Eur. J. Immunol. 1990, 20: 953-960

Lon A. Smolenski, Pravin KaumayaO, M. Zouhair AtassP and Susan K. Pierce Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Department of Obstetrics and Gynecology, The Ohio State University College of Medicineo, Columbus and Marre McLean Department of Biochemistry and Department of Microbiology and Immunology, Baylor College of Medicinen, Houston

Peptide competition for presented antigen

953

Characteristics of peptides which compete for presented antigen-binding sites on antigen-presenting cells* The T cell recognition of globular protein antigens requires the cell surface presentation of the protein by Ia-expressing antigen-presenting cells (APC). The mechanisms by which APC function remain to be elucidated. To gain a better understanding of association of antigen with APC surfaces, a large panel of peptides of diverse physicochemical properties was assayed for the ability to compete with presented antigen for binding sites on the APC surface. Competition was measured by the ability of a peptide to block the I-Ek-restrictedTcell response to pigeon cytochrome c (Pc) as presented by APC. The panel assayed included overlapping peptides representing the entire length of sperm whale myoglobin and the a and p chains of human adult hemoglobin as well as synthetic conformational peptides of lactate dehydrogenase C4 exhibiting stable secondary, a-helical structures. The results presented here show that several peptides of this group compete with the presented form of Pc for binding sites on the APC. However, there is no single biochemical property or amino acid sequence algorithm which predicts the blocking ability.The peptides which compete with presented Pc are not predicted to assume the amphipathic a-helical conformation hypothesized by De Lisi and Berzofsky (Proc. Natl. Acad. Sci. USA 1986. 82: 7048) for T cell antigenic peptides. However, peptides designed and synthesized to adopt a stable a-helical secondary structure show more potent blocking activity than the corresponding linear peptides, suggesting that the secondary structure may indeed be a contributing factor in the ability of presented antigenic peptides to be bound by the APC.The results with the myoglobin and hemoglobin peptides show no connection between any particular secondary structure of the peptide in the native proteins and the ability of the peptides to block presentation. Further, there is no correlation between the major histocompatibilitycomplex restriction of the competingpeptides and their ability to block the I-Ek-restrictedPc-specific Tcell response.This suggeststhat antigen presented by the APC may be bound to APC structures other than Ia prior to association with Ia. Such additional binding sites for presented antigen may be necessary to facilitate association with Ia.

1 Introduction To be recognized by antigen-specific T cells, globular protein antigens must be presented by Ia-expressing APC [l,21. The nature of the antigen presented by the APC and the mechanisms by which it is bound to Ia are not known. Syntheticpeptides of sperm whale myoglobin (Mb), hemoglobin (Hb) and pigeon cytochrome c (Pc) of approximately 10-14 amino acids are sufficient for specific T cell activation [3-71 and certain of these peptides, as well as others, have been demonstrated to bind to detergentsolubilized Ia in an MHC-restricted fashion [8, 91. However, several lines of evidence suggest that model synthetic [I 77711

* This work supported by the National Institutes of Health, grants AI 18939 and A1 23717 and by the Welch Foundation (grant Q994) and the award to M. Z. Atassi of the Robert A. Welch Chair of Chemistry. Correspondence: Susan K. Pierce, Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208, USA Abbreviations: Hb: Hemoglobin Mb: Sperm whale myoglobin Pc: Pigeon cytochrome c THMc: Tobacco hornworm moth cytochrome c 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990

peptides may not represent the form of the antigen presented by the APC and that presented antigen may differ from model peptides in binding to Ia andor the TcR . The length requirement for T cell antigenic peptides appears to be nonspecific and can be fulfilled by nonsense sequence beyond the essential contact residues of the site [4,5]. It has also been observed for Mb [lo-131 that the addition of amino acid residuesbeyond the minimal peptide required for T cell activation may markedly decrease their T cell-stimulating activity, reflecting interference in their ability to be presented with a particular Ia. Not all regions of proteins demonstrated to contain Tcell antigenic determinants, as represented by synthetic peptides, appear to be produced from the native protein by APC. For example, several regions of Mb which are highly immunogenic in mice as synthetic peptides do not elicit Tcell responses in mice immunized with the native protein [14], suggesting that peptides representing these regions are not presented by APC in vivo. Fox et al. [15] recently showed that APC process synthetic peptides and that the processed forms of these peptides are not identical to the corresponding minimal length synthetic peptide in their association with Ia. Studying the processing of OVA Michalek et al. [16] observed that an APC line which showed a selective inability to process and present native OVA to a particular OVA0014-2980/90/0505-0953$02.50/0

954

L. A. Smolenski, P. Kaumaya, M. Z. Atassi and S. K. Pierce

specificTcel1, presented a synthetic peptide containing the Tcell antigenic determinant to this, as well as to other OVA-specific T cells. It was concluded that the synthetic model peptide, representing the minimal stimulatory sequence,was not identicalto the structure produced by the MC. To gain a better understanding of the nature of the antigen presented to T cells by APC, a large panel of peptides of diverse physicochemical properties was assayed for the ability to block the Pc-specific T cell response to Pc as presented by APC. Previous studies [17] showed that nonstimulatory peptides of cytochrome c of several species, includingthe homologous mouse cytochrome c, block the Pc-specificT cell response in a peptide concentrationdependent fashion. Blocking was shown not to be due to toxic effects of the peptides as blocking is overcome by either increasing the concentration of the stimulatory Pc antigen, or by using the cytochrome c of tobacco hornworm moth (THMc) as a stimulatory antigen for which the Pc-specific T cell shows a heteroclitic, high-affinity response. Furthermore, blocking was shown to be due to a competition at the AF'C by nonstimulatory peptides for sites occupied by the presented antigen. Indeed, APC which were incubated with Pc and non-stimulatory peptides, and the excess peptides removed by washing, were unable to activate Tcells. Further analysis of the blocking ability of overlapping peptides representing staphylococcal nuclease showed that both I-E-restricted and I-A-restricted antigen-specificT cell responses were equally blocked by either I-E- or I-A-restricted immunodominant peptides [18]. In the present study,we extend this analysis and test a large panel of nonstimulatory overlapping peptides representing the entire length of Mb and the a and p chains of Hb, as well as conformational peptides which form stable a-helical structures in aqueous solution, for the ability to block the T cell response to Pc as presented by APC.

2 Materials and methods 2.1 Antigens and peptides Pc, Mc and THMc were isolated and purified according to Brautigan et al. [19]. The cyanogen bromide cleavage fragment of Mc (Mc 81-104) was prepared as previously described [20]. The overlapping synthetic peptides of Mb corresponding to residues 1-17, 13-29, 25-41, 37-53, 49-65, 61-77, 73-89, 85-101, 97-113, 109-125, 121-137, 133-149 and 141-153, were synthesized, purified to homogeneity and characterized as previously described [21,22]. The overlapping peptides of the Hb p chain residues 1-15, 11-25,21-35,31-45, 41-55,51-65,61-75,71-85, 81-95, 91-105, 101-115, 111-125, 121-135 and 131-146 and the overlapping peptides of the Hb a chain residues 1-15, 11-25,21-35,31-45, 41-55,51-65,61-75,71-85, 81-95, 91-105, 101-115, 111-125, 121-135 and 131-141 were synthesized, purified to homogeneity and characterized as described [23-251. Synthetic peptides of lactate dehydrogenase C4 were assembled by stepwise solid-phase synthesis using the Fmoc-t-butyl strategy as described [26]. In all cases purified peptides were stored as salt-free, freezedried powders and were dissolved immediately before use. Peptides which could not be solubilized were not tested as indicated in the Tables.

Eur. J. Immunol. 1990.20: 953-960

2.2 Animals, cell lines and preparation of APC CBA/J female mice, 5-6 weeks of age, were obtained from the Jackson Laboratory (Bar Harbor, ME). TPc9.1 is a mouse T cell hybridoma specific for Pc presented in the context of the I-Ekmolecule [17,18].The CTLL-2 line is an IL2-dependent cell line described by Gillis et al. [27]. B cells were isolated from the spleens of CBAN mice by removal of RBC by centrifugation on Ficoll-Hypaque (Pharmacia, Piscataway, NJ) gradients, and by removal of T cells by incubation with monoclonal antibodies directed toward L3T4, Thy-1 and Ly-2 and complement, as described previously [17, 181.

2.3 Activation and blocking of T cell responses The activation of the Tcell hybrids was measured by their ability to secrete IL2, 24 h after incubation with antigenpulsed B cells [17, 181. B cells (5 x 106 cells/ml) were incubated in complete DMEM containing 5% FCS and Pc (20 pM) or THMc (20 pM) overnight at 37 "C in 5% COz atmosphere. The cells were washed and cultured with irradiated (1300 rad) TPc9.1 cells (1 x 105-5 x 105 B cells15 x 10" T cells/0.2 ml culture) in the presence of graded concentrations of the nonstimulatory peptides. Culture SN were collected 24 h later and tested for their IL2 content by the ability to maintain the growth of the IL2-dependent CTLL cell line as measured by the incorporation of [3H]dThd.

3 Results 3.1 The effect of peptides of Mb on the I-Ehestricted Pc-specific T cell response TheT cell response to Mb has previously been described to be under Ir gene control [6,28-301. Mice of the H-2d and H-2S haplotypes are responders and mice of the H-2k haplotype nonresponders. The T cell antigenic determinants on Mb were identified in the H-2dand H-2Sstrains by testing the ability of mice immunized with the native Mb to proliferate in culture when provided with synthetic overlapping peptides representing the entire Mb chain [21,22]. These results are summarized in Table 1, where pluses indicate the degree to which the peptides stimulate Mbprimed T cells in culture and a minus indicates that no stimulatory activity was observed.The Mb 102-1 18peptide is recognized by T cells along with the I-Admolecule and Mb 133-149 is recognized only in the context of the I-Ed molecule [6,30]. The Mb 132-153 peptide has been reported to bind to purified I-Ed in detergent solution but not to the I-Akor I-Ekmolecules [9]. However, Mb 132-153 showed some weak ability to compete with Pc 88-104 in its binding to I-Ek [9]. Cells of the Pc-specific, I-Ek-restricted T cell hybridoma cell lineTPc9.1 were cultured with Pc-pulsed B cells in the presence of graded concentrations of nonstimulatory peptide (0-100 p ~and ) the culture SN tested 24 h later for the presence of IL 2 as a measure of Tcell activation.To avoid introducing bias, the peptides were coded by random numbers and the sequence deciphered after all blocking studies had been completed. Representative competition

Eur. J. Immunol. 1990. 20: 953-960

Peptide competition for presented antigen

955

Table 1. The ability of overlapping peptides representing the entire Mb protein to block the I-Ek-restricted Tcell response to processed Pea)

T cell stimulationb) Protein priming Single pept. priming BALBlc SJL BALB/c SJL

Peptide Mb 1- 17 Mb 13- 29 Mb 25- 41 Mb 37- 53 Mb 49- 65 Mb 61- 77 Mb 73- 89 Mb 85-101 Mb 97-113 Mb 109-125 Mb 121-137 Mb 133-149 Mb 141-153

-

-

-

+ + + ++++ + + +

+ +

-

++ + -

++ ++ +++ ++++ +++ +++ +++ ++++ ++++ -

Pept. mix. priming BALBlc SJL

-

++ +++ ++ ++ +++ ++ + ++++ -

++++ +

-

++ + ++ ++ ++ +++ + ++++ +++ ++++ +++

+ +++ + ++ ++ ++++ +++ +++ ++++

++++ ++++ ++++

Blocks I-Ekrestricted response to Pcc)

+ +-

+I-

Peptide required for maximal blocking (pM)

80

40 80

-

-

NT

-

-

-

+

-

-

+ -

80 -

80 -

a) B cells as APC were cultured for 18 h with Pc (20 KM), washed free of excess antigen and cultured withTPc9.1 cells in the presence of graded concentration of the Mb peptides. Culture SN were sampled 24 h later and the IL2 content determined as a measure of Tcell activation. b) The recognition in vifro of the Mb peptides by protein-primed Tcells [21, 221 and by peptide-primed Tcells [14] was previously reported. The number of (+) signs denotes extent of cell proliferative activation in vifro by the peptide. c) A plus (+) indicates that complete inhibition (>80%) of T cell response was observed at the peptide concentration shown. A pludminus ( + I - ) indicates that partial inhibition was achieved (> 50% and < 80%). A minus (-) indicates that no inhibition was observed with nonstirnulatory peptide concentrations up to 100 p.~. NT indicates that the peptide was not tested in the blocking assay due to the inability to completely solubilize the peptide.

curves for two of the Mb peptides, as well as for Mc 81-104, are shown in Fig. 1 and the results for all Mb peptides are summarized inTable 1. Five of the Mb peptides (Mb 1-17, Mb 13-29, Mb 25-41, Mb 97-113, and Mb 133-149) block the I-Ek-restricted Pc-specific T cell response to Pc as presented by APC. The remaining Mb peptides show no measurable effect. Comparison of these results with the ability of Mb-primed T cells to recognize these peptides in vitro and with the ability of these peptides to prime for in vivo peptide-specific or Mb-specific T cell responses

201

T

I

z

B

10 15t

n 10 0

100.0

[PEPTIOE] p M

Figure I . The blocking effect of nonstimulatory peptides on the Pc-specific T cell response to Pc as processed and presented by AF’C. B cells as APC were pulsed with Pc (20 PM) for 18 h, washed and co-cultured with TPc 9.1 cells (5 X 104) in the presence of graded concentrations of the nonstimulatory peptides Mc 81-104 ( O ) , H b a 11-25 (A)and Mb 1-17 (V)or without peptide (H). Culture SN were tested 24 h later for the presence of IL2 by the ability to maintain the uptake of [3H]dThd by CTLL cells.

[14,21, 221 or with the ability to bind I-Ek, in the case of Mb 133-149 [9], shows that none of these properties correlates with ability t o compete with an I-Ek-restricted antigen for association with APC.

3.2 The effect of peptides of H b a and Hbfl on the I-Ek-restrictedPc-specific T cell response Overlapping peptides of the a and $ chain of H b were assayed for their effect on the I-Ek-restricted Pc-specific T cell response as described above for Mb peptides and the results summarized inTable 2. The T cell antigenic peptides of the H b flchains were previously identified by the ability of T cells from BALBk (H-2d) and SJL (H-2S) mice immunized with H b to proliferate in vitro to overlapping peptides representing the entire Hb p chain [31]. A summary of these results is shown in Table 2 where pluses and minuses indicate the relative antigenicity of the peptides. Similar analyses have not been completed for the H b a chain. As shown, seven H b a and six H b p peptides block the Pc-specific T cell response to Pc-antigen-pulsed B cells. For the H b p peptides, eight are antigenic in mice of the H-2d and/or H-2S haplotypes and the remainder have not been found to contain Tcell epitopes. Thus, as was the case for Mb, the MHC restriction of the H b p peptides does not predict their ability t o block the I-Ek-restricted T cell response to Pc. The blocking effects observed for both the Mb and H b peptides cannot be attributed to toxicity of the peptides. As summarized in Tables 1 and 2, the Mb and H b peptides were previously used to map the T cell recognition of the respective protein [14,21, 22, 311.They

956

Eur. J. Irnmunol. 1990.20: 953-960

L. A. Smolenski, l? Kaumaya, M. Z. Atassi and S. K. Pierce

Table 2. The effect of overlapping peptides of the a and

peptide=)

T cell antigenb) BALBlc (H-2d) SJL(H-2s)

Hbfl 11- 25 Hbp 21- 35 Hbfl 31- 45 Hbfl 41- 55 Hbfl 51- 65 Hbfl 61- 75 Hbfl 71- 85 Hbfl 81- 95 H b p 91-105 Hb@101-115 H b p 111-125 Hb p 121-135 Hb B131-146

chain of Hb on the I-Ek-restrictedT cell recognition of presented Pc

Blocks I-Ek- Peptide required restricted for maximal response to Pcb) blocking (w) -

Hba 1- 15 Hba 11- 25 Hba 21- 35 Hba 31- 45 Hba 41- 55 Hba 51- 65 Hba 61- 75 Hba 71- 85 Hba 81- 95 Hba 91-105 Hba 101-115 Hba 111-125 Hba 121-135 H b a 131-141

Hbs 1- 15

fi

+ +

NT

-

+/-

80

-

+ +

+/-

NT -

+

NT +I-

++

-I+ -/+

+

-

+

++

+ ++ -

+ ++ ++

-

+NT -

-

+I+I-

++ +-

80 80

++

+I-

were used in the same concentration range employed here to stimulate the proliferation of lymph node cells from peptide- and protein-immunized mice, T cell lines and T cell clones in vitro [3-5, 14,21,22,31,32]. These results show that there is no relationship between the T cellstimulating activity of a given peptide and its blocking activity reported here. Furthermore, in the case of Mb, where protein-primed as well as peptide-primed (both as individual peptides and as an equimolar peptide mixture) T cells were studied, each of the Mb peptides was able to stimulate in vitro one or both types of Tcells (seeTable 1).If the results of the blocking studies reported here were due to any toxic effects of the peptides, then it would be expected that blocking would correlate with lack of Tcell-stimulating activity. Since this clearly is not the case, it can be concluded that the effects observed here are genuine biological activities of those peptide structures. Moreover, the blocking peptides are unble t o block the high-affinity crossreactivity of this Tcell hybrid to THh4c over the concentration range used here (data not shown). 3.3 The predicted amphiphic a-helical nature of Mb, Hb a and Hb flpeptides and their conformation in the native protein three-dimensional structure

Analysis of the influence of individual amino acid residues of antigenic peptides on their ability to interact with either the TcR or Ia have led to the conclusion that the most favored conformation for T cell antigens are a-helical

-

-

80 40 40 -

40 -

80 -

40 40 40

-

40 40

a) The antigenicity of the Hbp peptides are as described elsewhere [31]. b) Blocking activity was assessed as described in kible 1.

amphiphilic structures [7, 33, 341. Furthermore, based on computer-aided analysis of several known T cell antigenic peptides, De Lisi and Berzofsky [35] predicted that peptides which are capable of functioning asT cell antigens will show a tendency to form amphiphilic a-helical structures. Such a structure may be critical to the peptide's ability to associate with the APC,the Ia molecule, andor the TcR. From the three-dimensional structures of Mb [36-381 and Hb [39,40] the secondary structural feature (helical or otherwise) of the region represented by each peptide within the native protein is summarized in Table 3. In addition, amino acid sequences of the Mb and Hba and HbP peptides were analyzed by three predictive computer algorithms to determine if their blocking ability correlated with the potential to assume a defined secondary structure. The Stroud Fourier analysis [41] was used to examine the peptide amino acid sequences for intervals in hydrophobicity values fluctuating either with a periodicity of approximately 3.6 residues per cycle corresponding to an a-helix or with a periodicity of approximately 2.2 residues per cycle corresponding to a @-sheet.Similarly, the Eisenberg Hydrophobic Moment [42] and Amphi [43] analyses were applied to predict regions of high a-helical amphipathic nature. Based on these three analyses, peptides were judged to be highly, moderately or weakly amphiphilic or to show no tendency to adopt amphiphilic a-helical structures (Table 3). Helical-wheel representations [44]were carried out to further ascertain the validity of these analyses for amphiphilic a-helical segments. As shown, there is little correlation between the shape of the peptide region within '

Eur. J. Immunol. 1990. 20: 953-960

Peptide competition for presented antigen

the native protein or the predicted ability of the peptides to form an a-helical amphiphilic structure and the ability to block the I-Ek-restricted T cell response. This result suggests that sites on the APC which accomodate antigen are not restricted to binding a-helical amphiphilic structures. Finally, we examined whether the blocking H b peptides correspond to regions that occupy subunit interacting

957

surfaces in the H b tetramer. Peptides representing regions buried in subunit-interacting surfaces tend to be hydrophobic and might exhibit some nonspecific association tendencies. The levels of involvement, if any, of each H b peptide in subunit associations (which are summarized in Table 3) seem to have no correlation with the blocking activity.

Table 3. Comparison of peptide blocking activities with their conformation within the protein; involvement in a-P interactions (in Hb); and the predicted tendency to adopt amphiphilic a-helical secondary structures

Peptide

Mb 1- 17 Mb 13- 29 Mb 25- 41 Mb 37- 53 Mb 49- 65 Mb 61- 77 Mb 73- 89 Mb 85-101 Mb 97-113 Mb 109-125 Mb 121-137 Mb 133-149 Mb 141-153 H b a 1- 15 H b a 11- 25 H b a 21- 35 H b a 31- 45 H b a 41- 55 H b a 51- 65 H b a 61- 75 H b a 71- 85 H b a 81- 95 H b a 91-105 Hb a 101-115 Hb a 111-125 Hb a 121-135 H b a 131-141 Hbg 1- 15 Hbfi 11- 25 Hbfi 21- 35 Hbfi 31- 45 Hbp 41- 55 Hbg 51- 65 Hbfi 61- 75 Hbg 71- 85 Hbg 81- 95 Hbp 91-105 Hb p 101-115 Hbfi 111-125 Hb p 121-135 Hb f3 131-146

Blocks Tcell respond

+ +

+/-

NT -

+

-

+ -

-

+ +

NT

+/-

+ +

+/NT -

+

NT

-

+ -

NT -

+/+/-

+ +

+/-

Residues in Residues in subunit a helidtotal residues interfaceltotal resiof peptide dues of peptide

15/17 16/17 17/17 9/17 15/17 17/17 9117 12/17 14/17 11/17 14/17 17/17 9/13 13/15 14/15 15/15 12/15 6/15 14/15 11/15 6/15 10/15 12/15 12/15 10115 15/15 8/11 12/15 15/15 15/15 11/15 6/15 15/15 15/15 6/15 9/15 10115 15/15 10/15 13/15 13/16

Hydrophobic periodicity for an a helixb)

Helical wheel analysis"

-

-

-

+++ -

-

+++ ++ +++ +

+

-

+++ ++

-

0115 0115 3/15 10/15d) 3/15 0115 0115 0115 4/15 7/15d) 6/15d) 8/15d) 3/15 0111 0115 0115 4/15 7/15d) 0115 0/15

0115 0115 0115 5/15 5/15d) 8/15d) 7/15d) 3/16

++

-

-

+

-

-

+-

+++

+++ +

+++ -

+

-

-

-

+ +

++ +++

-

-

-

+++ ++ ++ + ++ ++ + + ++ ++ -

++ -

++ ++ -

++

-

a) A summary of the ability of peptides to block the response of the Pc-specificT cell hybnd,TPc 9.1, to Pc as processed and presented by APC is taken from results presented in Tables 1 and 2. b) The protein sequences of Mb, Hb a and Hb p were analyzed by Stroud-Fourier [41], Eisenberg Hydrophobic Moment [42], and Amphi [43]. Regions which are predicted to be highly (+++), moderately (++), or weakly (+) amphiphilic or to show no amphipathic character (-) are shown. c) Amino acid sequences of Mb, H b a and Hb P were analyzed according to Schiffer and Edmundson [44].A minus (-) indicates no amphiphilic character, pluses indicate that the sequence deviates from an idealized amphiphilicsequence by one (+ +), two (+ +), or three (+) amino acid residues. d) Note that this peptide corresponds to a region which is in fact completely buried in the Hb a-P interfaces.The remaining residues are involved in intrasubunit interactions [39,40].

+

L. A. Smolenski, €! Kaumaya, M. Z. Atassi and S. K. Pierce

958

3.4 The blocking ability of peptides designed and synthesized to adopt stable secondary a-helical amphiphilic structures As shown above, peptides which effectively block the Tcell response to Pc are not necessarily in an a-helical conformation in the native protein nor are they predicted to form amphiphilic a-helical structures. However, such predictive analyses are only of limited value and therefore we wished to test directly whether a peptide which was designed to take on a stable amphiphilic a-helical structure in aqueous solution is able to effectively compete for sites on the APC to which antigen is bound. A peptide representing an a-helical region of the protein lactate dehydrogenase C4 (LDH C4)was designed and synthesized to adopt a stable a-helical conformation in aqueous solution .The theoretical considerations underlying the design and synthesis strategy are detailed elsewhere [26]. Briefly, the peptide was modeled from an a-helical region of LDHC4, residues 310-327, designated a N (Fig. 2). Appropriate amino acid substitutions were made to yield an idealized amphiphilic a-helix, designated a1 (Fig. 2). This peptide has additional features which aid in stabilizing it in aqueous solution including an acidic residue (Glu310)at the N terminus and a basic residue (Lys”) at the C terminus allowing favorable interaction with the a-helical dipole [45], and appropriate electrostatic ion pairs [46] in positions i, i 3, i and i 4 stabilizing solvent-exposed hydrophilic residues. The a1

+

+

Eur. J. Immunol. 1990.20: 953-960

sequence was further stabilized into a supersecondary structure with an aa topology, designated a 2 (Fig. 2).Thus, two a1 sequences were connected in an antiparallel arrangement by a four-residue p-turn consisting of a Leu-Ser-Pro-Gly sequence. Four independent biophysical measurements: peptide monolayer studies, circular dichroism, Fourier transport infrared and low-angle scattering were used to characterize the conformation of these peptides as detailed elsewhere [26].The a N peptide showed a characteristic random coil structure in aqueous solution. In contrast, a1 was 14% helix in the monomeric state and 86% helix in the tetrameric form, and a2 was 24% helix in the monomeric state and 99% helix in the dimeric state as compared to poly-L-lysine-HC1. Thus, the a peptide is a considerably more stable a-helix in its monomeric form as compared t o al. As shown in Fig. 3 the a N and a1 peptides do not block theT cell response to Pc-pulsed APC while the a 2 peptide shows considerable blocking ability. Since a2 is more stable as an a-helix as compared to a l , this suggests that the binding sites on the APC for peptide may preferentially accommodate such structures. Alternatively, the blocking effect could be due to the p-turn in a 2 or to size differences since a 2 is larger. The analysis of additional conformational peptides should lend further insights into the contribution of secondary structure to peptide-APC association.

4 Discussion aN

The experiments described here were carried out to the sites on APC surfaces to which presented antigen is bound. Advantage was taken of the observation that peptides with are unable to activate an antigen-specific T cell, namely nonstimulatory peptides, block the T cell response to the antigen as processed and presented byAPC. Our previous results [ 17, 181 showed that all peptides representing the C-terminal regions of nonstimulatory cytochromes c, for example the homologous mouse cytochrome c , block the Tcell response to Pc as processed and presented by AFT. The blocking phenomenon appeared to be due to a competition between presented Pc and the nonstimulatory peptide for binding sites on the APC surface. Indeed, Pc-pulsed B cells incubated with nonstimulatory peptides remained unable to activate the Pc-specific T cell when the nonstimulatory peptides were removed by washing before addition of the Tcell. Subsequent studies assessing the ability of a panel of peptides representing the entire sequence of staphylococcal nuclease to block the I-Ek-restricted Pc-specific response and the I-Ak-restrictedT cell response to OVA showed that amino acid sequence similarity between the nonstimulatory peptide and the stimulatory antigen were not required for blocking [18]. A second important conclusion drawn from our previous studies was that the MHC restriction of the nonstimulatory peptide did not correlate with its blocking ability [HI. Thus, I-A-restricted peptides blocked I-Erestricted T cell responses and vice versa. Moreover, peptides restricted to I-E molecules of haplotypes other than H-2k, blocked I-Ek-restricted T cell responses.

310 327 characterize Glu-Glu-Glu-Gly-Leu-Leu-Lys-Lys-Ser-Ala~As~~r-Leu~Trp-Asn~Met-Glu-Lys Ill

310

327

a2

-Asp-mr-Leu-~~-Asn-Met-tBaLys

Pro

I

Ser

I

Leu-GIu~Glu-Gly-Leu-Leu-Lys-Lys-

Figure2. The amino acid sequences of the aN, a1 and a2 peptides.

0

:

:

:

:

:

,\A:

:

10.0

:

: i i l

100.0

[PEPTIDE]

pM

Figure 3. The ability of the conformational peptides of LDH C4, a N (+),a1 (V)and a2 (A),to block the T cell response to processed Pc. Blocking was assessed as described in Fig. 1.

The results reported here significantly extend our initial analysis to include a large number of overlapping peptides representing the entire sequence of Mb and the a and pchains of Hb, as well as two peptides of well-defined

Eur. J. Immunol. 1990.20: 953-960

secondary conformation. For two of the protein chains investigated, Mb [6, 14, 21, 221 and HbB [31], the Tcell antigenic determinants and MHC restrictions have been identified.The conclusions from results of the large panel of peptides studied here is that no single physicochemical property or amino acid sequence algorithm predicts the ability of a peptide to block the specific T cell response to antigen as presented by APC. In addition, as was already evident from our earlier analysis of a smaller number of peptides, the MHC restriction of the nonstimulatory peptides does not correlate with their blocking ability. If antigenic peptides restricted to a particular Ia bind only to that Ia molecule, as previously reported by Babbitt et al. [8] and Buus et al. [9], then the findings reported here indicate that presented antigen is initially associated at sites other than Ia. Published results of blocking studies by Guillet et al. [47] show that the MHC restriction of the nonstimulatory peptides predicts their ability to compete with synthetic peptides for Tcell activation. The interpretation of these results is that peptides are bound only to Ia. How might these results be reconciled with those presented here? One possible explanation is that the form of the antigen presented by the APC is not identical to the synthetic peptides studied by Guillet et al. [47] and by Buus et al. [9].The presented peptides and the synthetic peptides may initially bind to different sites on the APC, the latter exclusively on Ia. Alternatively, the MHC restriction of the presented form of the antigen may not be identical to that described for synthetic peptides and the lack of correlation described here may be a reflection of such differences. A key to understanding how antigen is bound t o the APC is an elucidation of the nature and form of the antigen presented by APC. Although such presented species of the antigen have not yet been identified, recent evidence supports the notion that it may not be identical to the minimal stimulatory synthetic peptides which are sufficient to activate T cells. Thus, Fox et al. [15], reported that a synthetic peptide of cytochrome c showed a different fine specificity pattern of T cell activation when presented by untreated APC o r by APC blocked in their processing function. This suggested that the processing of the peptide introduced changes which affected Ia association. Investigations of the antigenicity of various Mb peptides [lo-131 have shown that the addition of amino acid residues to the N or C terminus of the minimal antigenic peptide dramatically alters the antigenicity of the peptide. Thus, if peptides are produced by proteolytic cleavage of a protein antigen, it would not be possible to mimic their function unless identical synthetic peptides are studied. When information is available concerning the nature of the presented form of the antigen, it may be possible to reconcile differences in results studying presented antigen and synthetic peptides. An important unanswered question raised by the results reported here is where the presented form(s) of antigen might bind on the APC prior to binding to Ia for recognition by specific T cells. We would suggest that the APC use a number of cellular peptide transport mechanisms to display peptides in a concentrated form on the APC surface. For example, Falo et al. [48,49] have presented evidence that processed peptides initially associate with membranes. Indeed, treatment of APC which

Peptide competition for presented antigen

959

had processed antigen with lipases eliminated their presenting ability. Cellular proteins which bind peptides could play a similar role. We have described one such protein, a peptide-binding protein of 72/74 kDa M, (PBP72/74; [50]) which was shown to play a role in antigen presentation by the ability of antibodies raised against it to block presentation of antigen. PBP72/74 has recently been demonstrated to be a member of the heat shock family of related proteins [51]. This has interesting implications because several of the heat shock proteins bind to denatured or inappropriately folded or glycosylated cellular proteins and have been proposed to function in the renaturation or transport of these [52]. We would suggest that PBP72/74 binds processed antigen and concentrates it on membranes, either intracellularly or on the APC surface, for transfer to Ia. Two additional recent findings have intriguing implications for the potential role of heat shock-like proteins in the assembly of functional MHC-peptide complexes. Townsend et al. [53] reported on a mutant cell line defective in the association of P2-microglobulin with class I heavy chains which does not express class I molecules at the cell surface. The mutation is not in the genes encoding for the heavy chain or 02-microglobulin. It was found that class I cell surface expression could be stimulated by the presence of high concentrations of exogenous antigenic peptides. The implication of this binding is that the association of peptides with class I molecules may be required for correct folding of the heavy chain and association with P2microglobulin. The mutation in this cell line is presumably in a gene whose product facilitates class I folding or peptide binding, both functions related to heat shock proteins. In addition, Sargent et al. recently showed that the human MHC contains genes for the major heat shock protein, hsp70 [54]. Thus, there is a genetic link between MHC molecules and a protein which both binds peptide and facilitates protein assembly. Analyses of the physicochemical properties of knownT cell antigenic peptides have suggested that the ability of a peptide to form an a-helical amphiphilic structure correlates strongly with T cell antigenicity [7, 33-35]. This feature of T cell antigenic peptides presumably reflects a common mechanism for binding of the peptides to Ia or to theTcR.The results presented here indicate that the ability of nonstimulatory peptides to block T cell responses to presented antigen does not correlate with their predicted ability t o form amphiphilic a-helices, suggesting that this property alone is not sufficient to ensure antigen binding to the APC surface. However, we also show that a stable amphiphilic a-helical peptide representing a region of LDH C4 (a2) competes for processed antigen-binding sites, while a less stable a-helical peptide ( a l ) , and the corresponding linear peptide (aN) are ineffective. It should be considered that this is only one example. In addition, the engineering of the a 2 peptide introduced amino acid sequences conferring a 0-turn between two a1 peptides. It is possible that these sequences, rather than the resulting conformation per se, contribute to the blocking ability of a2. The a 2 peptide is larger than a N and steric obstruction might also contribute to its blocking activity.

Received July 7 , 1989; in final revised form January 11, 1990.

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L. A. Smolenski, I? Kaumaya, M. Z. Atassi and S. K. Pierce

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5 References

c.,