The Journal of Allergy and Clinical Immunology
Volume 97, Issue 1 , Pages 53-64, January 1996

Single amino acid substitutions on a Japanese cedar pollen allergen (Cry j 1)-derived peptide induced alterations in human T cell responses and T cell receptor antagonism☆☆☆★★★

Kumamoto, Japan

Received 1 November 1994; received in revised form 14 March 1995; accepted 17 March 1995.

Article Outline

Abstract 

We generated T cell clones specific to a Japanese cedar pollen allergen (Cry j 1) and investigated effects of altered T cell receptor (TCR) ligand on changes of T cell responses. One of these Cry j 1-specific T cell clones established from patients with Japanese cedar pollinosis, ST1.9, recognized an antigenic peptide Cry j 1 p335-346 in the context of HLA-DRA+DRB3*0301 molecules and secreted interleukin-4 dominantly, with a smaller amount of interferon-γ. ST1.9 represented one of the major T cell clones specific to Cry j 1 in the donor, because a short-term cultured polyclonal T cell line specific to Cry j 1 exhibited the same character as the ST1.9. We synthesized various analog peptides derived from Cry j 1 p335-346 with single amino acid substitutions and determined key residues for interactions between TCR of ST1.9 and HLA-DR molecules. We also analyzed changes in the responses of ST1.9 to Cry j 1 p335-346-derived analog peptides. Of interest was that the substitution of 339threonine to valine resulted in a significant increase in interferon-γ production, with no remarkable changes either in proliferative response or interleukin-4 production. Analog peptides carrying the substitutions of 339 threonine to glycine or glutamine revealed TCR antagonism, without changes in their binding affinities to the DR molecule. Therefore single amino acid substitutions on an allergen peptide carrying the T cell epitope may suppress helper-T-dependent class switch pressure to IgE in B cells either by inducing increased interferon-γ production or by inhibiting proliferative responses in helper-T cells. (J ALLERGY CLIN IMMUNOL 1996;97:53-64.)

Keywords:  T cell epitope, analog peptide, IFNγ, IL-4, TCR antagonism, HLA-DR, cedar pollen allergen

Abbreviations:  APC: , Antigen presenting cells, IC50: , Peptide concentration required for 50% inhibition of binding, IFNγ: , Interferon-γ, IL: , Interleukin, mAb: , Monoclonal antibody, MHC: , Major histocompatibility complex, PBMC: , Peripheral blood mononuclear cells, TCR: , T cell receptor

 

Exogenous antigens are engulfed and processed by endosomal proteases to peptides in antigen presenting cells (APC) and are presented to T cells by appropriate major histocompatability complex (MHC) class II molecules present on the cell surface. CD4+ helper T cells are activated upon recognition of these peptide-MHC complexes by T cell receptor (TCR) molecules and exert effector functions through various biologic activities of secreted lymphokines. Recent studies showed that T cell activation is not a simple on-off-type event; rather, qualitative changes in T cell responses can be induced by amino acid substitutions of either MHC molecules or antigenic peptides, that is, TCR ligands. Altered TCR ligands reportedly resulted in murine T cell anergy,1 TCR antagonism,2, 3 or the segregation of proliferative responses and lymphokine production.4, 5, 6 However, there has been no documentation of these observations in human systems.

Amino acid residues on antigenic peptides have been roughly divided into two groups: one that is important for binding to TCR (T cell epitope) and the other that is important for binding to MHC (MHC anchor). However, crystal structure of the human class II HLA-DR1 complexed with the influenza peptide recently reported by Stern et al.7 demonstrated that all the amino acid residues of the influenza virus peptide physically made contact with both HLA and TCR but that only one residue at the amino terminus is deeply buried into the groove of class II, which does not allow for interaction with TCR. Other residues anchored in pockets formed by polymorphic residues of HLA-DR1 contribute to existence of the structural motifs for DR1-binding peptides. However, most have a surface capable of interacting with HLA and also with TCR.

T cell epitopes of several major allergens have been reported.8, 9, 10, 11, 12 In a human model of allergy, detailed analyses of human T cell responses to single amino acid–substituted allergen peptides have not been made at clonal levels. Cry j 1 is a major allergen of Cryptomeria japonica (Japanese cedar) pollen, the most potent seasonal allergen in Japan; more than 10% of the population is affected by pollenosis. We generated T cell clones specific to Cry j 1 and examined restriction molecules, T cell epitopes, and lymphokine production. The important residues for interactions with TCR or HLA were determined in the Cry j 1 peptides. Effects of various analog peptides derived from Cry j 1 antigenic peptide with single amino acid substitutions on T cell responses were studied with this model.

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METHODS 

Preparation of allergen and synthesis of Cry j 1-derived peptides 

The major allergen of Cryptomeria japonica (Japanese cedar) pollen antigen, Cry j 1, was purified as described13, 14 but with some modifications. Briefly, dry pollen was defatted with ether, placed in 0.125 mol/L ammonium bicarbonate, and stirred for 2 days at 4° C. Solid ammonium sulfate was added to the extract to 100% saturation. The precipitate was dissolved in 0.01 mol/L Tris-HCl, pH7.8, and dialyzed against the same buffer. The sample was applied to a DEAE-cellulose (Whatman, Maidston, England) column and eluted with the same buffer. The major peak of the eluate was dialyzed against 0.01 mol/L acetate buffer (pH 5.0) and applied to a CM-cellulose (Whatman) column. The column was eluted first with 0.01 mol/L acetate buffer, then with 0.1 mol/L phosphate buffer, pH 7.0, and the second eluate was concentrated and purified by molecular sieving high-performance liquid chromatography (Bio Sil TSK-250; Bio-Rad, Hercules, Calif.). A fraction with the highest A280 value thus obtained contained the 41 kd and 46 kd proteins, as determined by SDS-PAGE analysis (data not shown).

Peptides were synthesized according to amino acid sequence of Cry j 1 reported by Griffith et al.15 and Sone et al.16 with a solid-phase simultaneous multiple peptide synthesizer PSSM-8 (Shimadzu, Kyoto, Japan) based on Fmoc strategy. All peptides were purified by C18 reverse-phase high-performance liquid chromatography (Millipore Corp., Bedford, Mass.).

Generation of antigen-specific T cell clones 

Cry j 1-specific T cell clones were established from peripheral blood mononuclear cells (PBMC) from five patients with type I allergy to Cryptomeria japonica pollen. Diagnosis was based on clinical symptoms, positive skin test results, and positive radioallergosorbent test results. T cell lines were generated by stimulating peripheral blood mononuclear cell (PBMC) (1 × 105/well) with 1 μg/ml Cry j 1 in RPMI 1640 medium (Gibco, Grand Island, N.Y.) supplemented with 2 mmol/L L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% pooled, heat-inactivated, normal human male plasma in 96-well flat-bottomed culture plates (Falcon; Becton Dickinson, Lincoln Park, N.J.). After 7 days, irradiated (3000 cGy) autologous PBMC (1.5 × 105/well), human interleukin-2 (IL-2) (50 U/ml), human rIL-4 (10 U/ml), and Cry j 1 (1 μg/ml) were added to the culture wells carrying T cell blasts and were maintained for 7 additional days. Cloning was performed in Terasaki plates (Nunc, Roskilde, Denmark) by limiting dilution at 0.3 cells/well in the presence of 3 × 104/well irradiated (3000 cGy) autologous PBMC, human rIL-2 (50 U/ml), human rIL-4 (10 U/ml), and Cry j 1 (1 μg/ml) in the same medium as described previously. Growing microcultures (approximately 5% of all culture wells) were then expanded at weekly intervals, first in a 96-well plate and then in a 24-well plate by feeding with irradiated feeder cells (1.5 × 106/well), rIL-2, rIL-4, and Cry j 1.

The cell surface markers of T cells were analyzed with anti-CD3, anti-CD4, anti-CD8, anti-CD28, and anti-T cell receptor (TCR) α/β monoclonal antibodies (mAbs) conjugated with either fluorescein isothiocyanate or phycoerythrin (Becton Dickinson Immunocytometry Systems, San Jose, Calif.) with the use of a FACScan (Becton Dickinson).

Antigen-induced proliferative responses of T cell clones 

Assays of antigen-induced proliferation of the T cell clones were done by culturing the T cells (2 × 104/well) in 96-well flat-bottom culture plates in the presence of Cry j 1 (1 μg/ml) and 3000 cGy-irradiated autologous PBMC (1.5 × 105/well). Cells were cultured for 72 hours in the presence of 1 μCi/well of [3H]TdR during the final 16-hour period, and the incorporated radioactivity was measured by liquid scintillation counting after harvesting. When stimulating with synthesized peptides, PBMC (3 × 105/well) were cultured in plates with peptides (various concentrations) in the culture medium for 2 hours at 37° C. Then excess peptides and nonadherent cells were removed by gently washing the plates three times with RPMI 1640 media containing 3% human serum. The remaining adherent cells were irradiated at 3000 cGy and used as APC. To investigate whether analog peptides act as an antagonist for TCR, T cell clones were incubated with irradiated PBMC prepulsed for 2 hours with the 0.125 μmol/L wild-type peptide and the soluble form of analog peptides at various concentrations.

To determine restriction molecules for antigen presentation, the T cell clones were cultured with irradiated autologous PBMC with or without saturating amounts of anti-HLA class II monoclonal antibodies HU-4 (anti-HLA-DRB1+DRB5 monomorphic),17, 18 HU-11 (anti-HLA-DQ1+DQ4),19 HU-18 (anti-HLA-DQ3),20 HU-46 (anti-HLA-DQ4),21 and B7/21 (anti-HLA-DP monomorphic).17 Allogeneic PBMC or mouse L cells transfected with HLA class II genes were also used as APC sources. The TR162-B2 cell line transfected with the DRA+DRB3*0301 genes isolated from the lymphoblastoid B cell line JY (DR4, w6)22 was a generous gift from Dr. H. Inoko, Tokai University, Isehara, Japan. All L cells, including those transfected with selection marker Neor gene alone (L-Neo) and used as a control, were treated with mitomycin C (20 mg/ml) before use, as described.23 Exposure to peptides was then performed for 2 hours.

Quantitation of IL-4, IL-2, and interferon (IFN)γ in supernatants of the T cell clones 

Culture supernatants of the T cell clones stimulated by APC plus antigen for the determination of proliferative responses were collected immediately before the addition of [3H]TdR and stored in aliquots at -80° C until determinations of lymphokine concentrations were done. Quantikine hIL-4, hIL-2 (R&D Systems, Minneapolis, Minn.), and hIFNγ ELISA kit (Otsuka, Tokyo, Japan) were used for the quantitation of IL-4, IL-2, and IFNγ in the supernatants according to manufacturer’s instructions.

HLA typing 

HLA class II (DR,DQ,DP) alleles were determined by hybridization of HLA-DR, DQ, DP genes amplified by polymerase chain reaction with sequence-specific oligonucleotide probes distributed in the Eleventh International Histocompatibility Workshop,24 as described elsewhere.25 The nomenclature of the HLA-DR, DQ, DP alleles was according to the WHO Nomenclature Committee for factors of the HLA system.26

Peptide binding assay 

The bindings of synthesized peptides to HLA-DRB3*0301 molecules were measured as follows. Briefly, the TR162-B2 cell line (3 × 105/well) was incubated with synthesized peptides with biotinylation on the N-terminus at various concentrations and in Dulbecco’s modified Eagle’s medium supplemented with 2 mmol/L L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% heat-inactivated fetal calf serum in 96-well flat-bottomed culture plates for 4 hours at 37° C. The cells were then harvested and washed three times with phosphate-buffered saline solution containing 0.01% NaN3 and 3% fetal calf serum. Flowcytometric analysis with FACScan (Becton Dickinson) was carried out followed by incubation with Streptavidin-R-Phycoerythrin (Gibco) or phycoerythrin-conjugated antiHLA-DR mAb (Becton Dickinson) for 0.5 hours at 4° C. Binding affinity was valued by binding index (BI), calculated with the following formula: BI = {Peak channel 1(A) - Peak channel 1(B)}/Peak channel 1(C) (A):peptide-pulsed L cell transfectant (B):peptide-unpulsed L cell transfectant (C):anti-HLA-DR.

Peptide binding inhibition assay 

To quantitate the binding affinity of each analog peptide with HLA molecules, we examined the inhibitory activity of each analog peptide against binding of the wild-type peptide to HLA molecule. Briefly, the TR162-B2 cell line (3 × 105/well) was incubated with several concentrations of unlabeled analog peptides in 96-well flat-bottomed culture plates. Four hours later 25 μmol/L of biotinylated wild-type peptide was added, and the preparation was further incubated for 4 hours. These cells were then harvested and stained with Streptavidin-R-Phycoerythrin (Gibco). The mean fluorescence intensity was determined with FACScan (Becton Dickinson). The inhibitory effect of analog peptides on binding of the wild-type peptide to HLA-DR molecules was estimated by measuring the peptide concentration required for 50% inhibition of binding (IC50).

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RESULTS 

Establishment of a Cry j 1-specific T cell clone ST1.9 

Sixteen T cell clones specific to Cry j 1 were generated from five donors allergic to Japanese cedar pollen. These clones proliferated specifically in response to Cry j 1 presented by autologous PBMC (data not shown). One of these clones, designated ST1.9, was generated from a short-term (2 weeks) cultured Cry j 1-specific T cell line ST1, which was tested for reactivity to a panel of overlapping synthetic peptides corresponding to Cry j 1 amino acid sequence. As shown in Fig. 1, ST1 reacted to a limited set of Cry j 1 peptides (Cry j 1 p327-346 and Cry j 1 p337-353 with 10-mer overlap). Similarly, the T cell clone ST1.9 proliferated in response only to these two peptides. Therefore it is highly conceivable that ST1.9 recognizes a major T cell epitope of Cry j 1 for the donor S.T. Based on the characteristics of ST1.9 and the availability of L cell transfectants expressing antigen-presenting HLA-DR molecules (see following) for the ST1.9, we extensively analyzed ST1.9. One of the other clones obtained from another patient responded to a Cry j 1 p151-170 in the context of HLA-DQ (data not shown). Flowcytometric analyses of clone ST1.9 revealed a CD3+, αβTCR+, CD4+, CD8– phenotype (data not shown).

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  • FIG. 1. 

    Proliferative responses of short-term cultured T cell line, ST1, to purified Cry j 1 protein and to synthesized overlapping peptides derived from Cry j 1. Peptides are designated by indicating N and C terminus amino acid residue numbers. Values shown are mean counts per minute of duplicate cultures. Standard error (SE) of duplicate response was <10%. Counts per minute of culture without antigen was 543 ± 5 cpm.

Antigen-presenting HLA molecules and a core peptide fragment recognized by ST1.9 

ST1.9 cells were cultured in the presence of Cry j 1 and autologous PBMC with anti-HLA class II mAbs. Unexpectedly, none of these mAbs inhibited the Cry j 1-driven proliferation of ST1.9, although the mAbs did block antigen-driven T cell responses in other systems (not shown). These observations led to the notion that the restriction molecule might be HLA-DRB3*0301, according to the HLA type of donor S.T.(HLA-DRB1*0101-DQA1*0101-DQB1*0501/DRB1*1202-DRB3*0301-DQA1*0601-DQB1*0301), because HU-4 recognizes DRB1 and DRB5 but not DRB3 nor DRB4 gene products.17, 18 Indeed, ST1.9 did proliferate in response to Cry j 1 presented by allogeneic PBMC, which share HLA-DRB3*0301 alone with the donor S.T., or L cell transfectant expressing DRB3*0301 molecules (data not shown). These observations indicate that the T cell clone ST1.9 recognizes Cry j 1 in the context of DRB3*0301.

To identify a core sequence of the peptide Cry j 1 p327-346 (FNVENGNATPQLTKNAGVLT) for the DRB3*0301-restricted antigen presentation to the ST1.9 cells, truncated peptides were synthesized, and reactivity of ST1.9 was tested. Proliferative responses of ST1.9 decreased significantly when the peptide Cry j 1 p327-346 was truncated either from the N-terminus to 337Q or from the C-terminus to 344V. However, the octamer peptide Cry j 1 p337-344 induced only marginal response of ST1.9. For the full response compared with Cry j 1 p327-346, two additional residues at both C- and N-termini were required (data not shown). Therefore Cry j 1 p335-346 (TPQLTKNAGVLT) was used for subsequent experiments.

Stimulatory activities to ST1.9 and HLA-DR binding of Cry j 1 p335-346-derived analog peptides 

To determine important amino acid residues on the antigenic peptide for the recognition of Cry j 1 p335-346, in the context of DRB3*0301 by ST1.9, we synthesized a series of analog peptides with nonconservative single amino acid substitution at every amino acid residue on Cry j 1 p335-346. Our previous study clearly demonstrated that not only the TCR-TCR ligand but also peptide-MHC interactions are significantly downmodulated when the polarity of important residues on an antigenic peptide is inversed.27 Thus each hydrophilic residue on TPQLTKNAGVLT was substituted with small hydrophobic alanine. To avoid introduction of either a large side chain that cannot be accommodated or charged side chain groups that might generate a repulsive interaction between the antigen-binding groove of HLA-DR molecule, each hydrophobic residue was substituted with small neutral hydrophilic serine residue.27 As demonstrated in Fig. 2, substitutions at five positions L338S (an analog peptide of Cry j 1 p335-346 with substitution to Ser at 338Leu), K340A, N341A, V344S, and L345S led to a markedly decreased proliferative response of ST1.9. Therefore it is likely that at least these five residues (338L, 340K, 341N, 344 V, and 345L) are important either for binding to HLA-DR molecules or for recognition by the T cell receptor of ST1.9. As shown in Fig. 2, of these five residues, 338L and 341N are likely to be critical HLA binding sites, because nonconservative residue substitutions to Ala or Ser at these positions significantly decreased peptide binding to HLA (IC50 values being greater than 1000 μmol/L).

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  • FIG. 2. 

    Identification of important amino acid residues on Cry j 1 p335-346 peptide for stimulation of ST1.9. Series of analog peptides with nonconservative single amino acid substitution at every amino acid residue on Cry j 1 p335-346 was synthesized; that is, T335A indicates that 335Thr on Cry j 1 p335-346 is substituted to Ala. - indicates residue without substitution. ST1.9 cells were cultured with each analog peptide (1 μmol/L)-pulsed PBMC for 72 hours. Proliferation was determined by [3H]TdR incorporation in duplicate, and Δ cpm means counts per minute with antigen subtracted with counts per minute without antigen. SE of duplicate response was <10%. Binding affinity of each peptide was assessed according to IC50, as described in Methods. NT, Not tested.

We synthesized a series of analog peptides with chemically conservative amino acid substitutions to examine the effects of minimal alterations in peptide sequence on T cell responses. To evaluate effects of single amino acid substitutions, proliferation and lymphokine production in response to analog peptides were determined and compared with those to the wild-type peptide. Because it was expected that not only important sites but also adjoining residues might affect the interaction between TCR and TCR ligand, conservative substitutions were made at each amino acid residue from 337Gln to 346Thr. As demonstrated in Fig. 3, analog peptides carrying a single amino acid substitution at two additional residues, Q337N and A342G, abrogated the proliferative response of ST1.9, thereby suggesting that these two residues 337 Gln and 342Ala may also be important for interactions with TCR or HLA.

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  • FIG. 3. 

    Proliferative responses versus lymphokine production patterns of ST1.9. ST1.9 cells were cultured in presence of peptide-pulsed (1 μmol/L) PBMC. After 48 hours of incubation, culture supernatants were collected immediately before addition of [3H]TdR for measurements of lymphokine production by ELISA. SE of duplicate response was <10%. Graphs in left column indicate proliferative responses valued by percent relative response to Cry j 1 p335-346 peptide calculated by following formula: 100 × (counts per minute of culture with analog peptide)/(counts per minute of culture with Cry j 1 p335-346 peptide). Value of 100% corresponded to 17,000-22,000 cpm. In right column, net lymphokine concentrations of culture supernatant fluids are expressed by mean values of duplicate cultures. Δ pg/ml means lymphokine concentration with peptide subtracted with lymphokine concentration without peptide; shaded bars, IL-4; hatched bars, IFNγ open bars, IL-2. Binding affinity of each peptide was assessed according to IC50 as described in Methods. NT, Not tested.

Some analog peptides carrying substitutions at 339Thr acted as TCR antagonists 

Analog peptides that induced no proliferative responses of ST1.9 were investigated to determine whether they functioned as TCR antagonists.2, 3 As shown in Fig. 4, two analogs, T339G and T339Q, revealed approximately a 70% inhibition of the T cell proliferation against the wild-type peptide. On the contrary, irrelevant peptides, p53p278- 291/282W and p53p236-253/245S, which bound to HLA-DRB3*0301 molecules equally to or more than T339G and T339Q judging from their DR binding indexes, showed no inhibition at the same concentration. Thus T339G and T339Q probably acted as TCR antagonists. In recognition of these two analogs, ST1.9 produced no IL-4, IL-2, or IFNγ.

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  • FIG. 4. 

    Analog peptides function as T cell receptor antagonists. PBMC were cultured with wild-type peptide (0.125 μmol/L) for 2 hours; then excess peptide was removed. ST1.9 cells were cultured in duplicate, with or without analog peptides (75 μmol/L) in presence of wild-type peptide-pulsed PBMC for 72 hours. Proliferation was determined by [3H]TdR incorporation in duplicate and was expressed by percent relative response to Cry j 1 p335-346 peptide as described in legend for Fig. 3. Value of 100% corresponded to 22,000-38,000 counts/min. SE of duplicate response was <10%. Binding affinity was assessed by binding index as described in Methods.

Increased production of IFNγ by ST1.9 in response to an analog peptide T339V 

Stimulation of ST1.9 with Cry j 1 p335-346 resulted in the production of IL-4 (320 pg/ml) in the supernatants, whereas there were lower amounts of IFNγ (75 pg/ml) and no IL-2 (Fig. 3). A similar pattern of cytokine secretion by ST1.9 was detected in response to Cry j 1 (data not shown). Thus stimulation with either core peptide sequence or Cry j 1 revealed the same lymphokine production patterns of ST1.9. At the range of peptide concentrations from 0.02 μmol/L up to 4 μmol/L, the same lymphokine pattern was observed, and with concentrations greater than 4 μmol/L, production of each lymphokine reached plateau levels.

Most of the analog peptides that stimulate ST1.9 showed a pattern of lymphokine production similar to that for the wild-type peptide, and the IL-4 production of ST1.9 for each analog peptide was in proportion to the proliferative response to each peptide. However, as shown in Fig. 3, the IFNγ production of ST1.9 in response to an analog peptide (T339V) was increased, whereas neither T cell proliferation nor production of other lymphokines showed any remarkable change; that is, only the production of IFNγ was affected in recognition of the analog peptide T339V. To determine whether the change of IFNγ production was due to differences in the HLA-peptide or TCR-TCR ligand avidity between T339V and the wild-type peptide, responses of ST1.9 to several different concentrations of T339V were compared with those of the wild-type peptide. As shown in Fig. 5, A, at the range of concentrations from 0.5 μmol/L up to 4 μmol/L, IFNγ production in response to T339V constantly exceeded that of the wild-type peptide. Moreover, the plateau level of T339V-driven IFNγ production was significantly higher. Mean IFNγ production of ST1.9 for T339V increased significantly over that for the wild-type (0.5 μmol/L: p = 0.0019, 1 μmol/L: p = 0.0018) (Fig. 5, B), whereas no statistical differences were noted in IL-4 production between T339V and the wild-type (0.5 μmol/L: p > 0.1, 1 μmol/L: p > 0.5) (data not shown). In light of the observation that substitution of the same residue, 339Thr, to glycine or glutamine induced TCR antagonism, it seems likely that this position might be a “hot spot” for the altered T cell recognition that leads to altered T cell response or to TCR antagonism.

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  • FIG. 5. 

    A, IFNγ production of ST1.9 in recognition of either wild-type peptide or T339V at different concentrations. ST1.9 cells were cultured in duplicate with peptide-pulsed PBMC at indicated concentrations. After 48 hours of incubation, mixed supernatants of duplicate culture were collected. Solid line, IFNγ concentration of supernatants cultured with the wild-type peptide; dotted line, cultured with T339V. B, Statistical differences in IFNγ production by ST1.9 between stimulation by wild-type peptide and T339V. ST1.9 cells were cultured in triplicate with peptide-pulsed PBMC at indicated concentrations. Closed bars, IFNγ concentration of supernatants cultured with wild-type peptide; open bars, cultured with T339V. SE of IFNγ concentrations of triplicate culture supernatants is indicated by bar, and statistical significance of difference was estimated with Student’s t test. *p = 0.0019, **p = 0.0018.

Binding to HLA-DRB3*0301 molecules of analog peptides inducing altered T cell response 

The wild-type peptide and the analog peptides were tested for their binding potential to the HLA-DRB3*0301 molecules. In various concentrations (100 μmol/L, 50 μmol/L, and 25 μmol/L) the analog peptide (T339V) bound to HLA molecules with the same affinity as the wild-type peptide (Table I). These data suggest that increase in IFNγ production of ST1.9 cannot be attributed to alteration in binding affinity between HLA and T339V. Although the analog peptides, T339G and T339Q, induced no proliferative responses at high concentrations and acted as TCR antagonists for ST1.9, these peptides revealed a sufficient binding affinity compared with the wild-type peptide (Table I).

Table I. Binding of a Cry j 1-derived peptide and its analogs to HLA-DRB3*0301 molecules
PeptideConcentration (μmol/L)Binding index
Cry j 1 p335-34610020.5
5012.4
258.7
T339V10018.6
5016.2
258.1
T339Q5012.4
T339G5010.6

L cell transfectant expressing HLA-DRB3*0301 molecule was incubated with each biotinylated peptide at the indicated concentrations. After 4 hours of incubation cells were harvested and incubated with Streptavidin-R-Phycoerythrin or phycoerythrin-conjugated anti HLA-DR mAb for 0.5 hours at 4° C. Peptide binding was analyzed by flowcytometer, and binding affinity was assessed by binding index as described in Methods.

Cry j 1 p335-346 did not carry an IgE B cell epitope for the donor ST 

To determine whether Cry j 1 p335-346 contains an IgE B cell epitope for the donor ST, immediate-type skin reaction was determined by intradermal injection of various doses given to donor ST (0.85 nmol/L, 8.5 nmol/L, 85 nmol/L, and 850 nmol/L of Cry j 1 and peptides), and after 15 minutes diameter of the erythema was measured. The diameters in injection of Cry j 1 protein were 5 mm with 8.5 nmol/L, 19 mm with 85 nmol/L, and 30 mm with 850 nmol, whereas Cry j 1 p335-346 or T339V produced no skin reactions regardless of the concentrations (data not shown). Thus donor ST exhibited immediate-type hypersensitivity to Cry j 1 in a dose-dependent manner but not to Cry j 1 p335-346 or T339V. These data suggested that Cry j 1 p335-346 and its derivative T339V do not contain an IgE B cell epitope in spite of recognition by T cells. Moreover, delayed-type skin reaction was not induced either by injection of Cry j 1 or by Cry j 1-derived peptides.

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DISCUSSION 

In this article we report that (1) a human CD4+T cell clone, ST1.9, a representative of the major T cell clones specific to Cry j 1 generated from a patient with type I allergy to Japanese cedar pollen produced IL-4 dominantly, with a little IFNγ and no detectable IL-2; (2) ST1.9 cells recognized a 12 mer peptide derived from Cry j 1 (Cry j 1 p335-346:TPQLTKNAGVLT) presented by HLA-DRA+DRB3*0301 molecules; (3) substitution of a single amino acid at seven residues, 337Q, 338L, 340 K, 341N, 342A, 344V, and 345L, abrogated proliferative responses of ST1.9, suggesting that at least these seven amino acid residues are involved in interactions with TCR, HLA-DR, or both; substitution at these residues may induce serious conformational changes that would impair proper interactions with TCR of ST1.9 or HLA-DR; (4) determination of binding affinities to HLA-DR molecules of analog peptides, by estimating IC50 values, indicated that both 338L and 341N were likely to be critical HLA-binding residues; (5) analog peptides with substitutions of 339Thr to Gly or Gln acted as TCR antagonists; and (6) ST1.9 cells revealed an increase in IFNγ production in recognition of the peptide T339V (an analog peptide of Cry j 1 p335-346 having substitution to Val at 339Thr) without alteration in IL-4 production or proliferative response, although T339V did bind to the HLA-DRB3*0301 molecules with the same affinity as seen with the wild-type peptide. Thus findings 5 and 6 suggest that the amino acid residue 339Thr may be a recognition site for TCR of ST1.9.

Recent studies showed that different DR molecules are capable of binding peptides with different structural motifs, because polymorphic residues of DR alleles are scattered within the peptide-binding grooves. To date, motifs for binding peptides have been reported in cases of several DR molecules.27, 28 Many of these motifs showed that the first anchors closest to the amino termini of the binding peptides need to be hydrophobic and that the second anchors are three residues apart in direction of the C-terminus from the first ones. Therefore 338L and 341N were likely to be critical HLA-binding residues in Cry j 1 p335-346 peptide. In comparison with DR motifs, it is highly conceivable that 338L is the first anchor and that 341N is the second one.

Because previous studies indicated that allergen-specific T cell clones established by atopic donors dominantly produced IL-4,29, 30 ST1.9 cloned from a Cry j 1 specific T cell line, ST1, secreted IL-4 with a little IFNγ in the recognition of Cry j 1 or Cry j 1-derived peptides. Some studies on the statistical association between IgE response to Cry j 1 and HLA allele determined at the DNA level showed no significant association between the two, although it was reported that HLA-DR molecules may present Cry j 1 to T cells, thus functioning as Ir-gene products for Cry j 1 responses.14 Indeed, ST1.9 responded to the Cry j 1-derived peptides close to the C-terminus of Cry j 1 (Cry j 1 p335-346) in the context of HLA-DRA+DRB3*0301 molecules. Of 18 patients with cedar pollenosis, seven had T cell proliferative responses specific to the C-terminal 23-mer peptides with a stimulation index of greater than five, indicating that this position is one of the major T cell epitopes (T. Sone, Meiji Institute of Health Science, personal communication).

One interesting observation was the dissociation between IFNγ production and T cell proliferation or IL-4 production in recognition of certain analog peptides. The production of IL-4 was induced parallel to the proliferative response but not to that of IFNγ. It is noteworthy that T339V increased the production of IFNγ but that other factors (proliferation and IL-4) remained unchanged. Recent studies demonstrated that altered TCR ligands induced qualitative changes of T cell responses such as T cell anergy,1 TCR antagonism,2, 3 or the segregation of proliferative responses from lymphokine productions.4, 5, 6 The current observation is the first report that an altered TCR ligand induces qualitative changes in T cell responses in human systems.

Stern et al.7 demonstrated that 30% of van der Waals surface on an antigenic peptide bound to HLA-DR1 molecules can be available for contact with TCR and that only the first hydrophobic anchor residue is completely buried in the peptide-binding groove of MHC. The other residues can physically contact TCR, raising the possibility that 339Thr can affect the TCR-TCR ligand interaction. The IC50 value of each peptide and the T cell response indicate that 340K is likely to be TCR-binding residues and that the other important residues (except 338L and 341N) are likely to influence both HLA and TCR binding. Other supportive evidence for this notion is that, as shown in Fig. 4, substitutions of 339Thr to Gly or Gln resulted in TCR antagonism. It might also be that conformational changes in the TCR ligand as induced by the substitution of 339Thr influence the interaction between TCR and its ligand, even if this position is not directly recognized by TCR.

The signaling pathway associated with TCR is possibly divergent between IFNγ and IL-4 production. In this connection, Gajewski et al.31 reported that different signaling pathways are used by Th1 and Th2 clones. Furthermore distinct CD3 moiety (ζ and ϵ) might be phosphorylated with different peptide stimulations. Recently Sloan-Lancaster et al.32 and Madrenas et al.33 reported that partial agonists induced partial phosphorylation of CD3 ζ chains, which induced no phosphorylation nor activation of ZAP-70. From this standpoint it is conceivable that the analog peptide, T339V, influences the signaling pathway only in IFNγ production, which results in increased production of IFNγ with no remarkable changes in either IL-4 production or proliferative response, although the concise signaling mechanism is unclear. Different conformational changes in the TCR molecule induced by analog peptides may induce qualitative differences in signaling pathways. The wild-type peptide, Cry j 1 p335-346, might act as a partial rather than a full agonist, and T339V fits the TCR more rigidly than does the wild-type peptide, which consequently leads to increased production of IFNγ. However, the significant difference of plateau levels of IFNγ production between the wild-type peptide and T339V cannot be fully explained by the difference of avidity between TCR and TCR ligand, because such a difference should be offset by dose of the antigen. Another possible explanation may be differences in threshold of activation signal through TCR required for plateau response between IFNγ and IL-4 productions. In this case wild-type Cry j 1 p335-346 gave a strong signal to ST1.9 for plateau response of IL-4 but not for that of IFNγ. The analog peptide T339V gave a stronger signal to ST1.9 for plateau response of both IL-4 and IFNγ.

If the affinity of T339V for HLA-DRB3*0301 molecules is higher than that for the wild-type peptide, T cell response might have been qualitatively changed, because it was reported that differentiation to Th1 or Th2-phenotype depends on either antigen dose or TCR ligand density.34 However, this was not the case, because T339V bound to HLA-DRB3*0301 molecules with the same affinity as the wild-type peptide. This observation suggests that the increased production of IFNγ cannot be attributed to alteration in TCR ligand density. Brown et al.35 demonstrated that the HLA-DR1 molecule crystallizes as a dimer of the αβ heterodimer. Although the functional significance of this dimerization is not clear, possible alteration of the dimerization of HLA molecules led by T339V may result in a qualitative change in the T cell response. Other workers suggested that prostaglandin E2 (PGE2) influences the production of IL-2 and IFNγ but not that of IL-4.36, 37 If the binding of T339V to HLA molecules leads to a decreased production of PGE2 from APC, then IFNγ production by T cells could increase. Therefore one may speculate that T339V induces (1) alteration of dimerization of HLA molecules through conformational changes that might also alter expression of costimulatory molecules on APC such as CD80 (B7/BB1, B7-1), and (2) decreased secretion of PGE2 from APC, thus leading to increases in IFNγ production.

Alterations in T cell responses induced by modified allergen peptide seen in our study not only facilitate understanding of lymphokine-specific signal transducing events but also will aid in developing therapeutics for allergy-related diseases. When analogs of allergen peptides act as a TCR antagonist, induce T cell anergy, or alter lymphokine production, especially decreased production of IL-4, increased IFNγ, or both, allergen-specific IgE would likely be reduced. The existence of diversity of individual HLA-TCR interactions toward an allergen will complicate applications for all cases. However, in the case of donor ST investigated in our study, reduction in IgE by stimulation with the analog peptide T339V may be feasible, because a short-term (3 weeks) cultured antigen-specific T cell line ST1 recognizes the limited part of allergen, which can be recognized by the T cell clone ST1.9.

Recent studies suggested the utility of allergen peptides containing the T cell epitope for immunotherapy.38, 39 These studies demonstrate that the administration of allergen peptide containing T cell epitope(s) induces T cell tolerance. In this study it is especially important to note that intracutaneous administration of Cry j 1-derived peptide containing T cell epitope did not induce an immediate-type hypersensitivity to donor ST. This means that the peptide may contain (1) no IgE B-cell epitope or (2) a single IgE B-cell epitope. It also means that the peptide does not aggregate to the extent to cross-link FcϵRI. In addition, an analog of this allergen peptide induces an increase in IFNγ production, which may downregulate IgE production. Thus effective and secure immunotherapy (i.e., anaphylaxis-free) with allergen-derived analog peptides that induce qualitative changes of T cell responses can be designed. Currently, a study is underway to examine intracellular events related to the altered TCR ligand in the T cell clone ST1.9.

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Acknowledgements 

We thank Dr. A. Wakisaka (Hokkaido University, Sapporo, Japan) for providing mAb; Dr. H. Inoko (Tokai University, Isehara, Japan) for TR162-B2; Drs. M. Suzuki (Ajinomoto Co., Kawasaki, Japan) and K. Hama (Ono Pharmaceuticals, Osaka, Japan) for human recombinant IL-2 and IL-4, respectively. We also thank Drs. H. Tsunoo, T. Sone (Meiji Institute of Health Science, Odawara, Japan), Dr. K. Komoriya (Teijin Institute for Biomedical Research, Hino, Japan) for their valuable discussions; and M. Ohara for helpful comments.

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☆ From athe Division of Immunogenetics, Department of Neuroscience and Immunology, Kumamoto University Graduate School of Medical Sciences; and bthe Department of Otorhinolaryngology, Kumamoto University School of Medicine.

☆☆ Supported in part by Grants-in-Aid 03452276, 05278118, 05272105, and 05272104 from the Ministry of Education, Science and Culture, Japan, Research Grant for Intractable Diseases from the Ministry of Health and Welfare, Japan, Ichiro Kanehara Foundation, Terumo Life Science Foundation, Kato Memorial Foundation, Japan Rheumatism Foundation, Meiji Institute of Health Science, Hokuriku Pharmaceutical Co. for Research of Allergy, and Mochida Memorial Foundation.

★ Reprint requests: Yasuharu Nishimura, MD, Division of Immunogenetics, Department of Neuroscience and Immunology, Kumamoto University Graduate School of Medical Sciences, Honjo 2-2-1, Kumamoto 860, Japan.

★★ 0091-6749/96 $5.00 + 0 1/1/64980

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The Journal of Allergy and Clinical Immunology
Volume 97, Issue 1 , Pages 53-64, January 1996

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