E. COLI-PRODUCED SOLUBLE MHC CLASS I PROTEINS INDUCE POTENT ALLOANTIGEN-SPECIFIC IN VIVO EFFECTS

Abstract

116 Because this method provides rapid production, we employed an E. coli protein expression system to produce soluble rat heavy chain class I MHC proteins. A PCR-based method with designed primers was used to introduce a six Histidine (6-His) tag and a stop codon after the α3 domain into RT1.An and RT1.Ac cDNAs. These constructs were expressed in bacterial pET-23a(+) vector with an ampicillin resistance gene and an isopropyl-b-D-thiogalactopyranoside (IPTG)-inducible viral T7 promoter. The produced proteins were solubilized in 6M urea and purified on a Ni2+ affinity column. Untreated Lewis (LEW, RT1.Al) recipients rejected Brown-Norway (BN, RT1.An) heart allografts at a mean survival time (MST) of 7.5±0.5 days. Although subcutaneous (SC) immunization at day -4 with 1 µg of RT1.An was ineffective (8.0±1.0 days; NS), either 50 µg or 100 µg RT1.An protein induced accelerated rejection of BN heart allografts at 5.0±0.0 days (p<0.05) and 5.0±0.0 days, (p<0.05), respectively. This effect was antigen-specific; immunization of LEW rats with self-RT1.Al protein failed to induce accelerated rejection of BN heart allografts (MST 8.0±1.0 days; NS). In another strain combination, untreated PVG (RT1c) recipients rejected BN heart allografts at 9.1±1.0 days. Although SC administration (day -7) with 10 µg RT1.An was ineffective (10.0±0.0 days, NS), either 50 µg or 100 µg of RT1.An induced accelerated rejection of BN heart allografts at 7.8±0.6 days (p=0.004) and 6.1±1.0 days (p=0.0004), respectively. Immunization of the PVG rats with self-RT1.Ac protein failed to cause accelerated rejection of BN heart allografts (8.0±0.0, NS). In another control experiment, immunization with RT1.An did not induce an accelerated rejection of third-party control LEW heart allografts (11.0±1.0 days, NS). In a tolerogenic protocol, 50 µg RT1.An injected by portal vein (PV) at the time of transplantation to PVG rats in combination with a 7-day course of cyclosporine (CsA, 4 mg/kg/day) prolonged the survival of BN heart allograft to a MST of 33.0 days (13×3, 19, 20, >50, >100 days; p=0.07). Administration of RT1.An protein alone produced a MST of 9.75±0.25 days, and CsA alone, a MST of 11.0±1.0 days. Thus, the bacterial expression system may be used to produce class I MHC proteins that are active in vivo in immunogenicity and graft prolongation protocols.