Biological assay using T cell response for Cry-consensus peptide designed for the peptide-based immunotherapy of Japanese cedar pollinosis.
BACKGROUND
Cry-consensus peptide is a linearly linked peptide of T-cell epitopes for the management of Japanese cedar (JC) pollinosis and is expected to become a new drug for immunotherapy. However, the mechanism of T-cell epitopes in allergic diseases is not well understood, and thus, a simple in vitro procedure for evaluation of its biological activity is desired.


METHODS
Peripheral blood mononuclear cells (PBMC) were isolated from 27 JC pollinosis patients and 10 healthy subjects, and cultured in vitro for 4 days in the presence of Cry-consensus peptide and (3)H-thymidine. The relationship between growth stimulation (stimulation index; SI) and antigen-specific IgE levels in serum was also investigated in JC pollinosis patients. Moreover, to confirm the importance of the primary sequence in Cry-consensus peptide, heat-treated Cry-consensus peptide and a mixture of the amino acids of which Cry-consensus peptide is composed, and their (3)H-thymidine uptake was compared with Cry-consensus peptide. Finally, whether Cry-consensus peptide stimulates PBMCs from healthy subjects was investigated.


RESULTS
The mean SI of JC patients showed a good correlation with Cry-consensus peptide concentration in the culture medium; however, the SI was independent of the anti-Cry j 1 IgE level. Heat-denatured Cry-consensus peptide retained a PBMC proliferation stimulatory effect comparable to the original Cry-consensus peptide, while the mixture of amino acids constituting Cry-consensus peptide did not stimulate PBMC proliferation. PBMCs from healthy subjects did not respond to Cry-consensus peptide at all.


CONCLUSIONS
These data indicate that the PBMC response of patients suffering from JC pollinosis to Cry-consensus peptide is specific for the sequence of T cell epitopes thereof and may be useful for the evaluation of the efficacy of Cry-consensus peptide in vivo.
Kozutsumi
Daisuke
D
Research and Development Section I, Pharmaceuticals Development Department, Meiji Dairies Corporation, 540 Naruda, Odawara, Kanagawa 250-0862, Japan. daisuke_kozutsumi@meiji-milk.com
Tsunematsu
Masako
M
Yamaji
Taketo
T
Kino
Kohsuke
K
D000328:Adult; D000485:Allergens / Q000276:immunology*; D000595:Amino Acid Sequence; D001681:Biological Assay / Q000379:methods*; D049109:Cell Proliferation; D002478:Cells, Cultured; D032485:Cryptomeria / Q000276:immunology*; D018984:Epitopes, T-Lymphocyte / Q000276:immunology; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008875:Middle Aged; D011058:Pollen / Q000276:immunology; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000627:therapeutic use*; D006255:Rhinitis, Allergic, Seasonal / Q000276:immunology / Q000628:therapy*; D012680:Sensitivity and Specificity; D013936:Thymidine
D000485:Allergens; D018984:Epitopes, T-Lymphocyte; D011994:Recombinant Proteins; C518796:cry-consensus peptide; D007073:Immunoglobulin E; D013936:Thymidine