Journal of Immunological Methods, 139 (1991) 41-47
41
© 1991 Elsevier Science Publishers B.V. 0022-1759/91/$03.50 ADONIS 0022175991001588 JIM 05907
Soluble HLA-A2.1 restricted peptides that are recognized by influenza virus specific cytotoxic T lymphocytes Maria A. Bednarek, Sara A. Engl, Maureen C. Gammon, Jonathan A. Lindquist *, Gene Porter, Alan R. Williamson and Hans J. Zweerink Merck Sharp and Dohme Research Laboratories, P.O. Box 2000, Rahway, NJ 07065, U.S.A.
(Received 11 October 1990, revised received 18 January 1991, accepted 18 January 1991)
The influenza A virus matrix protein derived peptide with amino acids 57-68 (Lys-Gly-Ileu-Leu-GlyPhe-Val-Phe-Thr-Leu-Thr-Val) is recognized by influenza virus H L A - A 2 restricted CTL. Because of the large number of hydrophobic residues this peptide is very insoluble. Substitution with a number of polar amino acids resulted in a soluble peptide (Lys-Lys-Ala-Leu-Gly-Phe-Val-Phe-Thr-Leu-Asp-Lys) that was very effective in sensitizing HLA-A2 positive target cells. Further substitution of threonine in position 65 with lysine resulted in a soluble antagonist peptide that inhibited sensitization. Both agonist and antagonist peptides retained 20% of their biological activity when tyrosine was added at the N terminus. Soluble radio-iodinated peptides can now be prepared that will be useful reagents to study the interaction of peptides and class I molecules. Key words: Peptide; Major histocompatibility complex class I; HLA-A2; T cell recognition
Introduction Structural analyses of HLA-A2 support the hypothesis that M H C class I molecules bind peptide
Correspondence to: H.J. Zweerink, Department of Autoimmune Diseases Research, Immunology and Inflammation Research, Merck Sharp and Dohme Research Laboratories, Rahway, NJ 07065, U.S.A. * Present address: Department of Biochemistry, Oregon State University, Corvallis, OR 97331, U.S.A. Abbreviations: CTL, cytotoxic T lymphocytes; RPMI-H10 or RPMI-FBS10, RPMI containing 50/tg/ml gentamycin, 0.01 M Hepes buffer (pH 7.4), 4 mM glutamine and 10% pooled human plasma (H10) or fetal bovine serum (FBS10); PBMN, peripheral blood mononuclear cells; HMY/A2 cells, HMY2. CIR cells that express transfected genomic HLA-A2.1; TFA, trifluoroacetic acid; RP-HPLC, reverse phase high pressure liquid chromatography; DMSO, dimethyl sulfoxide.
epitopes in a groove formed by two a helices and a fl pleated sheet (Bjorkman et al., 1987a,b; Garett et al., 1989). Experimental systems employing influenza virus specific C T L and target cells that are sensitized with synthetic peptides have provided a great deal of insight into the amino acid motifs necessary for binding to class I molecules and to T cell receptors (Gotch et al., 1988; Hogan et al., 1988; Shimojo et al., 1989). However, additional quantitative and kinetic studies on the interactions between peptides and class I molecules require in vitro binding assays. Attempts to develop a soluble assay, as described for M H C class II molecules (Babbitt et al., 1985; Buus et al., 1986; Roche and Cresswell, 1990) have met with limited success (Chen and Parham, 1989). An alternate assay, employing peptides bound to plates and 125iodine-labeled affinity purified M H C class I mole-
42 cules, has been published (Bouillot et al., 1989; Chen and Parham, 1990; Choppin et al., 1990; Frelinger et al., 1990). However, this kind of assay does not allow for quantitative analysis. Two peptides have been described in the literature that are recognized in association of HLA-A2 by influenza virus specific CTL: one derived from the influenza B nucleoprotein, residues 82-94 (BNP82-94; Robbins et al., 1989) and the other from the influenza A matrix protein, residues 5768 (M57-68; Gotch et al., 1987). However, both BNP82-94 and M57-68, as well as their biologically active analog peptides, are very insoluble. This makes them poor reagents to study the interaction between peptides and HLA-A2 molecules. Given the relative ease with which HLA-A2 restricted CTL that recognize peptide M57-68 can be generated we decided to focus on this peptide and design analog peptides that are soluble under physiological conditions. In this paper we describe soluble peptides that either sensitize HLA-A2 positive target cells for lysis by influenza virus specific CTL, or inhibit sensitization.
Materials and methods
Generation of M57-68 specific cytotoxic T cells (CTL) CTL lines specific for the influenza virus type A matrix peptide 57-68 (M57-68) were generated as described (Hogan et al., 1988; Bodmer, 1989). Briefly, PBMN from HLA-A2 positive donors were separated on Hypaque-Ficoll and incubated in RPMI-H10 at 106/ml in the presence of 5 / z g / m l peptide M57-68. After 3 days recombinant IL-2 (Amgen, Thousands Oaks, CA) was added (2 U / m l ) . 7 days later cells were centrifuged (800 x g for 10 r a i n ) a n d resuspended at 5 x 105 viable cells per ml in the presence of 5 x 105/ml peptide pulsed PMN and IL-2, Peptide pulsed PMN were prepared as follows: 107 cells were centrifuged and resuspended in 100 /~1 PBS containing 1 m g / m l M57-68. After 1 h at 37 ° C the cells were irradiated (2000 R), washed once and added to the cultures. When cell densities exceeded 106/ml, cells were diluted to 5 X 105/ml by centrifugation and resuspending in RPMI-H10 with IL-2. Every
10 days the cells were pulsed with M57-68 as described above. After approximately 20 days in culture, cells were tested for cytotoxic activity against HLA-A2 positive cells in the presence and absence of M5768 (see below). Cultures that lysed at least 40% of peptide pulsed cells and less than 10% of nonpeptide pulsed cells were maintained for further experiments. Since one donor gave the most consistently active CTL cultures, all experiments described in this paper were carried out with CTL derived from this individual. Generally, CTL cultures remained cytotoxic for at least 3 weeks after the initial assay at 20 days. HLA-DR1 restricted CTL specific for influenza virus A matrix peptide amino acids 17-31 and HLA-DR1 positive K4B target cells were obtained from Dr. William E. Biddison, National Institutes of Health, Bethesda, MD.
Generation of allospecific CTL PBMN from a HLA-A2 negative donor at 106/ml were incubated in R P M I / F B S 1 0 in the presence of 106/ml irradiated (6000 R) H M Y / A 2 cells (see below). Cultures were restimulated every 5 days and tested for cytotoxic activity against H M Y / A 2 cells after three stimulations. Over 90% of the target cells were lysed at E / T ratios between 1 and 2.5. Cytotoxic assays Target cells were H M Y 2 . C I R cells that express transfected genomic HLA-A2.1 D N A (Hogan et al., 1988). They were obtained from Dr. William E. Biddison at the National Institutes of Health, Bethesda, MD, and they will be referred to as H M Y / A 2 cells. For reasons not related to this paper these cells also contained an episomal plasmid p8901 with an E. coli hygromycin B phophotransferase gene which enabled the cells to grow in 150 /~g/ml hygromycin (Maniatis et al., 1982). The presence of this vector did not affect expression of HLA-A2 or the ability of these cells to be lysed by CTL described above (data not shown). Target cells were labeled overnight (2 x 1 0 6 cells in 2 ml R P M I / F B S and 100 /zCi 51-chromium), washed three times and suspended at 5 × 104 cells/ml in R P M I / F B S 1 0 . Aliquots of 100 /~1
43
were added per well, followed by 50 /~1 peptide solution and 50 /~l CTL (centrifuged and resuspend at appropriate concentrations in R P M I / FBS10). After 4 h incubation 100 ~1 supernatant was collected, counted and percent lysis calculated from the formula: 100 x ( E - M / D - M ) , where E = experimental release; M - - r e l e a s e in the absence of CTL; D = release in 1 M HCI. For each CTL culture, cytotoxic assays were carried out beforehand at different effector to target cell ratios in the presence of 0.5 ffg/ml M57-68; E / T ratios that gave near maximum lysis (generally 5) were then choosen for peptide sensitization or competition experiments. For peptide sensitization experiments, peptides (4 x concentrated in 50 ffl) were added to targets followed by the addition of CTL. In competition experiments, competing and sensitizing peptides were mixed, added to targets (50 /~1 4 x concentrated), followed by the addition of CTL. Peptides were dissolved in 50% DMSO at concentrations of 5 m g / m l and diluted into R P M I / FBS just prior to use in cytotoxic assays.
Peptide synthesis Peptides were synthesized by solid-phase techniques (Barany and Merrifield, 1979) on an Applied Biosystems 431A peptide synthesizer, using commercially available t-BOC and F M O C protected amino acids and resins. After each coupling step resins were treated with acetic anhydride to block free, unreacted amino groups. Deprotection and cleavage of peptides from resins were performed as recommended by Applied Biosystems (431A peptide synthesis manual). Crude products were dissolved in either water-acetic acid or DMSO-acetic acid mixtures. Solutes were filtered and injected on C18 semipreparative reverse phase HPLC columns (Delta-Prep 4000 Waters System). Columns were eluted isocratically or with linear water-acetonitrile gradients (in 0.1% TFA). Fractions were checked for purity on analytical C18 and C4 reverse phase columns. Identity and purity of the final materials (as DMSO solutions) was established by amino acid analysis, fast atom bombardment mass spectrometry and analytical RP-HPLC. Purification was continued until single peaks were obtained by analytical RP-HPLC and one molecular ion was observed by fast atom
TABLE I PEPTIDE USED IN THIS STUDY Amino acid sequence
Short hand indication
KGILGFVFTLTV
M57-68
........ K---
M57-68K65
KKA ....... DK
KKAM1
KDA ....... DK
KDAMI
KKA ..... K-DK
KKAK65
YKKA ....... DK
YKKAMI
YKKA ..... K-DK
YKKAK65
bombardment mass spectrometry. Recoveries of peptide M57-68 and its analog with lysine in position 65 (M57-68K65) was much lower than those of the other modified peptide (see Table I) due to poor solubility in the HPLC solvents.
Peptide solubility Peptides were diluted from 5 m g / m l in 50% DMSO to 100 ~ g / m l in PBS, incubated at room temperature for 2 h and centrifuged at 10,000 × g for 10 min. Supernatants were diluted five-fold in 6 M guanidine and pelleted material was solubilized in 6 M guanidine. Samples were analyzed by RP-HPLC using a Vydac C18 column (5/~m particle size, 150 mm long) and a 5-80% acetonitrile gradient in water and 0.1% trifluoroacetic acid. Peak heights were measured and peptide concentrations in the pellets and supernatants calculated relative to the original PBS solution.
Results
Peptide solubility Table I lists the peptides that were synthesized for this study. Solubility of each peptide was determined operationally by diluting 5 m g / m l solutions in 50% DMSO into PBS to a final concentration of 100 btg/ml. After 2 h at room temperatures samples were centrifuged at 10,000 x g and peptide distribution in pellet and supernatant measured by RP-I-IPLC. Greater than 90% of peptides M57-68 and M57-68K65 was recovered from the pellets, whereas greater than 90% of the other peptides remained in the supernatants. Similar results were obtained when 1% BSA or 0.05%
44
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10 100 1000 10000 nM Peptide Fig. l. Sensitization of HMY/A2 target cells with peptides M57-68, KKAM1 or KDAM1. Target cells were incubated with peptides at the concentrations indicated and with CTL specific for M57-68 (E/T= 5). ©, M57-68; D, KKAM1; zx, KDAM1. Tween 20 were added to the buffer. Clearly, peptides M57-68 and M57-68K65 are not truly in solution in PBS whereas the other peptides appear to be (see also Fig. 6).
Peptide sensitization of H L A - A 2 positive target cells Peptides KKAM1 and K D A M 1 were compared with M57-68 for their ability to sensitize HLA-A2 positive target cells for lysis with CTL that were generated against the influenza virus M57-68 peptide. K K A M 1 was at least 100-fold more potent than M57-68 (Fig. 1); significant stimulation was observed at concentrations below 1 nmol KKAM1. K D A M 1 on the other hand was less potent than M57-68. We also observed (data not shown) that peptides M57-68 and K D A M 1 at 5 /~M or higher were cytotoxic as judged by the release of 51Cr from targets in the absence of CTL. KKAM1 at 30 /tM (the highest concentration tested) did not show such cytotoxicity.
Threonine to lysine substitution in position 65 generates antagonist peptides It has been reported by Gotch et al. (1988) that substitution of threonine in position 65 of peptide M57-68 by lysine resulted in a peptide that inhibits target cell sensitization with M57-68. The same threonine to lysine substitution was made in
peptide KKM1. We found that this analog (KKAK65) would not sensitize target cells (data not shown). Next, K K A K 6 5 was compared with M57-68K65 for its ability to inhibit sensitization with M57-68 at 0.8/~M. M57-68K65 appeared to be a more effective inhibitor than KKAK65 (Fig. 2): a concentration of less than 10 # M M57-68K65 reduced lysis by 50% whereas greater than 20/~M of K K A K 6 5 was required. However at M5768K65 concentrations of 15 /~M this peptide induced spontaneous lysis of target cells. Thus at least part of the inhibitory effect of M57-68K65 may have been nonspecific. N o such toxicity of KKAK65 was observed at the highest concentrations tested (150/~M). Further experiments established that KKAK65 also antagonized KKAM1. Fig. 3 shows a sensitization dose response curve of KKAM1 (Fig. 3A) and M57-68 (Fig. 3B) in the absence or presence of K K A K 6 5 (28 /~M). The latter peptides shifted the K K A M 1 and M57-68 titration curves over by 2 and 1 logs respectively. The addition of 28/~M KKAK65 to 0.01 btM K K A M 1 reduced lysis from 55 to 10%. The need for this 2800-fold excess can be explained by differences in the affinities of both peptides for HLA-A2. A number of additional experiments were carried out to establish that KKAK65 had a specific
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Fig. 2. Inhibition of target cell sensitization with M57-68 by M57-68K65 and KKAM1. HMY/A2 cells were incubated with M57-68 (at 0.8 ~M), with competing peptides at concentrations indicated and with M57-68 specific CTL. n M5768K65; ©, KKAK65.
45 inhibitory effect on the binding of sensitizing peptides to HLA-A2 . (1) Preincubation of C T L with K K A K 6 5 up to 100 /~M followed by centrifugation to remove unbound peptide had no effect on their cytotoxic activity (not shown). (2) Peptide K K A K 6 5 did not inhibit lysis of H M Y / A 2 cells by alloreactive HLA-A2 specific C T L whereas M57-68K65 was inhibitory (Fig. 4; M57-68K65 could not be assayed above 15 # M due to toxicity to target cells). Presumably, inhibition by M 5 7 68K65 is non-specific and due to toxicity. (3) K K A K 6 5 did not inhibit lysis of H L A - D R 1 cells
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Fig. 4. Inhibition of allospecific CTL by M57-68K65 and KKAK65. HMY/A2 cells were incubated with allospecific CTL (E/T= 2) in the presence of M57-68K65 or KKAK65 at concentrations indicated, o, plus KKAK65; [3, plus M5768K65.
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that were sensitized with the matrix peptide amino acids 17-31 (Shimojo et al., 1989; data not shown).
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Tyrosinated KKAM1 and KKAK65 K K A M 1 and K K A K 6 5 were modified further by adding tyrosine at the N terminus so that they could be iodinated. Both tyrosinated peptides retained significant biological activity. Fig. 5A shows that Y K K A M 1 sensitized target cells although 3-4-fold less efficient compared to K K A M 1 (EDs0 values were approximately 1.5 and 5 nM). YKK A K 6 5 inhibited sensitization of cells with K K A M 1 (at 7 nM) approximately 4-fold less efficient than K K A K 6 5 (Fig. 5B).
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Fig. 3. Inhibition of target cell sensitization with peptides M57-68 and KKAM1 by KKAK65. HMY/A2 cells were incubated with agonist peptides (KKAM1 (A) or M57-68 (B)) at concentrations indicated with or without 28 laM KKAK65 and with CTL at E / T = 5. o, no competing peptide; [3, plus competing peptide.
Peptide M 5 7 - 6 8 is insoluble in aqueous buffers because it contains a significant number of hydrophobic amino acids. The opportunity for substitution with polar residues is limited because a minimal five amino acid motif with hydrophobic side chains in position 6 0 - 6 4 is necessary for binding to H L A - A 2 and T cell receptor recognition (Gotch et al., 1988; Shimojo et al., 1989). Single amino acid changes in positions 57, 58, 59,
46
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Fig. 5. Biological activities of peptides Y K K A M 1 and Y K K A K 6 5 . A: H M Y / A 2 cells were incubated with K K A M 1 ( o ) or Y K K A M 1 (t3) at the concentrations indicated and with C T L at E / T = 5. B: H M Y / A 2 cells were incubated with 7 n M sensitizing peptide K K A M 1 and competing peptides K K A K 6 5 ( o ) or Y K K A K 6 5 (D) at concentrations indicated and with C T L at E / T = 5.
67 and 68 on the other hand had little effect on recognition (Gotch et al., 1988). Furthermore, H M Y / A 2 cells transfected with a minigene that codes for a modified M57-68 peptide (the amino acids in positions 57-59 and 65-68 were substituted with alanine and amino acids 60-64 remained unchanged) were recognized by CTL specific for M57-68 (submitted for publication). Therefore, the strategy in designing more soluble peptides that retained biological activity was to leave amino acids 60-64 unchanged and to reduce hydrophobicity by making substitutions elsewhere in the molecule. Changing valine to lysine in position 68 does not affect recognition (Gotch et al., 1988); the threonine to aspartic acid change in position 67 was made because of the steric resemblance between both residues; and the isoleucine to alanine change in position 59 to reduce hydrophobicity. Glycine 58 was replaced either by positively charged lysine or by negatively charged aspartic acid. Fig. 1 shows that the lysine substitution resulted in a more potent sensitizing peptide (KKAM1) than the aspartic acid substitution (KDAM1). The precise alignment of the matrix protein derived peptide in the HLA-A2 binding groove is not known. Thus one can only speculate as to whether the charge reversal in position 58 results in reduced peptide binding to HLA-A2.
Alternatively, difference in target cell sensitization may be due to T cell receptor recognition. Tyrosinated KKAM1 and KKAK65 (with significant biological activity) can be radio-iodinated and will be valuable tools in developing assays to quantitate peptide binding to HLA-A2 molecules. Competitive binding experiments with synthetic peptide analogs, using wild type HLA-A2 as well as site specific -A2 mutants, should lead to significant insight in the structural requirements for peptide-protein interactions in the class I binding groove. Most class I molecules at the cell surface are occupied with endogenous peptides but the presence of empty molecules is likely (Ljunggren et al., 1990; Townsend et al., 1990). Presumably, exogenous peptides bind to these molecules a n d / o r exchange endogenous peptides that are bound with low affinity. Fewer than 200 peptide/class II complexes on the surface of antigen presenting cells will activate T cells (Demotz et al., 1990; Harding and Inanue, 1990). The need for binding of a small number of peptides to class I molecules sufficient for CTL recognition and lysis has been suggested (Bodmer et al., 1989) but could not be formally determined because of the lack of suitable reagents. The use of radio-iodinated peptide KKAM1 will allow us to address this issue.
47
Peptides M57-68, M57-68K65 and KDAM1 at 5 ~M or above were toxic to cells. We believe that this cytotoxicity was a property of the peptides themselves because truncated, deleted or otherwise modified peptides derived from parent sequences and other potentially toxic molecules were not detected (see materials and methods section; peptide synthesis). However, the presence of trace amounts of cytotoxic contaminants can not be ruled out.
Acknowledgements We thank Dr. William E. Biddison at the National Institutes of Health, Bethesda, MD, for providing us with H M Y / A 2 cells, with HLA-DR1 restricted CTL specific for the matrix peptide 17-31 and with the latter peptide.
References Babbit, B.P., Allen, P.M., Matsueda, G., Haber, E. and Unanue, E.R. (1985) Binding of immunogenic peptides to Ia histocompatibility molecules. Nature 317, 359-360. Barany, G. and Merrifield, R. (1979) In: E. Gross and J. Meienhofer (Eds.), The Peptides. Academic Press, New York, pp. 1-284. Bouillot, M., Choppin, J., Cornille, F., Martinon, F., Papo, T., Gomard, E., Fournie-Zaluski, M.-C. and Levy, J.-P. (1989) Physical association between MHC class I molecules and immunogenic peptides. Nature 339, 473-475. Bjorkman, P.J., Saper, M.A., Samraoui, B., Bennett, W.S., Strominger, J.L. and Wiley, D.C. (1987a) Structure of the human class I histocompatibility antigen, HLA-A2. Nature 329, 506-512. Bjorkman, P.J., Saper, M.A., Samraoui, B., Bennett, W.S., Strominger, J.L. and Wiley, D.C. (1987b) The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens. Nature 329, 512-518. Bodmer, H.C., (1989) Ph.D. Thesis, University of Oxford. Bodmer, H.C., Bastin, J.M., Askonas, B.A. and Townsend, A.R.M. (1989) Influenza-specific cytotoxic T-cell recognition is inhibited by peptides unrelated in both sequence and MHC restriction. Immunology 66, 163-169. Buus, S., Colon, S., Smith, C., Freed, J.H., Miles, C. and Grey, H.M. (1986) Interaction between a "processed" ovalbumin peptide and Ia molecules. Proc. Natl. Acad. Sci. U.S.A. 83, 3968-3971. Chen, B.P. and Parham, P. (1989) Direct binding of influenza peptides to class I HLA molecules. Nature 337, 743-745. Chen, B.P., Rothbard, J. and Parham, P. (1990) Apparent lack
of MHC restriction in binding of class I HLA molecules to solid-phase peptides. J. Exp. Med. 172, 931-936. Choppin, J., Martinon, F., Gomard, E., Bahraoui, E., Connan, F., Bouillot, M. and Levy, J.-P. (1990) Analysis of physical interactions between peptides and HLA molecules and application to the detection of human immunodeficiency virus 1 antigenic peptides. J. Exp. Med. 172, 889-899. Demotz, S., Grey, H.M. and Sette, A. (1990) The minimal number of class II MHC-antigen complexes needed for T cell activation. Science 249, 1028-1030. Frelinger, J.A., Gotch, F.M., Zweerink, H., Wain, E. and McMichael, A.J. (1990) Evidence of widespread binding of HLA class I molecules to peptides. J. Exp. Med. 172, 827-834. Garrett, T.P.J., Saper, M.A., Bjorkman, P.J., Strominger, J.L. and Wiley, D.C. (1989) Specificity pockets for the side chains of peptide antigens in HLA-Aw68. Nature 342, 692-696. Gotch, F., Rothbard, J., Howland, K., Townsend, A. and McMichael, A. (1987) Cytotoxic T lymphocytes recognize a fragment of influenza virus matrix protein in association with HLA-A2. Nature 326, 881-882. Gotch, F., McMichael, A. and Rothbard, J. (1988) Recognition of influenza A matrix protein by HLA-A2-restricted cytotoxic T lymphocytes. Use of analogues to orientate the matrix peptide in the HLA-A2 binding site. J. Exp. Med. 168, 2045-2057. Harding, C.V. and Unanue, E.R. (1990) Quantitation of antigen-presenting cell MHC class II/peptide complexes necessary for T-cell stimulation. Nature 346, 574-576. Hogan, K.T., Shimojo, N., Walk, S.F., Englehard, V.H. Maloy, W.L., Coligan, J.E. and Biddison, W.E. (1988) Mutations in the a2 helix of HLA-A2 affect presentation but do not inhibit binding of influenza virus matrix peptide. J. Exp. Med. 168, 725-736. Ljunggren, H.-G., Stam, N.J., Ohlen, C., Neefjes, J.J., Hoglund, P., Heemels, M.-T., Bastin, J., Schumacher, T.N.M., Townsend, A., Karre, K. and Ploegh, H.L (1990) Empty MHC class I molecules come out in the cold. Nature 346, 476-480. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New York, p. 86. Robbins, P.A., Lettice, L.A., Rota, P., Santos-Aguado, J., Rothbard, J., McMichael, A.J. and Strominger, J.L. (1989) Comparison between two peptide epitopes presented to cytotoxic T lymphocytes by HLA-A2. Evidence for discrete locations within HLA-A2. J. Immunol. 143, 4098-4103. Roche, P.A. and Cresswell, P. (1990) High-affinity binding of an influenza hemagglutinin-derived peptide to purified HLA-DR. J. lmmunol. 144, 1849-1856. Shimojo, N., Maloy, W.L., Anderson, R.W., Biddison, W.E. and Coligan, J.E. (1989) Specificity of peptide binding by the HLA-A2.1 molecule. J. Immunol. 143, 2939-2947. Townsend, A., Elliott, T., Cerundolo, V., Foster, L., Barber, B. and Tse, A. (1990) Assembly of MHC class I molecules analyzed in vitro. Cell 62, 285-295.
41
© 1991 Elsevier Science Publishers B.V. 0022-1759/91/$03.50 ADONIS 0022175991001588 JIM 05907
Soluble HLA-A2.1 restricted peptides that are recognized by influenza virus specific cytotoxic T lymphocytes Maria A. Bednarek, Sara A. Engl, Maureen C. Gammon, Jonathan A. Lindquist *, Gene Porter, Alan R. Williamson and Hans J. Zweerink Merck Sharp and Dohme Research Laboratories, P.O. Box 2000, Rahway, NJ 07065, U.S.A.
(Received 11 October 1990, revised received 18 January 1991, accepted 18 January 1991)
The influenza A virus matrix protein derived peptide with amino acids 57-68 (Lys-Gly-Ileu-Leu-GlyPhe-Val-Phe-Thr-Leu-Thr-Val) is recognized by influenza virus H L A - A 2 restricted CTL. Because of the large number of hydrophobic residues this peptide is very insoluble. Substitution with a number of polar amino acids resulted in a soluble peptide (Lys-Lys-Ala-Leu-Gly-Phe-Val-Phe-Thr-Leu-Asp-Lys) that was very effective in sensitizing HLA-A2 positive target cells. Further substitution of threonine in position 65 with lysine resulted in a soluble antagonist peptide that inhibited sensitization. Both agonist and antagonist peptides retained 20% of their biological activity when tyrosine was added at the N terminus. Soluble radio-iodinated peptides can now be prepared that will be useful reagents to study the interaction of peptides and class I molecules. Key words: Peptide; Major histocompatibility complex class I; HLA-A2; T cell recognition
Introduction Structural analyses of HLA-A2 support the hypothesis that M H C class I molecules bind peptide
Correspondence to: H.J. Zweerink, Department of Autoimmune Diseases Research, Immunology and Inflammation Research, Merck Sharp and Dohme Research Laboratories, Rahway, NJ 07065, U.S.A. * Present address: Department of Biochemistry, Oregon State University, Corvallis, OR 97331, U.S.A. Abbreviations: CTL, cytotoxic T lymphocytes; RPMI-H10 or RPMI-FBS10, RPMI containing 50/tg/ml gentamycin, 0.01 M Hepes buffer (pH 7.4), 4 mM glutamine and 10% pooled human plasma (H10) or fetal bovine serum (FBS10); PBMN, peripheral blood mononuclear cells; HMY/A2 cells, HMY2. CIR cells that express transfected genomic HLA-A2.1; TFA, trifluoroacetic acid; RP-HPLC, reverse phase high pressure liquid chromatography; DMSO, dimethyl sulfoxide.
epitopes in a groove formed by two a helices and a fl pleated sheet (Bjorkman et al., 1987a,b; Garett et al., 1989). Experimental systems employing influenza virus specific C T L and target cells that are sensitized with synthetic peptides have provided a great deal of insight into the amino acid motifs necessary for binding to class I molecules and to T cell receptors (Gotch et al., 1988; Hogan et al., 1988; Shimojo et al., 1989). However, additional quantitative and kinetic studies on the interactions between peptides and class I molecules require in vitro binding assays. Attempts to develop a soluble assay, as described for M H C class II molecules (Babbitt et al., 1985; Buus et al., 1986; Roche and Cresswell, 1990) have met with limited success (Chen and Parham, 1989). An alternate assay, employing peptides bound to plates and 125iodine-labeled affinity purified M H C class I mole-
42 cules, has been published (Bouillot et al., 1989; Chen and Parham, 1990; Choppin et al., 1990; Frelinger et al., 1990). However, this kind of assay does not allow for quantitative analysis. Two peptides have been described in the literature that are recognized in association of HLA-A2 by influenza virus specific CTL: one derived from the influenza B nucleoprotein, residues 82-94 (BNP82-94; Robbins et al., 1989) and the other from the influenza A matrix protein, residues 5768 (M57-68; Gotch et al., 1987). However, both BNP82-94 and M57-68, as well as their biologically active analog peptides, are very insoluble. This makes them poor reagents to study the interaction between peptides and HLA-A2 molecules. Given the relative ease with which HLA-A2 restricted CTL that recognize peptide M57-68 can be generated we decided to focus on this peptide and design analog peptides that are soluble under physiological conditions. In this paper we describe soluble peptides that either sensitize HLA-A2 positive target cells for lysis by influenza virus specific CTL, or inhibit sensitization.
Materials and methods
Generation of M57-68 specific cytotoxic T cells (CTL) CTL lines specific for the influenza virus type A matrix peptide 57-68 (M57-68) were generated as described (Hogan et al., 1988; Bodmer, 1989). Briefly, PBMN from HLA-A2 positive donors were separated on Hypaque-Ficoll and incubated in RPMI-H10 at 106/ml in the presence of 5 / z g / m l peptide M57-68. After 3 days recombinant IL-2 (Amgen, Thousands Oaks, CA) was added (2 U / m l ) . 7 days later cells were centrifuged (800 x g for 10 r a i n ) a n d resuspended at 5 x 105 viable cells per ml in the presence of 5 x 105/ml peptide pulsed PMN and IL-2, Peptide pulsed PMN were prepared as follows: 107 cells were centrifuged and resuspended in 100 /~1 PBS containing 1 m g / m l M57-68. After 1 h at 37 ° C the cells were irradiated (2000 R), washed once and added to the cultures. When cell densities exceeded 106/ml, cells were diluted to 5 X 105/ml by centrifugation and resuspending in RPMI-H10 with IL-2. Every
10 days the cells were pulsed with M57-68 as described above. After approximately 20 days in culture, cells were tested for cytotoxic activity against HLA-A2 positive cells in the presence and absence of M5768 (see below). Cultures that lysed at least 40% of peptide pulsed cells and less than 10% of nonpeptide pulsed cells were maintained for further experiments. Since one donor gave the most consistently active CTL cultures, all experiments described in this paper were carried out with CTL derived from this individual. Generally, CTL cultures remained cytotoxic for at least 3 weeks after the initial assay at 20 days. HLA-DR1 restricted CTL specific for influenza virus A matrix peptide amino acids 17-31 and HLA-DR1 positive K4B target cells were obtained from Dr. William E. Biddison, National Institutes of Health, Bethesda, MD.
Generation of allospecific CTL PBMN from a HLA-A2 negative donor at 106/ml were incubated in R P M I / F B S 1 0 in the presence of 106/ml irradiated (6000 R) H M Y / A 2 cells (see below). Cultures were restimulated every 5 days and tested for cytotoxic activity against H M Y / A 2 cells after three stimulations. Over 90% of the target cells were lysed at E / T ratios between 1 and 2.5. Cytotoxic assays Target cells were H M Y 2 . C I R cells that express transfected genomic HLA-A2.1 D N A (Hogan et al., 1988). They were obtained from Dr. William E. Biddison at the National Institutes of Health, Bethesda, MD, and they will be referred to as H M Y / A 2 cells. For reasons not related to this paper these cells also contained an episomal plasmid p8901 with an E. coli hygromycin B phophotransferase gene which enabled the cells to grow in 150 /~g/ml hygromycin (Maniatis et al., 1982). The presence of this vector did not affect expression of HLA-A2 or the ability of these cells to be lysed by CTL described above (data not shown). Target cells were labeled overnight (2 x 1 0 6 cells in 2 ml R P M I / F B S and 100 /zCi 51-chromium), washed three times and suspended at 5 × 104 cells/ml in R P M I / F B S 1 0 . Aliquots of 100 /~1
43
were added per well, followed by 50 /~1 peptide solution and 50 /~l CTL (centrifuged and resuspend at appropriate concentrations in R P M I / FBS10). After 4 h incubation 100 ~1 supernatant was collected, counted and percent lysis calculated from the formula: 100 x ( E - M / D - M ) , where E = experimental release; M - - r e l e a s e in the absence of CTL; D = release in 1 M HCI. For each CTL culture, cytotoxic assays were carried out beforehand at different effector to target cell ratios in the presence of 0.5 ffg/ml M57-68; E / T ratios that gave near maximum lysis (generally 5) were then choosen for peptide sensitization or competition experiments. For peptide sensitization experiments, peptides (4 x concentrated in 50 ffl) were added to targets followed by the addition of CTL. In competition experiments, competing and sensitizing peptides were mixed, added to targets (50 /~1 4 x concentrated), followed by the addition of CTL. Peptides were dissolved in 50% DMSO at concentrations of 5 m g / m l and diluted into R P M I / FBS just prior to use in cytotoxic assays.
Peptide synthesis Peptides were synthesized by solid-phase techniques (Barany and Merrifield, 1979) on an Applied Biosystems 431A peptide synthesizer, using commercially available t-BOC and F M O C protected amino acids and resins. After each coupling step resins were treated with acetic anhydride to block free, unreacted amino groups. Deprotection and cleavage of peptides from resins were performed as recommended by Applied Biosystems (431A peptide synthesis manual). Crude products were dissolved in either water-acetic acid or DMSO-acetic acid mixtures. Solutes were filtered and injected on C18 semipreparative reverse phase HPLC columns (Delta-Prep 4000 Waters System). Columns were eluted isocratically or with linear water-acetonitrile gradients (in 0.1% TFA). Fractions were checked for purity on analytical C18 and C4 reverse phase columns. Identity and purity of the final materials (as DMSO solutions) was established by amino acid analysis, fast atom bombardment mass spectrometry and analytical RP-HPLC. Purification was continued until single peaks were obtained by analytical RP-HPLC and one molecular ion was observed by fast atom
TABLE I PEPTIDE USED IN THIS STUDY Amino acid sequence
Short hand indication
KGILGFVFTLTV
M57-68
........ K---
M57-68K65
KKA ....... DK
KKAM1
KDA ....... DK
KDAMI
KKA ..... K-DK
KKAK65
YKKA ....... DK
YKKAMI
YKKA ..... K-DK
YKKAK65
bombardment mass spectrometry. Recoveries of peptide M57-68 and its analog with lysine in position 65 (M57-68K65) was much lower than those of the other modified peptide (see Table I) due to poor solubility in the HPLC solvents.
Peptide solubility Peptides were diluted from 5 m g / m l in 50% DMSO to 100 ~ g / m l in PBS, incubated at room temperature for 2 h and centrifuged at 10,000 × g for 10 min. Supernatants were diluted five-fold in 6 M guanidine and pelleted material was solubilized in 6 M guanidine. Samples were analyzed by RP-HPLC using a Vydac C18 column (5/~m particle size, 150 mm long) and a 5-80% acetonitrile gradient in water and 0.1% trifluoroacetic acid. Peak heights were measured and peptide concentrations in the pellets and supernatants calculated relative to the original PBS solution.
Results
Peptide solubility Table I lists the peptides that were synthesized for this study. Solubility of each peptide was determined operationally by diluting 5 m g / m l solutions in 50% DMSO into PBS to a final concentration of 100 btg/ml. After 2 h at room temperatures samples were centrifuged at 10,000 x g and peptide distribution in pellet and supernatant measured by RP-I-IPLC. Greater than 90% of peptides M57-68 and M57-68K65 was recovered from the pellets, whereas greater than 90% of the other peptides remained in the supernatants. Similar results were obtained when 1% BSA or 0.05%
44
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10 100 1000 10000 nM Peptide Fig. l. Sensitization of HMY/A2 target cells with peptides M57-68, KKAM1 or KDAM1. Target cells were incubated with peptides at the concentrations indicated and with CTL specific for M57-68 (E/T= 5). ©, M57-68; D, KKAM1; zx, KDAM1. Tween 20 were added to the buffer. Clearly, peptides M57-68 and M57-68K65 are not truly in solution in PBS whereas the other peptides appear to be (see also Fig. 6).
Peptide sensitization of H L A - A 2 positive target cells Peptides KKAM1 and K D A M 1 were compared with M57-68 for their ability to sensitize HLA-A2 positive target cells for lysis with CTL that were generated against the influenza virus M57-68 peptide. K K A M 1 was at least 100-fold more potent than M57-68 (Fig. 1); significant stimulation was observed at concentrations below 1 nmol KKAM1. K D A M 1 on the other hand was less potent than M57-68. We also observed (data not shown) that peptides M57-68 and K D A M 1 at 5 /~M or higher were cytotoxic as judged by the release of 51Cr from targets in the absence of CTL. KKAM1 at 30 /tM (the highest concentration tested) did not show such cytotoxicity.
Threonine to lysine substitution in position 65 generates antagonist peptides It has been reported by Gotch et al. (1988) that substitution of threonine in position 65 of peptide M57-68 by lysine resulted in a peptide that inhibits target cell sensitization with M57-68. The same threonine to lysine substitution was made in
peptide KKM1. We found that this analog (KKAK65) would not sensitize target cells (data not shown). Next, K K A K 6 5 was compared with M57-68K65 for its ability to inhibit sensitization with M57-68 at 0.8/~M. M57-68K65 appeared to be a more effective inhibitor than KKAK65 (Fig. 2): a concentration of less than 10 # M M57-68K65 reduced lysis by 50% whereas greater than 20/~M of K K A K 6 5 was required. However at M5768K65 concentrations of 15 /~M this peptide induced spontaneous lysis of target cells. Thus at least part of the inhibitory effect of M57-68K65 may have been nonspecific. N o such toxicity of KKAK65 was observed at the highest concentrations tested (150/~M). Further experiments established that KKAK65 also antagonized KKAM1. Fig. 3 shows a sensitization dose response curve of KKAM1 (Fig. 3A) and M57-68 (Fig. 3B) in the absence or presence of K K A K 6 5 (28 /~M). The latter peptides shifted the K K A M 1 and M57-68 titration curves over by 2 and 1 logs respectively. The addition of 28/~M KKAK65 to 0.01 btM K K A M 1 reduced lysis from 55 to 10%. The need for this 2800-fold excess can be explained by differences in the affinities of both peptides for HLA-A2. A number of additional experiments were carried out to establish that KKAK65 had a specific
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Fig. 2. Inhibition of target cell sensitization with M57-68 by M57-68K65 and KKAM1. HMY/A2 cells were incubated with M57-68 (at 0.8 ~M), with competing peptides at concentrations indicated and with M57-68 specific CTL. n M5768K65; ©, KKAK65.
45 inhibitory effect on the binding of sensitizing peptides to HLA-A2 . (1) Preincubation of C T L with K K A K 6 5 up to 100 /~M followed by centrifugation to remove unbound peptide had no effect on their cytotoxic activity (not shown). (2) Peptide K K A K 6 5 did not inhibit lysis of H M Y / A 2 cells by alloreactive HLA-A2 specific C T L whereas M57-68K65 was inhibitory (Fig. 4; M57-68K65 could not be assayed above 15 # M due to toxicity to target cells). Presumably, inhibition by M 5 7 68K65 is non-specific and due to toxicity. (3) K K A K 6 5 did not inhibit lysis of H L A - D R 1 cells
120 100
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Fig. 4. Inhibition of allospecific CTL by M57-68K65 and KKAK65. HMY/A2 cells were incubated with allospecific CTL (E/T= 2) in the presence of M57-68K65 or KKAK65 at concentrations indicated, o, plus KKAK65; [3, plus M5768K65.
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that were sensitized with the matrix peptide amino acids 17-31 (Shimojo et al., 1989; data not shown).
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Tyrosinated KKAM1 and KKAK65 K K A M 1 and K K A K 6 5 were modified further by adding tyrosine at the N terminus so that they could be iodinated. Both tyrosinated peptides retained significant biological activity. Fig. 5A shows that Y K K A M 1 sensitized target cells although 3-4-fold less efficient compared to K K A M 1 (EDs0 values were approximately 1.5 and 5 nM). YKK A K 6 5 inhibited sensitization of cells with K K A M 1 (at 7 nM) approximately 4-fold less efficient than K K A K 6 5 (Fig. 5B).
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Fig. 3. Inhibition of target cell sensitization with peptides M57-68 and KKAM1 by KKAK65. HMY/A2 cells were incubated with agonist peptides (KKAM1 (A) or M57-68 (B)) at concentrations indicated with or without 28 laM KKAK65 and with CTL at E / T = 5. o, no competing peptide; [3, plus competing peptide.
Peptide M 5 7 - 6 8 is insoluble in aqueous buffers because it contains a significant number of hydrophobic amino acids. The opportunity for substitution with polar residues is limited because a minimal five amino acid motif with hydrophobic side chains in position 6 0 - 6 4 is necessary for binding to H L A - A 2 and T cell receptor recognition (Gotch et al., 1988; Shimojo et al., 1989). Single amino acid changes in positions 57, 58, 59,
46
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Fig. 5. Biological activities of peptides Y K K A M 1 and Y K K A K 6 5 . A: H M Y / A 2 cells were incubated with K K A M 1 ( o ) or Y K K A M 1 (t3) at the concentrations indicated and with C T L at E / T = 5. B: H M Y / A 2 cells were incubated with 7 n M sensitizing peptide K K A M 1 and competing peptides K K A K 6 5 ( o ) or Y K K A K 6 5 (D) at concentrations indicated and with C T L at E / T = 5.
67 and 68 on the other hand had little effect on recognition (Gotch et al., 1988). Furthermore, H M Y / A 2 cells transfected with a minigene that codes for a modified M57-68 peptide (the amino acids in positions 57-59 and 65-68 were substituted with alanine and amino acids 60-64 remained unchanged) were recognized by CTL specific for M57-68 (submitted for publication). Therefore, the strategy in designing more soluble peptides that retained biological activity was to leave amino acids 60-64 unchanged and to reduce hydrophobicity by making substitutions elsewhere in the molecule. Changing valine to lysine in position 68 does not affect recognition (Gotch et al., 1988); the threonine to aspartic acid change in position 67 was made because of the steric resemblance between both residues; and the isoleucine to alanine change in position 59 to reduce hydrophobicity. Glycine 58 was replaced either by positively charged lysine or by negatively charged aspartic acid. Fig. 1 shows that the lysine substitution resulted in a more potent sensitizing peptide (KKAM1) than the aspartic acid substitution (KDAM1). The precise alignment of the matrix protein derived peptide in the HLA-A2 binding groove is not known. Thus one can only speculate as to whether the charge reversal in position 58 results in reduced peptide binding to HLA-A2.
Alternatively, difference in target cell sensitization may be due to T cell receptor recognition. Tyrosinated KKAM1 and KKAK65 (with significant biological activity) can be radio-iodinated and will be valuable tools in developing assays to quantitate peptide binding to HLA-A2 molecules. Competitive binding experiments with synthetic peptide analogs, using wild type HLA-A2 as well as site specific -A2 mutants, should lead to significant insight in the structural requirements for peptide-protein interactions in the class I binding groove. Most class I molecules at the cell surface are occupied with endogenous peptides but the presence of empty molecules is likely (Ljunggren et al., 1990; Townsend et al., 1990). Presumably, exogenous peptides bind to these molecules a n d / o r exchange endogenous peptides that are bound with low affinity. Fewer than 200 peptide/class II complexes on the surface of antigen presenting cells will activate T cells (Demotz et al., 1990; Harding and Inanue, 1990). The need for binding of a small number of peptides to class I molecules sufficient for CTL recognition and lysis has been suggested (Bodmer et al., 1989) but could not be formally determined because of the lack of suitable reagents. The use of radio-iodinated peptide KKAM1 will allow us to address this issue.
47
Peptides M57-68, M57-68K65 and KDAM1 at 5 ~M or above were toxic to cells. We believe that this cytotoxicity was a property of the peptides themselves because truncated, deleted or otherwise modified peptides derived from parent sequences and other potentially toxic molecules were not detected (see materials and methods section; peptide synthesis). However, the presence of trace amounts of cytotoxic contaminants can not be ruled out.
Acknowledgements We thank Dr. William E. Biddison at the National Institutes of Health, Bethesda, MD, for providing us with H M Y / A 2 cells, with HLA-DR1 restricted CTL specific for the matrix peptide 17-31 and with the latter peptide.
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of MHC restriction in binding of class I HLA molecules to solid-phase peptides. J. Exp. Med. 172, 931-936. Choppin, J., Martinon, F., Gomard, E., Bahraoui, E., Connan, F., Bouillot, M. and Levy, J.-P. (1990) Analysis of physical interactions between peptides and HLA molecules and application to the detection of human immunodeficiency virus 1 antigenic peptides. J. Exp. Med. 172, 889-899. Demotz, S., Grey, H.M. and Sette, A. (1990) The minimal number of class II MHC-antigen complexes needed for T cell activation. Science 249, 1028-1030. Frelinger, J.A., Gotch, F.M., Zweerink, H., Wain, E. and McMichael, A.J. (1990) Evidence of widespread binding of HLA class I molecules to peptides. J. Exp. Med. 172, 827-834. Garrett, T.P.J., Saper, M.A., Bjorkman, P.J., Strominger, J.L. and Wiley, D.C. (1989) Specificity pockets for the side chains of peptide antigens in HLA-Aw68. Nature 342, 692-696. Gotch, F., Rothbard, J., Howland, K., Townsend, A. and McMichael, A. (1987) Cytotoxic T lymphocytes recognize a fragment of influenza virus matrix protein in association with HLA-A2. Nature 326, 881-882. Gotch, F., McMichael, A. and Rothbard, J. (1988) Recognition of influenza A matrix protein by HLA-A2-restricted cytotoxic T lymphocytes. Use of analogues to orientate the matrix peptide in the HLA-A2 binding site. J. Exp. Med. 168, 2045-2057. Harding, C.V. and Unanue, E.R. (1990) Quantitation of antigen-presenting cell MHC class II/peptide complexes necessary for T-cell stimulation. Nature 346, 574-576. Hogan, K.T., Shimojo, N., Walk, S.F., Englehard, V.H. Maloy, W.L., Coligan, J.E. and Biddison, W.E. (1988) Mutations in the a2 helix of HLA-A2 affect presentation but do not inhibit binding of influenza virus matrix peptide. J. Exp. Med. 168, 725-736. Ljunggren, H.-G., Stam, N.J., Ohlen, C., Neefjes, J.J., Hoglund, P., Heemels, M.-T., Bastin, J., Schumacher, T.N.M., Townsend, A., Karre, K. and Ploegh, H.L (1990) Empty MHC class I molecules come out in the cold. Nature 346, 476-480. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New York, p. 86. Robbins, P.A., Lettice, L.A., Rota, P., Santos-Aguado, J., Rothbard, J., McMichael, A.J. and Strominger, J.L. (1989) Comparison between two peptide epitopes presented to cytotoxic T lymphocytes by HLA-A2. Evidence for discrete locations within HLA-A2. J. Immunol. 143, 4098-4103. Roche, P.A. and Cresswell, P. (1990) High-affinity binding of an influenza hemagglutinin-derived peptide to purified HLA-DR. J. lmmunol. 144, 1849-1856. Shimojo, N., Maloy, W.L., Anderson, R.W., Biddison, W.E. and Coligan, J.E. (1989) Specificity of peptide binding by the HLA-A2.1 molecule. J. Immunol. 143, 2939-2947. Townsend, A., Elliott, T., Cerundolo, V., Foster, L., Barber, B. and Tse, A. (1990) Assembly of MHC class I molecules analyzed in vitro. Cell 62, 285-295.
