occurring low-affinity MART-1(26– 35) epitope (bottom right). (b) Flow cytometric analysis of peripheral blood mononuclear cells from an HLA-A2.1–positive individual stained with classical MHC tetramers containing the CMV pp65(495–503) epitope (top left) or influenza A M1(58–66) epitope (top right), or with MHC exchange tetramers containing a control peptide HY(311–319) (bottom left), the CMV pp65(495– 503) epitope (bottom middle) or the influenza A M1(58–66) epitope (bottom right). (c) Flow cytometric analysis of MHC tetramer staining of peripheral blood mononuclear cells of a C57BL/6 mouse (top) or a C57BL/6 mouse 8 d after intranasal infection with influenza A/HKx31, encoding the A/PR8/34 NP(366–374) ASNENMETM epitope (bottom). Analysis was performed with H-2Db exchange tetramers containing the A/PR8/34 NP(366–374) epitope (left), a control peptide (SMCY(738–746) bottom middle), or H-2Db tetramers prepared from biotinylated H-2Db–ASNENJETM monomers that had not undergone exchange reactions (bottom right). Cartoon depicts the process used to generate MHC exchange tetramers. The UV light–sensitive peptide is indicated by an asterisk. I and IV refer to the UV light–sensitive ligands for HLA A2.1 and H2-Db. Db-(IV) UV NP(366–374)-PE cytometry. MHC exchange tetramers containing the high-affinity (A2L) variant of the A/HK × 31Melan-A/MART-1(26–35) epitope23 stain a infected MART-1–specific cytotoxic T lymphocyte (CTL) clone as efficiently as conventional MHC class I tetramers (Fig. 3a). Likewise, Db-(IV) UV SMCY(738–746)-PE Db-(IV)-PE Db-(IV) UV NP(366–374)-PE MHC exchange tetramers can be used to detect low-magnitude T-cell responses in perHLA-A2.1 corresponds to the mass of the CMV pp65(495–503) ipheral blood samples (Fig. 3b and Supplementary Fig. 3 online). UV epitope (Fig. 2). Collectively, these experiments indicate that we can light–induced cleavage of MHC-bound ligands also allows the synthproduce conditional MHC ligands that are released from MHC esis and use of MHC tetramers that contain the naturally occurring molecules upon exposure to UV light and that MHC molecules MART-1(26–35) peptide that binds to MHC class I molecules with generated in this process can be loaded with epitopes of choice. To low affinity, because of the absence of a leucine or methionine residue extend these data to other conditional HLA-A2.1 ligands, we synthe- at the anchor site at position 2 (Fig. 3a). This indicates that UV light– sized a peptide that is predicted to bind avidly to HLA-A2.1 (ref. 22), induced peptide exchange is sufficiently robust to screen collections of with the UV light–sensitive building block J incorporated at position 8 putative T-cell epitopes without a priori knowledge of MHC binding (ILAETVAJV). Refolding reactions with this conditional ligand gave affinities. Notably, MHC tetramers prepared from HLA-A2.1– high yields of folded HLA-A2.1–peptide complexes and, analogous to GILGFVFJL complexes or A2Kb-GILGFVFJL complexes (in which the data obtained with HLA-A2.1–GILGFVFJL complexes, these the HLA-2.1 a3 domain is replaced by the mouse MHC H-2Kb a3 complexes disintegrated after exposure to UV light (data not shown). domain) that have not been exposed to UV light, and that are therefore uniformly occupied by the UV light–sensitive influenza A MHC exchange tetramers M1(58–66) epitope variant, do not stain polyclonal influenza A To test the potential value of this MHC exchange technique for the M1(58–66) epitope–specific T cells (Supplementary Fig. 3 online). visualization of antigen-specific T cells, we performed MHC exchange This indicates that either the alteration in the peptide backbone as a reactions with biotinylated HLA-A.2.1– GILGFVFJL or HLA-A.2.1– result of the introduction of an unnatural b-amino acid, or the ILAETVAJV complexes. Then we added phycoerythrin-streptavidin replacement of the threonine side chain is incompatible with T-cell and used the resulting MHC tetramers (hereafter referred to as recognition by M1(58–66)–specific T cells. Consequently, even in MHC exchange tetramers) to detect antigen-specific T cells by flow settings in which the release of conditional ligand would not be 248 1.87% 0.07% 0.07% VOLUME 12 [ NUMBER 2 [ FEBRUARY 2006 NATURE MEDICINE optimal, MHC exchange reagents are not expected to have background reactivity because of the presence of residual conditional ligand. Should conditional ligand-MHC complexes for other alleles display background reactivity, it should be straightforward to prevent such reactivity by modification of T-cell receptor–exposed side chains. Notably, MHC tetramers generated by UV light–mediated exchange compare favorably with MHC-immunoglobulin dimers generated by passive peptide exchange24,25, both with respect to signal intensity and signal-to-noise ratios (Supplementary Fig. 4 online). To test whether conditional ligands may readily be identified for other MHC class I alleles, we synthesized four variants of the influenza A NP(366–374) epitope (sequence, ASNENMETM) that is presented by the mouse MHC class I allele H-2Db. Two of those variants, IV and V (Supplementary Fig. 1 online), fulfilled both criteria in that H2-Db complexes could be generated with these conditional ligands and that these ligands could be cleaved in the MHC-bound state. Consistent with the data obtained for HLA-A2.1, H-2Db exchange tetramers prepared from UV light–sensitive H-2Db–ASNENJETM complexes stained antigen-specific T cells with high specificity (Fig. 3c). Furthermore, as is the case for HLA-A2.1–GILGFVFJL tetramers, H-2Db–ASNENJETM tetramers that are uniformly occupied by the UV light–sensitive variant of NP(366–374) did not stain influenza A NP(366–374)–specific T cells (Fig. 3c). High-throughput screening with MHC exchange reagents As a first test of the potential of MHC exchange for high-throughput epitope mapping, we cloned and sequenced the four genes encoding the immunodominant proteins of influenza A/Vietnam/1194/04, an influenza A H5N1 strain isolated from an individual with a fatal case of influenza in Vietnam, and scanned the encoded proteins for potential HLA-A2.1–binding peptides. Within the genes encoding hemagglutinin, neuraminidase, M1 and nucleoprotein (NP), we identified 132 potential epitopes with a score for predicted MHC binding of Z20 (ref. 22), and these peptides were produced by microscale synthesis. In parallel, we used the same set of genes to prepare vectors for DNA vaccination and vaccinated groups of HLAA2.1 transgenic mice26 by DNA tattoo27. At the peak of the vaccination-induced T-cell response, we generated a collection of MHC exchange tetramers by performing 132 parallel UV light–mediated NATURE MEDICINE VOLUME 12 [ NUMBER 2 [ FEBRUARY 2006 a b A2Kb-(I) UV M1 58–66 A2Kb-(I) UV NP 373–381 0.03% A2Kb-(I) UV NP(373–381)-PE Percent MHC tetramer CD8+ cells + 15 12 9 6 3 0 9.9% – + – + – + + 15 A2Kb-(I) UV NP(373–381)-PE Percent MHC tetramer CD8+ cells Figure 4 High-throughput screen of H5N1 T-cell epitopes. (a) Flow cytometric analysis of mouse peripheral blood mononuclear cells stained with A2Kb-NP(373–381) exchange tetramers of either a nonvaccinated HLA-A2.1 transgenic mouse (top) or of an HLA-A2.1 transgenic mouse (bottom) vaccinated at days 0, 3 and 6 with 20 mg of influenza A/Vietnam/ 1194/04 NP-encoding DNA. (b) Top, T-cell responses of individual mice (closed squares) and average T-cell responses (stripes) of nonvaccinated mice (–) and mice vaccinated with the influenza A/Vietnam/1194/04 M1 (left, +) and NP (right, +) genes, analyzed with the indicated A2Kb exchange tetramers at day 13 after primary vaccination. At the peak of the vaccination-induced T-cell response, peripheral blood was drawn. Blood of mice in each of the four groups was pooled and analyzed by MHC tetramer staining. MHC tetramers that scored positive were used for reanalysis of individual mice. Bottom, T-cell responses of individual mice (closed squares) and average T-cell responses (stripes) of nonvaccinated mice (–) and mice vaccinated with the influenza A/Vietnam/1194/04 M1 (left, +) and NP (right, +) genes, analyzed with the indicated A2Kb exchange tetramers at day 11 after secondary vaccination. The identity of the NP(373–381) epitope was confirmed by synthesis of this sequence on a preparative scale followed by high-performance liquid chromatography purification and screening of vaccinated mice by MHC exchange tetramer staining and intracellular interferon-g staining (data not shown). NP, nucleoprotein; M1, matrix protein 1. anti–CD8-APC © 2006 Nature Publishing Group http://www.nature.com/naturemedicine TECHNICAL REPORTS 12 9 6 3 0 – Vaccination + exchange reactions on A2Kb-GILGFVFJL complexes28 in microtiter format, and used the resulting MHC tetramer collection to screen peripheral blood samples of vaccinated mice. This analysis showed the presence of two T-cell epitopes within these four gene products of influenza A/Vietnam/1194/04. Specifically, this screen confirmed the immunogenicity of the known influenza A M1(58–66) epitope that is conserved between the majority of influenza A strains. In addition, this scan indicated the presence of a previously unknown HLA-A2.1– restricted T-cell epitope located in the influenza A/Vietnam/1194/04 NP (Fig. 4). Notably, in this HLA-A2.1 transgenic mouse model, the immunogenicity of this epitope (NP(373–381)) is substantially higher than that of the classical influenza A M1(58–66) epitope (M1(58–66), one out of five responding mice after primary vaccination, three out of five responding mice after secondary vaccination; NP(373–381), five out of five responding mice after primary vaccination). This previously unknown T-cell epitope is shared between H5N1 strains of the past years but is distinct in older influenza A strains. DISCUSSION Here we have described conditional MHC ligands that can disintegrate in the MHC-bound state under conditions that do not affect the integrity of the MHC molecule, thereby permitting the reloading of assembled MHC molecules with epitopes of choice. The peptide ligands that are bound to the MHC after exchange are not modified, nor is the MHC backbone altered. In line with this, MHC complexes generated by this exchange technology have the predicted binding specificity in all cases tested. This strategy and related chemical cleavage strategies should be of substantial use in the high-throughput identification of both MHC ligands and cytotoxic T-cell responses. In this strategy, a single batch of UV light–sensitive MHC complex is prepared by the classical in vitro MHC class I refolding and purification protocols, and this UV light–sensitive MHC complex is subsequently used to generate large arrays of desired peptide-MHC complexes in 1-h exchange reactions. MHC-immunoglobulin dimers purified from eukaryotic cells have previously been used to generate peptide-MHC reagents, by performing exchange reactions with exogenously added peptide24,25. Although the overall goal of this technology is similar to that of UV light– induced peptide exchange, the technologies differ at essential points. Specifically, whereas the MHC-immunoglobulin dimer technology 249 © 2006 Nature Publishing Group http://www.nature.com/naturemedicine TECHNICAL REPORTS depends on the slow release of a pool of unknown endogenous peptides and is facilitated by conditions (for example, low or high pH) that also destabilize the MHC molecule, UV light–induced peptide exchange is based on the release of a single ligand, by exposure to a defined trigger that does not affect the integrity of the MHC molecule. Furthermore, the capacity of MHC-immunoglobulin–based reagents to identify antigen-specific T-cell responses is limited as compared to MHC exchange tetramers (Supplementary Fig. 4 online). The observation that ligands that disintegrate on command could be readily identified for both tested MHC alleles suggests that it will be straightforward to identify 2-nitrophenyl–based conditional ligands for other MHC class I alleles. Conditional ligands can be designed by replacement of amino acids in either a known peptide ligand, or in a predicted high-affinity ligand based on the peptide binding motif for this allele, and for HLA-A2.1 both approaches have been successful. For MHC alleles for which structural information is available, the water-accessibility of side chains may also be used as a criterion to select positions at which the UV light–sensitive building block can be incorporated. Various types of functional assays for detection of antigen-specific T cells, such as intracellular cytokine staining and cytokine capture, have been developed in the past few years and these assays may be used for high-throughput analysis of T cells that have a given effector function. MHC exchange multimer technology should complement these technologies by allowing high-throughput analysis of T-cell responses, irrespective of the capacity of T cells to produce a given cytokine. Also the combination of the two technologies, in which high-complexity MHC multimer arrays are used to probe T-cell reactivity by monitoring cytokine production, may be particularly useful. On-command cleavage of MHC ligands also seems attractive for the production of clinical-grade MHC reagents for adoptive T-cell therapy13. Specifically, the generation of a single large batch of clinicalgrade MHC molecules complexed with conditional ligand may allow the straightforward assembly of an MHC reagent desired for clinical use, by performing MHC exchange reactions with the relevant peptide ligands. Such high-grade MHC reagents should be particularly attractive for the isolation of melanoma-specific T cells29, and for the isolation of defined minor histocompatibility antigen-specific T cells12. Finally, in addition to the use of conditional MHC class I ligands for high-throughput diagnostic screening and for adoptive T-cell therapy, we speculate that cleavage of ligands bound to MHC molecules in the crystalline state30 might be used to obtain the elusive structure of the empty MHC class I molecule. METHODS Peptide synthesis and preparation of recombinant MHC. We obtained the UV light–sensitive building block for peptide synthesis (N-fluorenylmethyloxycarbonyl 3-amino-3-(2-nitro)phenyl-propionic acid) by protection of 3-amino-3-(2-nitro)phenyl-propionic acid (Lancaster) with fluorenylmethyl chloroformate (Sigma-Aldrich) in dioxane–10% aqueous Na2CO3 3/2 (vol/ vol) according to a published procedure21. We synthesized naturally occurring peptides and UV light–sensitive peptide variants by standard Fmoc synthesis. We performed MHC class I refolding reactions as previously described20, and we purified refolded MHC class I molecules by gel-filtration chromatography on a Phenomenex Biosep SEC S3000 column (Phenomenex) in 20 mM TrisHCl (pH 7.0), 150 mM NaCl. We stored purified MHC class I complexes at –20 1C in 20 mM Tris-HCl (pH 7.0), 150 mM NaCl and 16% glycerol. MHC exchange reactions. To produce single or small sets of MHC reagents by MHC exchange, we exposed biotinylated HLA-A2.1–GILGFVFJL, HLA-A2.1– ILAETVAJV or H-2Db–ASNENJETM complexes (0.5 mM in 20 mM Tris-HCl (pH 7.0), 150 mM NaCl and 0.5 mM dithiothreitol (DTT)) to UV light 250 (366-nm UV lamp; Camag) in the presence of 50 mM of the indicated peptides for 1–2 h on ice. After exchange, we spun samples at 16,000g for 5 min, added PE-streptavidin and used the resulting MHC exchange tetramers for T-cell staining without further purification. To generate the collection of H5N1-A2Kb tetramers, we predicted potential peptide epitopes within four gene segments of influenza A/Vietnam/1194/04 using the SYFPEITHI prediction program22 and all peptides with a SYFPEITHI score Z20 were produced by microscale (60 nmol) synthesis (JPT Peptide Technologies, GmbH). Of the 132 potential epitopes, 116 terminated in either a valine, leucine or isoleucine residue and these were synthesized with the naturally occurring C-terminal amino acid. The remaining 16 peptides, terminating in various nonaliphatic amino acids, were all synthesized with a C-terminal isoleucine to facilitate peptide production. We prepared MHC exchange tetramers by performing parallel small-scale exchange reactions on biotinylated A2Kb-GILGFVFJL class I complexes (0.5 mM)28 with the 132 candidate influenza A/Vietnam/1194/04 epitopes and a set of control peptides (all peptides at 50 mM), by exposure to UV light for 1 h on ice in 20 mM Tris-HCl (pH 7.0), 150 mM NaCl, and 0.5 mM DTT in 96-well tissue-culture V-bottom polystyrene plates (NUNC). Then we spun the samples at 3,300g for 5 min, added PE-streptavidin (final concentration, 10 mg/ml) and used the resulting MHC exchange tetramers for T-cell staining without further purification. Mice and vaccinations. We obtained C57BL/6 mice and mice transgenic for the HLA-A2Kb fusion gene26 from the animal department of The Netherlands Cancer Institute. All animal experiments were carried out in accordance with institutional and national guidelines and were approved by the Experimental Animal Committee of the Netherlands Cancer Institute (DEC). For live influenza A infections, we intranasally administered 50 ml of HEPESbuffered saline solution (Life Technologies) containing 200 hemagglutinating units influenza A/HKx31 virus to anesthetized mice. For vaccination of HLAA2 transgenic mice with H5N1 gene segments, we obtained the indicated gene segments from influenza A/Vietnam/1194/04, isolated from an individual in Vietnam and cloned these into pVAX. Groups of four to six mice were vaccinated by DNA tattoo27 on days 0, 3 and 6 with 20 mg of pVAX expressing either the influenza A/Vietnam/1194/04 M1, hemagglutinin, neuraminidase or NP gene under control of the CMV promoter. Cells and flow cytometry. For analysis of MHC multimer binding and T-cell responses in mouse samples, we obtained peripheral blood and removed erythrocytes by incubation in erythrocyte lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA (pH 7.4)) on ice. Cells were stained with antibody to CD8a (BD Biosciences) and the indicated MHC tetramers for 10–15 min at 15–25 1C. For analysis of MHC multimer binding and T-cell responses in human samples, we obtained peripheral blood mononuclear cells of healthy volunteers by Ficoll gradient separation. We obtained the MART-1(26–35)–specific CTL clone by repetitive stimulation and cloning of tumor-infiltrating lymphocytes of an individual with melanoma. We stained cells with the indicated MHC tetramers for 5 min at 37 1C. Then we added CD8-specific antibody (BD Biosciences) and incubated cells for 10–15 min at 15–25 1C. Data acquisition and analysis was carried out on a FACSCalibur (Becton Dickinson) using CellQuest software. Note: Supplementary information is available on the Nature Medicine website. ACKNOWLEDGMENTS We would like to thank V. Cerundolo (Weatherall Institute of Molecular Medicine) for the A2Kb expression construct and F. Lemonnier (Institut Pasteur) for HLA-A2.1-transgenic mice. We would like to thank A. Pfauth and F. van Diepen for flow cytometry assistance, I. Blonk for help in generation of H5N1 DNA vaccines and H. Hilkmann for peptide synthesis. This work was funded by Netherlands Organization for Scientific Research (NWO) Pioneer (to T.S.) and Vidi (to H.O.) grants. COMPETING INTERESTS STATEMENT The authors declare competing financial interests (see the Nature Medicine website for details). VOLUME 12 [ NUMBER 2 [ FEBRUARY 2006 NATURE MEDICINE TECHNICAL REPORTS © 2006 Nature Publishing Group http://www.nature.com/naturemedicine Published online at http://www.nature.com/naturemedicine/ Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/ 1. Ljunggren, H.G. et al. Empty MHC class I molecules come out in the cold. Nature 346, 476–480 (1990). 2. Schumacher, T.N. et al. Direct binding of peptide to empty MHC class I molecules on intact cells and in vitro. Cell 62, 563–567 (1990). 3. 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