640. E1 Bp-Specific CD8+ T Lymphocytes Are Highly Induced in the Liver and Spleen Eight Days after Adenoviral Infection

Abstract

Top of pageAbstract The use of Adenovirus (Ad) as a gene delivery vector is limited by transient gene expression due to the host cytotoxic T lymphocyte (CTL) response and innate responses. We and others have shown that the cytotoxic response to Ad is only partially inhibited by administration of Ad to mutant mice deficient in producing a cytotoxic immune response, or by treatment with TNF inhibitors. However, analysis of the specific CTL response to Ad has not been possible due to the lack of reagents that recognize the rearranged T cell receptor expressed on Ad-specific cytotoxic T cells. We therefore developed a tetramer that recognizes the immunodominant epitope of E1B (192 VNIRNCCYI), referred to as E1Bp, in the context of class Ib MHC. The tetramer consists of H2-Db, β2-microglobulin and the peptide epitope, and is referred to as Db-E1Bp. First, to test the specificity of this peptide-MHC complex, C57BL/6 (H-2b; B6) mice were injected with 8×108i.u. of wild type (wt) Ad5. As a control, wt Ad5 injected DBA/2 (H-2d; D2) were analyzed. Lymphocytes (105 )isolated 8 days later from spleen or liver were restimulated with E1Bp in vitro and analyzed for IFN-γ production by an ELISPOT assay. There was a significant increase in spots (6 and 63 spots, respectively) compared to cells stimulated with human influenza virus peptide M1(58 GILGFVFTL) (0 spot, p 98%) of the Db-E1Bp+CD8+ T cells are CD44+. No Db-E1Bp+CD8+ T cells were found in immunized D2 or naive B6 mice. To determine peptide-specific lysis of target cells in vivo, we labeled target cells from syngeneic mice with either high (2μM; CFSEHi) or low (0.2μM; CFSELo) concentration of CFSE and then pulsed CFSEHi cells with E1Bp and CFSELo cells remained unpulsed. Target cells were adoptively transferred into B6 or D2 mice that were challenged 8 days earlier with wt Ad5, and the percentage of specific killing was determined 6 hours later. As a peptide specificity control, CFSEHi cells pulsed with M1 peptide and unpulsed CFSELo cells were transferred to identically immunized B6 mice. E1Bp-specific lysis was significantly increased in B6 mice compared to control mice or immunized D2 mice (p<0.001). There was a significant correlation between E1Bp-specific lysis and the frequency of Db-E1Bp+ CD8+ T cells in B6 mice (p<0.01). We next used these methods to measure the decreased Ad-specific CTL response in aged mice. Compared with 2-mo-old mice, there was a 30% decrease in killing efficiency in 18-mo-old mice and a 58% decrease in Db-E1Bp+CD8+ T cells. Our results indicate that this tetramer is novel, sensitive and specific tool to detect and follow the E1Bp-specific CTL response against Ad infection.