Analysis of CD8 T-cell response by IFNgamma ELISPOT and H-2L(d)/pRL1a tetramer assays in pRL1a multiple antigen peptide-immunized and RL male 1-bearing BALB/c and (BALB/c x C57BL/6) F(1) mice.

We previously identified an H-2L(d)-binding peptide pRL1a (IPGLPLSL) 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 x C57BL/6)F(1) mice by using IFNgamma ELISPOT and H-2L(d)/pRL1a tetramer assays. A few IFNgamma 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 IFNgamma ELISPOTs/10(5) 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, CD62L(low) and CD62L(high). Secondary in vitro stimulation expanded CD62L(high) cells more efficiently than CD62L(low) cells. Furthermore, a higher frequency of IFNgamma-producing and tetramer-positive CD8(+) T-cells was detected ex vivo in RL male 1-bearing semi-allogeneic (BALB/c x C57BL/6)F(1) than in BALB/c mice on day 14 after tumor inoculation.