Vaccination with tumor cells pulsed with foreign peptide induces immunity to the tumor itself

Abstract

EMT-6 mammary carcinoma and B16 melanoma (B16M) cells are lethal and barely immunogenic in syngeneic BALB/c and C57BL/6 mice, respectively. We show that mice vaccinated with tumor cells pulsed with a MHC class I-restricted peptide develop a T cell response, not only to the peptide, but also to the unpulsed tumor. These mice display protective immunity against the unpulsed tumor, and their T cells adoptively transfer tumor-specific protection to immunodeficient SCID mice. Our data have implications for cancer vaccine strategies. Grafting a single well-defined foreign peptide on tumor cells might suffice to trigger anti-tumor immunity.

Figure 1

Lethality and immunogenicity of EMT-6 breast cancer cells and B16M cells in syngeneic mice. A, EMT-6 cells were injected i.p. into BALB/c mice (n=16, open squares). B16M cells were injected i.v. into C57BL/6 mice (n=16, open circles). The survival rate of the mice was monitored daily. The data were reproduced in 3 independent experiments. B, BALB/c or C57BL/6 mice were injected with EMT-6 or B16M tumor cells as above or remained uninjected, 4 mice in each group. A third group of mice was immunized with CSp or LCMVp in IFA with CpG oligonucleotide. Fourteen days later, single cell suspensions of spleen cells were tested in an IFN-γ ELISPOT assay as specified under “in vitro recall”. The data shown are the mean values and standard error of the frequencies of IFN-γ-producing cells from duplicate cultures of representative mice. The data were reproduced in three independent experiments.

Figure 2

Pulsing of EMT-6 or B16M cells with foreign antigenic peptide induces protective immunity and determinant spreading to tumor antigens. A, EMT-6 or B16M cells were cultured with CSp or LCMVp and injected into BALB/c (i.p.) or C57BL/6 (i.v.) mice (closed squares or circles), respectively. Unpulsed EMT-6 or B16M cells were injected at the same numbers (open squares or circles). Mice were monitored daily for survival. The survival rates shown are for 4 mice in each group. The data were reproduced in three independent experiments. B, Fourteen days after the injection of peptide-loaded tumor cells, spleen cells of respective mice were tested in IFN-γ ELISPOT assays for the response elicited by the peptide-pulsed and unpulsed tumor as well as by peptide alone. Mean values from duplicate wells are shown for representative mice. The data were reproduced in two independent experiments.

Figure 3

The T cell response engaged by epitope spreading to tumor antigens is protective. EMT-6 (A) or B16M cells (B) were pulsed with CSp or LCMVp and injected into BALB/c or C57BL/6 mice as described in legend to Fig. 2A. Fourteen days later, CD3⁺, CD4⁺ or CD8⁺ subpopulations were isolated at >95% purity by negative selection on affinity columns and were injected into congenic SCID mice as specified by the symbols (the numbers of T cells injected are stated in the text). 24 hours later, the unpulsed tumor was injected, and the survival rate of the recipient mice was monitored. Each group involved 6 mice, and the shown results were reproduced in two separate experiments.

Figure 4

The protection induced by peptide-pulsed tumor cells is tumor-specific. The CD3⁺ cell-grafted BALB/c SCID mice that survived the challenge with EMT-6 tumor in Fig. 3A (closed triangles) and untreated BALB/c SCID mice (open triangles) were injected with a lethal dose of LR lymphoma cells (2 × 10⁵ cells per recipient i.p.). The C57BL/6 SCID mice that survived the challenge with B16M in Fig. 3B were challenged 50 days later with RMA tumor cells (1 × 10³ cells per recipient i.p.) (closed diamonds). Naïve C57BL/6 SCID mice (open diamonds) received the same challenge. Survival of the mice was monitored.