Inducing mammaglobin-A (mamA) specific HLA class I tetramer positive CD8+cytotoxic T-lymphocytes (CTL) against breast cancer by DNA vaccination

Abstract

2522 Background: We recently reported that DNA vaccination using MamA, a highly expressed human (hu) breast cancer specific protein, leads to immunity against breast cancer. Here we demonstrate that the induction of breast cancer immunity following MamA DNA vaccination occurs through the expansion of CD8+ CTL by using HLA class I MamA peptide tetramers. Methods: HLA-A2+huCD8+ double transgenic mice received four (100μg) two-weekly IM injections of PCI-neo vector cloned with hu MamA gene. HLA-A2 tetramers (tet) carrying an immunodominant Mam2.1 peptide (LIYDSSLCDL) were used to monitor development of mam-A specific CD8+ T cells. Specificity of tetramers was tested against CD8+ CTL lines developed in vitro by stimulation of normal hu HLA-A2+ peripheral blood lymphocytes with TAP-2 deficient T2 cells pulsed with pooled MamA HLA-A2 binding peptides. Results: There was a sequential expansion of Mam2.1 tet+CD8+ T cells during stimulation of hu PBLs with MamA peptides in vitro (<1% pre-stimulation to >15% after six stimulations). These Mam2.1 tet+CD8+ T cells were HLA-A2 restricted and MamA specific since they revealed cytotoxicity only towards UACC-822 (HLA-A2+MamA+, 75% lysis) but not MCF-7 (A2+MamA−) or DU-4475 (A2−MamA+) breast cancer cell lines. In contrast, there was no binding of MamA peptide tetramers to control Flu-specific CD8+ CTL lines (<1%). MamA DNA vaccination, but not PCI-neo vector alone, of HLA-A2+huCD8+ mice lead to the expansion of Mam2.1 tet+CD8+ T cells (<0.7% pre-vaccination to >2.5% post-vaccination) in the peripheral blood that revealed in vitro cytotoxicity against UACC-822 (70% lysis) but not against DU-4475 or MCF-7 breast cancer cell lines. Adoptive transfer of both whole splenocytes or fractionated CD8+ T cells from vaccinated mice into immunodeficient SCID-beige mice with previously established subcutaneous hu breast cancer colonies lead to tumor infiltration of tet+CD8+ CTLs and, at 4-weeks, at least 80% tumor regression of UACC-822 but not DU4475 or MCF-7 colonies. Conclusions: MamA DNA vaccination resulted in the expansion of CD8+ CTL. The CD8+ CTL were HLA class I MamA peptide tetramer positive, HLA class I restricted and specifically lysed mamA+ human breast cancer cells both in vitro and in vivo. No significant financial relationships to disclose.