Eur. J. Immunol. 1990. 20: 2691-2697

M. I q m e Tcell epitope conformation and activity

2601

Dave C. Anderson, Wim C. A.Van Schooten, Michael E. Barry, Anneke A. M. Janson and RenC R. P. DeVries

Use of flanking sequences to study secondary structure-activity correlations of a Mycobacterium Zeprae T cell epitope*

Department of Pathobiology, University of Washington, Seattle, and Department of lmmunohematology and Blood Bank, University Hospital Leiden, Leiden

The 65-kDa protein of the intracellular pathogen M . leprue is prominent in the immune response to this mycobacterium, and individual Tcell epitopes from this protein sequence have been defined. We have tested the stimulatory activity of extended analogs of the minimal peptide representing one such epitope, LQAAPALDKL, with a variety of tetrapeptide extensions added to enhance or destabilize a helix formation. The conformational potential of the peptides was measured by circular dichroism using aqueous trifluoroethanol as a secondary structure inducer. Although analogs with high helical potential activated Tcells at low concentrations, a less helical variant was similarly potent. Activity also did not correlate with predicted overall a helical amphipathicity. One analog was found which stimulated T cell proliferation in the 50 pM range. The effect of tetrapeptide extensions on epitope activity is not consistent with the importance in activity of only a single stable secondary structure such as an a helix.

1 Introduction

helical, and found both preferentially adopted nonhelical conformations under a variety of solution conditions.

Foreign proteins can be presented as antigens to Tcells in the form of peptides produced by fragmentation of the proteins. Th cells are activated by peptides bound to Ia or HLA proteins, which are the “receptors” for these peptides [l], in the membrane of an APC such as an M Q or B cell [2]. There has been considerable interest in potential peptide conformational preferences [3-61 or sequence motifs [7-91 which allow T cell activation. The importance of peptide a helical secondary structure in Tcell stimulation was initially hypothesized by Pincus et al. [ 101,who suggested that cytochrome c peptides stimulated responding Tcells in an a helical conformation. DeLisi and Berzofsky [3] and Spouge et al. [ 111 suggested that this may be true for almost all Tcell epitopes, and that the helices are amphipathic. Fasman and colleagues [12] found that Tcell clones could respond to peptides from herpes simplex virus glycoprotein D which were nonhelical, and Carbone et al. [13]and Bhayani et al. [ 141have suggested that mouseTcell recognition of pigeon cytochrome c peptides may depend more on membrane-binding ability of the peptide than on helical potential. Sette et al. [15] found that Ia(d) binding of 22 analogs of an OVA Tcell epitope did not correlate with any regular secondary structure predicted by the ChouFasman algorithm. Abergel et al. [16] studied the solution conformation of twoTcell epitopes, both predicted to be a

[I 86591

*

This research was supported by grants from the University of Washington Graduate Student Research Fund (1986-1987), the Netherlands Leprosy Relief Association, the United Nations Development Program/World Ban W o r l d Health Organization Special Program for Research and Training in Tropical Diseases, and the Dutch Foundation for Medical and Health Research.

Correspondence: Dave Anderson, c/o Somatogen Inc., 350 Interlocken Parkway, Broomfield. CO 80021, USA Abbreviations: CD: Circular dichroism TFE: Trifluoroethanol, [ O ] :Mean residue ellipticity, degree-cm2/dmol 0 VCH Vcrlagsgesellschaft mbH, D-6940 Weinheim. 1990

The immunodominant 65-kDa heat-shock protein from Mycobucterium leprue [17] contains nine T cell epitopes with different HLA restrictions [18], including an M . leprue-specific, HLA-DRZrestricted epitope, whose minimal strongly stimulating sequence is LQAAPALDKL [ 191. We have mapped the residues critical for Tcell activation in this sequence with two different responding T cell clones, 2F10 and 2B6, to include 7-8 interior residues [20], and have examined the effects of substitutions in these core residues on activity [21]. Here we have designed and tested extended peptide analogs with different secondary structure potentials using differing tetrapeptide flanking sequences attached to a shorter active peptide. These longer peptide analogs, in which trifluoroethanol (TFE)-induced a helicity is enhanced by residues added to both ends of the peptide, are active in stimulating T cells. Correlation of their TFEinduced secondary structure with their activity tests hypotheses for the active conformation of these peptides.

2 Materials and methods 2.1 Proliferation assays Tcell clones 2F10 and 2B6 were obtained from a tuberculoid leprosy patient as described previously [22] and were activated by peptides as described [21]. Briefly, lo4 Tcells and 5 x loJ (40 Gy-irradiated) DR-matched allogeneic PBMC as APC were cultured in 200 yl Iscove’s modified Dulbecco’s medium with 10% human serum in the presence or absence of variable concentrations of peptides in 96-well, flat-bottom microtiter plates. The cultures were set up in triplicate and incubated as described above for 72 h. Eighteen hours before termination, 1.0 yCi = 37 kBq of [3H]dThd (New England Nuclear, Boston, MA) was added. The samples were harvested on glass-fiber filters using a semiautomatic sample harvester. [3H]dThd incorporation was assessed by counting in a liquid scintillation counter.

+

00 14-2980/90/1212-2691$3.50 .25/0

2692

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D. C. Anderson,W. C. A.Van Schooten, M. E. Barry et al.

The results are expressed as the mean of triplicate cultures. Paraformaldehyde fixation of cells was carried out as described [23] and HLA-DRZtransfected mouse L cells were constructed as described [24]. 2.2 Circular dichroism (CD) spectra and analysis CD spectra were obtained on a Cary 61 spectrometer, with Online Instrument Systems (Jefferson, GA) electronics and a model 4000 data system. Data was generally collected between 190-260 nm, with 1data point/0.2 nm, in a 0.1-nm path length cell (Varian Associates, Palo Alto, CA). The peptides were dissolved at ca. 1 mg/ml in pH 7.0, 0.01 M phosphate buffer, in 90%-100% trifluoroethanol (TFE; Aldrich, Milwavkee, WI; reagent grade) or in mixtures of both. Buffer solutions were made from HPLC-grade water (J. T. Baker, Phillipsburg, NJ), and filtered and degassed under vaccum before use. Data recalculated as mean residue ellipticity was fit to a linear combination of CD spectra from 16 proteins of known structure [25] using the program CONTIN [26]. The three secondary structures used in this fit are a helix, p sheet and “remainder”.

solution they show single minima around 200 nm, characteristic of peptides having a significant amount of disordered conformation [32]. Nearly every synthetic peptide tested showed this feature. To determine secondary structure preferences, TFE, an organic solvent which induces secondary structure in peptides by favoring intramolecular hydrogen bonds, and thus induces structure as well as an a helix [33], was added to each peptide. The peptides with lysine and alanine extensions showed evolution of negative peaks or shoulders around 207nm and 220nm with increasing TFE addition, indicative of formation of an a helix in this solvent [32]. The transition into an a helical form occurred at lower concentrations of TFE for the lysine extension peptide than for the alanine extensions or for the analogous proline peptide. Thus, this peptide appears to have a greater preference for an a helix. For all three peptides the transition to the a helix form saturated at about 50% TFE. The tetraproline extension peptide showed little evidence of TFE-induced a helicity, even in 100% TFE, and little induction of any additional secondary structure not present in aqueous solution. An approximate estimate of the a helical content of this peptide can be obtained from the mean residue ellipticity at 222 nm. For 100% helix, this peptide length-dependent [34] value is

2.3 Peptide synthesis Peptides were synthesized using the solid-phase peptide synthesis methodology of Merrifield [27] and the tea bag method of Houghten [28]. Peptides were made as amides except as noted, using 0.3 meq/g 4-methylbenzhydrylamine resin (Colorado Biotechnology Inc., Laramie, WY). Peptides were cleaved from the resin using HF and either the high or low-high protocols of Tam and Merrifield [29]. Peptides were dissolved in 20%-40% acetic acid and lyophilized, and then checked by reverse-phase HPLC with aVydac C4 column [20]. For peptides which were not white powders after the first lyophilization this step was repeated. Most peptides gave a single main peak; those which did not were repurified by reverse-phase chromatography. Peptides were hydrolyzed by gas-phase hydrolysis and analyzed as PTC amino acids using a modified procedure [20].

3 Results 3.1 Peptides with extensions of helix-forming and helix-breaking residues

u -5

.

-10

. c

-

T

CD spectra for three representative peptides, containing tetrapeptide extensions at each end of the strong helixforming residues lysine (K), alanine (A) or the helixbreaker proline (P) [31], are shown in Fig. 1. In aqueous

/SO% TFE

I

.:[

I

I

A AAAALLOAAP4LDKLAAAAIOX TFE /2OX T F E C 1502 T F E

B x

-

-10

-m

I

1EO

200

I

220 WLVELENGTH. nm

I

A

240

260

I

3.1.1 General remarks Our previous work with this epitope has shown that peptides longer than the minimal length were somewhat more potent than the minimal peptide [19].Thus, we added non-native N- and C-terminal extensions to affect peptide secondary structure or amphipathicity without disturbing the core residues, modification of which usually destroyed potency [20]. The effect of the extensions o n induced secondary structure was measured by CD spectroscopy [301.

I

.-lj

-5 F P P P P L L Q A A P A L D K L P P P P / O ~ TFE /loo: TFE -1c

c

1SL

29:

-4G

-1

L-.

nLl’ELENGTh,

--r -5-

rl

Figure 1. CD spectra of extension peptide analogs in 10 rnM phosphate buffer, pH 7.0, titrated with TFE to induce their preferred secondary structures.The spectral changes saturated by 50% TFE. Peptides with a higher a helical propensity exhibited appearance of a distinct negative shoulder in the spectrum at around 222 nm at lower percentage of TFE.

Eur. J. Immunol. 1990. 20: 2691-2697

M . leprue Tcell epitope conformation and activity

determined structure is the a helix and the spectra do not possess enough information t o determine more than three classes of secondary structure [36]. The best fit spectra are shown in Fig. 2, and the derived structure compositions are listed inTable 1. These results are consistent with the rank order of a helix content in 50% TFE for the tetrapeptide extensions shown in Fig. 1.These are E4 > K4 > G4 > A4 > P4. Except for the G4 peptide, in whichTFE appears to induce an increased fi sheet character, these results are consistent with the rank order of statistical preferences of these residues for an a helix [31]. In each peptide except G4 and P4, addition of TFE appears to induce a significant

- 36 000 for a 17mer [35]. A random coil can have a baseline value of - 1400 to - 3300 deg cm2/dmol. By this method, the TFE-induced a helicity of this peptide may be negligible.The extended peptides can thus be ranked by the extent of a helix formation in aqueous TFE solution.

3.1.2 Best-fit CD Spectra An alternate method involves fitting the entire CD spectrum to a linear combination of secondarv structures. For CD spectra between 190-240nm the most accurately

-

EEEELLOAAPALDKLEEEE

KKKKLLOAAPALDKLKKKK 20007

ir

0

-2030 4000

-6020 .BOO0

-1mo 200

210

220

230

240

-12000 185

205

215

225

235

WAVELENGTH, nm

WAVELENGTH. nm

GGGGUOAAPALDKLGGQG

AAAAUOAAPALDKIAAAA

0-

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-0

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195

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,

.

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PPPPLLOAAPALDKLPPPP

PPLLOAAPALDKLPP

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205 215 225 WAVELENGTH, nm

WAVELENGTH, nm

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.

190

WAVELENGTH, nm

I

200

.

I

I

'

210 220 WAVELENGTH, nm

I

.

I

230

240

LOAAPALDKL 0

Figure2. Best fit of peptide CD spectra to a linear combination of secondary structures. Data c points from the spectra are shown in 10mM z phosphate buffer, pH 7.0 (dark squares) and in 50% TFE (light squares), and the best fit curves E are shown as solid lines from the program CONTIN. Qualitatively, only the P4 extension peptide fails to show development of a significant negative shoulder at 222 nm. The best-fit parameters are listed in n b l e 1.

-2000 -4wo

-6000

-8000

- 1 ~ ~ 0

-12wo 195

2693

205

215 225 WAVELENGTH, nm

235

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Table 1. Secondary structure content of peptide analogs with variable tetrapeptide flanking sequences Data fit with CONTINb) Helix Beta sheet Other (”/. 1 (Yo 1 (% 1

Peptideal extensions E4 in 50% TFE in HzO K4 i n T F E in H20 A4 inTFE in H20 G4 inTFE H20 P4 i n T F E in H2O €9 inTFE in H20 LQA APALDKL-amide in TFE in H20

42 17 34 7 17 0 29 51 12 14 30 24

18 57 24 61 38 64 26 21 52 64 22 14

40

10 5

49 58

40

includes random configurations. This is not surprising since short peptides are known generally to contain a significant amount of random structure at room temperature. 3.1.3 Activity Fig. 3 shows activation of clone 2F10, fixed by 0.1% paraformaldehyde to block antigen internalization and processing. Under these conditions peptides may bind directly to surface HLA-DR2 in antigen presentation. The most potent peptide is that with a tetraproline extension, which appears approximately 10-fold more active than the more a helical tetraglutamate extension peptide. A similar experiment was done with paraformaldehyde-fixed DR2B1 chain mouse L cell transfectants (Fig. 2), in which the only antigen-presenting DR molecule contains the B-1 chain. Similar results are seen. Fixed cells such as these are fully functional in antigen presentation [23].The P4 peptide here also appears more potent than the more a helical analogs G4, A4 and E4. Again, there is no correlation between helix-forming potential in TFE and potency in activating the T cell clone 2F10.

26 42 32 45 36 45 28 36 22 49 62 37

a) Extensions are added to both ends of the peptide LLQAAPALDKL-amide. b) Secondary structure was determined by the best-fit linear combination of structures using CONTIN (26). Induced secondary structure was measured in 50% TFEdO% 10 mM phosphate buffer, pH 7.0, while structure in aqueous solution was measured in the above buffer.

increase in a helicity; in the latter two, the a helical content decreases. Most of the peptides are also fit with a significant fraction of ‘‘6 sheet” structure and “other” structure, which

A similar experiment was done with unfixed APC for clones 2F10 and 2B6 (Fig. 4). In this system the peptide potency may reflect processing steps inside APC as well as HLA peptide-T cell interactions. For both clones the peptides with helix-forming extensions are now more potent than the proline-extension peptide. This raises the possibility that an a helical conformation may be preferred at some step involved in antigen processing, but not necessarily in binding to HLA-DR2 and steps in antigen presentation.

100 1

I

-

P4- -P4 P2- -P2

I G4--G4

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-1 1

-9

E4--E4 LQAAPALDKL

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LOG [PEPTIDE, M I

I

I

-. -9

-8

-7 -6 LOG [PEPTIDE, MI

-5

-4

K4- -K4 P4- -P4

Figure 3. Concentration dependence of activation of T cell clone 2F10 by extension peptides presented by 0.1% paraformaldehydcfixcd monA4--A4 onuclear cells (a) and 1% paraformaldehydeE4--E4 fixed mouse L cell HLA DR2 transfectants (b). L Q M P A ~ K L Activation by the minimum-length epitope is shown for comparison. At levels about tenfold above saturation of activation, inhibition of Tcell proliferation is apparent. The data are expressed as mean cpm for triplicate determinations. The SEM did not cxceed 10% of the mean. P2--P2 G4--G4

M. leprue Tcell epitope conformation and activity

Eur. J. Immunol. 1990. 20: 2691-2697

120 100 80 60 40 20 140

-

-

7

.

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3.2 Amphipathic analogs E4--E4 K4--K4 A4--A4 P4--P4 LQAAPALDKL

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3.2.1 General description

.

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.

To examine the possibility that the activity of the M. leprue epitope or analogs might depend on the overall amphipathicity of the analogs, we compared four extension peptides with changes in the extensions which would affect overall amphipathicity. We have previously found that the activity of analogs with internal mutations does not correlate with the magnitude of their predicted a helical amphipathicity [21]. Helical amphipathicity is represented using Edmundson helical wheels [37] shown at the bottom of Fig. 5. This method of representing the induced secondary structure is reasonable since these peptides show significant induced a helical structure when in 50% TFE at room temperature. The calculated percent a helix varies from 36% for the peptide with extensions of AAAK and 38% for peptides with extensions AKKA and AKKK to 44% for the peptide with the extension AAKA. When completely helical, the parent peptide AKKALLQAAPALDKLAKKA would form a helix with nearly complete segregation of polar and nonpolar amino acids. In contrast the analog AAAKLLQAAPALDKLKAAA would have four alanines distributed along the polar face of the helix, and two lysines on the nonpolar face. The peptides AKKKLLQAAPALDKLKKKA and AAKALLQAAPALDKLAKAA will be intermediate between the first two peptides.

1

-8

-10

LOG (PEPTIDE, 6o

-6

M)

E4- -E4

(b)

h

K4--K4 A4--A4

,,,hJ

-Y-

~ 4 - - ~ 4

LQAAPALDKL

3.2.2 Activity

LOG (PEPTIDE, M)

Figure 4. Activation of T cell clones 2F10 (a) and 2B6 (b) by different concentrations of extension peptides presented by unfixed peripheral blood mononuclear cells. The core of the peptides is LLQAAPALDKL. The activity of the minimal active epitope, LQAAPALDKL, is presented for comparison. Data are presented as mean cpm for triplicate determinations.The SEM did not exceed 10% of the mean. 150

-

125I

0

r

x

5

100

-

75

-

50

-

25

-

u I

--

AAAK-DeD-KAAA AKKK-DeD-KKKA -0- AAKA-DeD-AKAA -0- AKKA-DeD-AKKA LQAAPALDKL

0-12

-10

- 8

- 6

Fig. 5 shows the activity of the amphipathic peptide analogs stimulating the clone 2F10. All peptides are more potent than the minimal stimulatory peptide, although their potency does not correlate with overall amphipathicity-The least amphipathic peptide, AAAKLLQAAPALDKLKAAA, is remarkably potent, with a half-stirnulatory

Figure 5. Activity of extended peptides with flanking sequences designed to vary the amphipathicity of the peptide.The changes in amphipathicity, assuming the peptide is in the form of an a helix, are shown at the bottom using the helical wheel representation. Stimulation of Tcell clone 2F10 is shown at the top as a function of peptide concentration. Data are presented as mean cpm for triplicate determinations, and the SEM did not exceed 10% of the mean. - 4

- 2

0

2

LOG (PEPTIDE, pg/ml) AAAK-

2695

-KAAA

AKKK-

-KKKA

AAKA-

-AKAA

AKKA-LLQAAPALDKL-AKKA A19

2696

D. C. Anderson, W. C. A.Van Schooten, M. E. Barry et al.

concentration of 50 PM. The high potency of these analogs suggests they are not compromised during antigen processing by the added extensions, and that flanking sequences may be used to construct considerably more potent analogs.The data do not support a correlation of overall helical amphipathicity for these extension peptides in enhancingT cell activation.

4 Discussion We have used TFE to probe the secondary structural preferences of the various peptide analogs of the parent epitope. This is a standard methodology for examining induced peptide secondary structure by CD, since many short peptides have minimal amounts of a single regular secondary structure in aqueous solution. They fluctuate between a variety of different conformations, the most populated of which are not necessarily the “active”ones. It is possible that the peptide conformations assigned here only approximate the actual conformation(s) in solution, since the standard CD spectra used t o fit the data are derived from the crystal structures of larger, more conformationally stable proteins. It is also possible that solutions with high concentrations of TFE do not adequately mimic the peptide-binding site in HLA or the HLA-TcR complex, although it has been argued that the decrease in solvent polarity due to TFE addition may mimic the dehydrated environment of the peptide bound to HLA [16]. A popular conception concerning the effect of a proline in the middle of an a helix is that it will break the helical structure. The minimal epitope examined here, LQAAPALDKL, has such a proline, flanked by a number of helix-forming residues [31]. At least 19 other known Tcell epitopes also contain a single internal proline [7-91. Barlow and Thornton examined the structure of 291 a helices found in proteins or peptides of known crystal structure and found 10 which included an internal proline [38]. In most of these helices prolines occurred at a kink in the structure of approximately 26 degrees. Thus the presence of a proline makes an a helix less likely to form, but does not necessarily eliminate this possibility, although it may change the details of the structure somewhat. Our CD data demonstrate that the minimal peptide can be induced by TFE t o form some a helical structure. Some of the longer analogs studied here form considerably more a helical structure, up to about 40% in the case of the E4 extension peptide. Thus, this peptide system is appropriate for addressing questions of the necessity of this secondary structure for T cell activation. If the active, HLA DR2-bound form of T cell-stimulating peptides is required to be an a helix, it would be expected by mass action that peptide potency should be enhanced as conformational equilibria are shifted to the helical form by extensions of helix-forming residues. Results presented here establish that, in assays with fixed APC, the helixforming potential of peptide analogs of LQAAPALDKL does not correlate with activation of the responding Tcell clone 2F10. In the case of fixed mononuclear cells, the most potent peptide, with four prolines added to each end of a minimal stimulating peptide, is the one least likely to form an a helix in TFE. A stronger correlation with a helical potential was noted for unfixed APC, suggesting that a

Eur. J. Immunol. 1990. 20: 2691-2697

structure containing a helical character may be important for a step in antigen processing which is not used in presentation by fixed cells. It is possible that this peptide is induced instead into a polyproline helix, due to the presence of 9 prolines in 19 residues. However, the stable form of this helix in aqueous solution, the polyproline type I1 helix, is left handed with three residues per turn [32] and, therefore, distinct from a right-handed a helix. Our observed CD spectra for this peptide in TFE, which correspond well to those of standard secondary structures, also do not contain a positive band around 230 nm. characteristic of a polyproline I1 helix [32, 391. The observed lack of correlation of experimentally induced a helicity with peptide activity, similar to that we have previously seen comparing the activity of internal epitope substitutions with predicted a helicity, including the observed activity of mutants with added internal helix breakers [21], suggests that (a) factors other than peptide conformation alone are important for Tcell activation, and (b) although analogs of this peptide can clearly be induced into a structure containing a helix inTFE, the active form may not be required to be a helical. Since the peptides exhibit a significant fraction of disordered or extended structure in aqueous solution and in aqueousTFE, it is also possible that the peptides may bind in an extended form. Another important factor for activity is the length of the peptide. Although the activity of the minimal peptide decreases drastically with further deletions, inclusion of more of the native sequence causes more stimulation of the responding T cell clones [19]. It is also possible that removing the N-terminal charge in LQAAPALDKL amide by addition of other residues removes a repulsive interaction with an HLA residue. Given the native sequence from the 65-kDa M . leprae protein of GGVTLLQAAPALDKLKLTG, the extension peptides containing Gly (G) or Lys (K) will be reconstructing part of the native sequence. However, the extension peptides containing Ala or Glu are similarly active with fixed cells. Another possible explanation is that enhanced membrane solubility may increase activity of the extended peptides [13].The (Glu)J and ( L y s ) ~peptides should, however, result in a very hydrophilic peptide, and these are the most active extended peptides with unfixed APC. An additional feature previously hypothesized to be important in the action of Tcell epitopes is amphipathicity when in a helical form. For example, this could be important for interaction with either of the two hypothesized HLA a helices delimiting the potential peptide binding groove [40]. Here we have shown that analogs of differing helical amphipathicity can be induced into a helices in TFE, but their activity does not strictly correlate with their overall amphipathicity. Using different internal replacement analogs, we have previously shown that their activity also does not correlate with predicted a helical amphipathicity

P11. Our results may require a more complex picture of the interaction between active peptides and HLA-DR proteins in Tcell activation. Since different active extended peptide analogs examined here appear in both a helical and non-a helical conformations in TFE, it appears possible that

Eur. J. Irnmunol. 1990. 20: 2691-2697 peptides m a y be able t o interact with APC and/or HLA in different b o u n d conformations, a possibility consistent with th e hypothesized structure of this protein [40].

We thank Rachel Klevit arid Grace Parraga (Deppt. of Biochemistry, University of Washington)for their kind assistance in the use of the CD spectrorneter; Richurd Houghten for instruction in tea hag peptide synthesis methodology. and Gene Merutka for helpful discussions.

M . Ieprae T cell epitope conformation and activity

17 Young. D.. Lathigra. R., Hendrix. R.. Swcetser. D. and Young. R. A.. Proc. Natl. Acad. Sci. USA 1988. 85: 4267. 18 Van Schooten, W. C. A.. Elferink, D. G.. Van Embden. J.. Anderson, D. C. and DeVries, R. R. P., Eur: J. Immuriol. 1989. 19: 2075. 19 Anderson. D. C.. W. C. A. van Schooten. M. E. Barry. A.

20 21

Receivcd June 19. 1990: in revised form July 31. 1990. 22

5 References 1 Babbitt. B. P., Allen. P. M.. Matsueda, Haber. E. and Unanue. E. R.. Nature 1985. 317: 359. 2 Buus. S.. Sette, A. and Grey, H. M., Immuriol. Rev. 1987. 98:

115. 3 DeLisi. C. and Berzofsky, J. A . . Proc. Natl. Acad. Sci. U S A 1985. 82: 7048. 4 Allen. P. M.. Matsueda. G. R., Evans, R. .I., Dunbar, J. B.. Marshall. G. R. and Unanue. E . R . , Nature 1987. 327: 713. 5 Sette. A . . Buus. S., Colon. S., Smith, J. A. , Miles. C. and Grey, H. M.. Irnrnunol. Rev. 1987. 98: 395. 6 Rothbard. J. B.. Lechler. R . I . . Howland. K., Bal, G., Eckels, D. D.. Sekaly, R . , Long. E. O..Taylor,W. R. and Lamb. J. B., Cell 1988. 52: 515. 7 Rothbard. J. B. and Taylor, W. R., EMBO J. 1988. 7: 93. 8 Sette. A.. Buus. S., Colon. S.. Miles. C. and Grey, H . M.. .I. Immunol. 1988. 141. 45. 9 Claverie, J. M.. Kourilsky. P.. Langlayde-Demoycn, P.. Chalufour-Prochnicka, A,. Dadaglio, G., Tekaia. E , Plata. E and Bougueleret, L.. Eur. J. Immunol. 1988. 18: 1547. 10 Pincus. M. R.. Gcrcwitz. F., Schwartz, R. H. and Scheraga, H . A.. Proc. Natl. Acad. Sci. USA 1Y83. 80: 3297. 11 Spouge, J. L.. Guy. H. R.. Cornette. J. L., Margalit, H . , Cease. K.. Berzofsky. J. A. and DeLisi. C., J. lmrnunol. 1987. 138:

23 24

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Janson, R. A. Young. T. Buchanan and De Vrics. R. R. P.. Science 1988. 242: 259. Anderson, D. C.,Van Schooten, W., Janson. A , , Barry. M. A. and De Vrics, R. R. P., J. Immunol. 1990. 144: 2459. Anderson, D. C..VanSchooten,W. C. A., Barry, M.. Janson. A . A. M. and DeVries, R. R. F!, in Synthetic Peptides: Aproachrs to Biological Prohlerns,Tam, J. P. and Kaiser, E.T. (Eds.). Alan, R. Liss. New York 1989, p. 199. Haanen. J.. Ottenhoff. T., Voordouw. A , , Elfcrink. B. G.. Klatser. P. R., Spits, H. and De Vries, R. R. P.. Scrrrid. J. Immunol. 1986. 23: 101. Lechler, R. I., J. Imrnunol. 1985. 135: 2914. Wilkinson, D., DeVries. R. R. P.. Madrigal, J. A,. Lock. C. B.. Morgenstern, J. P.,Trowsdale, J. and Altmann. D. M.. J. Exp. Med. 1988. 167: 1442. Provenchcr, S. W. and Glockner, J., Biochemistry 1981. 20:

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