Analysis of CD8 T-cell response by IFNγ ELISPOT and H-2Ld/pRL1a tetramer assays in pRL1a multiple antigen peptide-immunized and RL male 1-bearing BALB/c and (BALB/c × C57BL/6)F1 mice

Abstract

We previously identified an H-2Ld-binding pepticle pRL1a (IP-GLPLSL) on RL male 1 that is predominantly recognized by cytotoxic T-lymphocytes (CTLs). MAP is a multibranched lysine core with antigenic peptides. Immunization of BALB/c mice with pRL1a MAP effectively induced pRL1a CTLs. Here, we demonstrate the presence of pRL1a-recognizing CD8+ T-cells in pRL1a MAP-immunized and RL male 1-bearing BALB/c and (BALB/c×C57BL/6)F1 mice by using IFNγ ELISPOT and H-2Ld/pRL1a tetramer assays. A few IFNγ ELISPOTs and no tetramer-positive cells were detected ex vivo in spleen cells from BALB/c mice immunized with pRL1a MAP. After a single in vitro stimulation with RL male 1, 432 and 741 IFNγ ELISPOTs/105 cells were detected and tetramer-positive CD8+ T-cells occurred at relative frequencies of 5.7% and 30.8% in splenic CD8+ T-cells from mice that had been doubly and triply immunized, respectively, against pRL1a MAP. Tetramer-positive cells displayed two distinct cell populations, CD62Llow and CD62Lhigh. Secondary in vitro stimulation expanded CD62Lhigh cells more efficiently than CD62Llow cells. Furthermore, a higher frequency of IFNγ-producing and tetramer-positive CD8+ T-cells was detected ex vivo in RL male 1-bearing semi-allogeneic (BALB/c×C57BL/6)F1 than in BALB/c mice on day 14 after tumor inoculation.