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

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.

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.

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.

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.

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.

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.