Localization of cryptic tolerogenic epitopes in the alpha1-helical region of the RT1.Au alloantigen.
BACKGROUND
Transplantation tolerance is induced by perioperative administration of host class I major histocompatibility complex proteins bearing donor-type amino acid (a.a.) epitopes substituted for native residues. Herein we demonstrate that two cryptic tolerogenic a.a. epitopes are localized in the alpha1-helical region of the rat RT1.Au class I major histocompatibility complex alloantigen.


METHODS
Three allochimeric proteins were produced by superimposing the nucleotides encoding donor-type RT1.Au a.a. onto the host RT1.Aa backbone using the polymerase chain reaction-based method of gene splicing with overlap extension. We substituted nucleotide sequences encoding all nine (Arg62, Glu63, Gln65, Gly66, Gly69, His70, Val73, Asn74, and Asn77; alpha1h u62-77-RT1.Aa), the first four (Arg62, Glu63, Gln65, and Gly69; alpha1h u62-69-RT1.Aa), or the last four (His70, Val73, Asn74, and Asn77; alpha1h u70-77-RT1.Aa) alpha1-helical RT1.Au polymorphic a.a. in RT1.Aa cDNA. A baculovirus/Spodoptera frugiperda (Sf9) expression system was harnessed for production of the proteins.


RESULTS
Untreated ACI (RT1a) rats reject Wistar-Furth (WF; RT1u) heart allografts at a mean survival time of 8.9+/-1.0 days. A single portal vein injection of 10 microg of alpha1h u62-77-RT1.Aa protein had no effect on the survival of WF heart allografts (10.5+/-0.6 days) in ACI hosts. Interestingly, portal vein administration of 10 microg of alpha1h u70-77-RT1.Aa induced transplantation tolerance toward WF grafts in four of six untreated ACI recipients (>100 days; P<0.01). In contrast, 10 microg of alpha1h u62-69-RT1.Aa only prolonged the survival of WF heart allografts in ACI hosts (14.0+/-0.8 days; P<0.01). However, when combined with a 7-day course of cyclosporine (4.0 mg/kg; oral gavage), alpha1h u62-69-RT1.Aa induced tolerance toward WF allografts in five of seven ACI recipients (>170 days; P<0.01). Long-term survival of WF grafts was not achieved when a 7-day course of cyclosporine was administered alone (14.3+/-3.0 days) or with 10 microg of alpha1h u62-77-RT1.Aa (14.6+/-0.6 days; NS).


CONCLUSIONS
These findings suggest that the use of allochimeric proteins may provide a novel approach to the induction of tolerance.
Wang
M
M
Department of Surgery, The University of Texas Medical School at Houston, 77030, USA.
Stepkowski
S M
SM
Yu
J
J
Wang
M
M
Kahan
B D
BD
D000818:Animals; D000939:Epitopes / Q000032:analysis*; D006085:Graft Survival / Q000276:immunology; D016027:Heart Transplantation / Q000276:immunology; D015395:Histocompatibility Antigens Class I / Q000096:biosynthesis; D007108:Immune Tolerance; D007114:Immunization; D007519:Isoantigens / Q000737:chemistry / Q000276:immunology*; D017433:Protein Structure, Secondary; D051381:Rats; D011912:Rats, Inbred ACI; D011914:Rats, Inbred BN; D011917:Rats, Inbred Lew; D011920:Rats, Inbred WF; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
D000939:Epitopes; D015395:Histocompatibility Antigens Class I; D007519:Isoantigens; D011993:Recombinant Fusion Proteins