Spleen cells from young but not old immunized mice eradicate large established cancers

Abstract

Purpose: Solid tumors that have grown two weeks or longer in mice and have diameters larger than 1 cm are histologically indistinguishable from autochthonous human cancers. When experimental tumors reach this clinically relevant size, they are usually refractory to most immunotherapies but may be destroyed by adoptive T-cell transfer. However, TCR-transgenic T cells and/or tumor cells overexpressing antigens are frequently used in these experiments. Here we studied the requirements for destroying clinical size, unmanipulated 8101 tumors by adoptive cell therapy.

Experimental design: 8101 arose in an old mouse after chronic exposure to UV light. A cancer line was established, which was never serially transplanted. The immunodominant CD8(+) T cell-recognized antigen of this tumor is caused by a somatic tumor-specific mutation in the RNA helicase p68. 8101 tumors were treated with spleen cells from young naive, or young and old immunized mice to ascertain the characteristics of immune cells that lead to rejection.

Results: Here we show that the mutant p68 peptide has an exceptionally high affinity to the presenting MHC class I molecule K(b) and that spleen cells from immunized young syngeneic mice adoptively transferred to Rag(-/-) or cancer-suppressed euthymic mice eradicate 8101 tumors larger than 1 cm in average diameter and established for several weeks. Spleen cells from naive young mice or from old and boosted (reimmunized) mice were ineffective.

Conclusions: Relapse-free destruction of large and long-established tumors expressing a genuine very high-affinity tumor-specific antigen can be achieved by using adoptive transfer of lymphocytes from immunized young individuals.

Figure 1

Cancer progressor variants are selected by young but not old naïve mice. A. Experimental design. Cryopreserved fragments of the autochthonous 8101 tumor were first adapted to culture and then injected into a nude C57BL/6 mouse that developed a tumor. Fragments of this tumor were transplanted into one old (2 year-old) and twenty young (2-3 month-old) normal euthymic C57BL/6 mice. Five of the twenty young and the old mouse failed to reject the tumor challenge. B. Analysis of the transplant behavior of each tumor found to be progressively growing in A by fragment transplantation into a new set of young naïve mice. Every tumor that had developed in a young naïve mouse grew again, whereas the tumor that grew in the old mouse was rejected.

Figure 2

Adoptive transfer of spleen cells from young immune but not naïve or old immune mice leads to the eradication of large established 8101 tumors in Rag1^-/-mice. A. Experimental design. B. Tumor-bearing Rag1^-/- C57BL/6 mice were treated by adoptive transfer of spleen cells (one spleen per recipient, around 1×10⁸ cells) from young naïve donors (3-4 month-old), young donors (3-4 month-old) immunized once with 2×10⁷ live 8101 cancer cells at the age of 2 months, or old immune mice (29 month-old) immunized when 4 month-old and boosted 2, 12 and 19 months later. Results are pooled from several experiments, one of which is sharing all three experimental groups and two sharing the young naïve and young immune groups. The ⊕ symbol in the right panel designates a tumor that was reisolated and found to express the mutant p68 gene by RT-PCR. * The average tumor size and duration of growth of 8101 (mean ± SD) at time of treatment was: 1117 ± 339 mm³ and 41 ± 11 d for the “naïve young” group; 1219 ± 315 mm³ and 39 ± 7 d for the “young immune”; 1203 ± 961 mm³ and 30 ± 6 d for the “old immune”.

Figure 3

Adoptive transfer of spleen cells from young immune but not naïve or old immune mice leads to the eradication of large 8101 tumors grown in euthymic B6C3F1 mice. A. Experimental design. B. 8101-bearing euthymic B6C3F1 mice were treated by adoptive transfer of spleen cells (one spleen per recipient, around 1×10⁸ cells) from young naïve donors (3-4 month-old), young donors (4-8 month-old) immunized once with 2×10⁷ live 8101 cancer cells 2 months before transfer, or old immune mice (9-16 month-old) immunized when 2-3 month-old and boosted 2 months before transfer. Results are pooled from several experiments, one sharing all three experimental groups and two sharing the young naïve and young immune group. The ⊕ symbols in the right and left panels designate tumors that were reisolated and found to express the mutant p68 gene by RT-PCR; the antigen-loss variant is designated with Ø. ^a The pre-existent PRO4L tumor burden at time of 8101 inoculation was 539 ± 165 mm³ (mean ± SD) and had grown for an average of 21 ± 8 days. ^b The average size of PRO4L was 1577 ± 988 mm³ when strung at day 38 ± 10 of growth 8101 had grown for an average of 18 ± 10 days when the PRO4L tumor burden was removed. ^c The average tumor size and duration of growth of 8101 at time of treatment was: 738 ± 206 mm³ and 19 ± 6 d for the “naïve young” group; 845 ± 328 mm³ and 29 ± 9 d for the “young immune”; 909 ± 286 mm³ and 28 ± 19 d for the “old immune”.

Figure 4

Old and young mice have similar numbers of mp68-specific CD8⁺ T cells but differ in number of CD4⁺ T cells and in the ability to increase the percentage of effector memory cells after boosting. A. Peripheral blood cells were isolated from naïve and immune young or old mice (5 mice per group) and the binding of mp68 peptide-loaded tetramers to CD8⁺ T cells was measured. The young (6 month-old) immune mice had been primed once at the age of 2 months whereas the old (16 month-old) immune mice had been primed at 2 months of age and boosted at 5 and 12 months of age. Day 0 of analysis corresponds to 4 months after immunization/last boosting respectively. The results are representative for 3 mice each, for young and old. M-mp68 is a cell line transfected to express very high levels of mp68 antigen. B. Absolute numbers of CD4⁺ and CD8⁺ T cells were determined in peripheral blood from old (14 month-old) and young (4 month-old) mice. An experiment representative of two is shown with data from 5 mice per group. C. The percentages of central memory (CM: CD62L^hi/CD44^hi), and effector memory (EM: CD62L^lo/CD44^hi) CD8⁺ T cells were determined in peripheral blood from old (16 month-old) and young (6 month-old) mice before (pre) and on day 9 after boosting (post) as in A. 4-5 mice per group were analyzed in total in two experiments pooled here. *p < 0.05; **p ≤ 0.01; ns, no significant.