MUC1 is a mucin over-expressed in breast cancer and a proposed target for immunotherapy. By immunising mice with MUC1 conjugated to mannan (M-FP), CD8(+) MHC-class-I restricted cytotoxic T lymphocytes (CTL), of high CTL precursor (CTLp) frequency (1/8000) and with significant tumour protection, can be induced. The effect of various cytokines [interleukin-2 (IL-2), IL-4, IL-6, IL-7, interferon gamma (IFNgamma), and granulocyte/macrophage-colony-stimulating factor (GM-CSF)] on the MUC1 CTL immune response was investigated (a) by measuring the frequencies of CTLp in mice immunised with vaccinia virus constructs containing recombinant cytokines and M-FP, or (b) by immunising cytokine- or cytokine-receptor-knockout (-/-) mice with M-FP. Vaccinia virus (VV) constructs containing recombinant cytokines were used either individually or in combination in vivo with M-FP immunisation. M-FP immunisations combined with VV-IL-2, VV-IL-7 and VV-GM-CSF, and combinations of VV-IFNgamma + VV-IL-2, VV-IFNgamma + VV-IL-4 or VV-GM-CSF + VV-IL-7 increased CTLp frequencies up to threefold (1/17 666: M-FP + VV-GM-CSF + VV-IL-7) compared to M-FP (1/77 500) alone. By contrast, M-FP combined with VV-IL-4 decreased the CTLp frequency threefold whereas VV-IL-6 and VV-IFNgamma had no effect. Studies in cytokine- and cytokine-receptor-gene-knockout (-/-) mice demonstrated that mice that are IL-2 -/- and IL-7 receptor -/- produce the same CTLp response to M-FP as do control mice, whereas responses in the IL-6 -/-, IL-10 -/- and IFNgamma -/- mice were marginally improved and responses to M-FP in IL-4 -/- and tumour necrosis factor receptor 2 -/- mice were weaker. In spite of the increase in CTLp frequency, this was not reflected in an in vivo tumour model. Tumour challenges using MUC1(+) P815 cells, demonstrated that the addition of cytokines had little additive effect on the already effective tumour-regression capabilities of M-FP alone.

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Immunotherapy with oxidized mannan-MUC1 fusion protein (M-FP) leads to a T1 immune response characterized by the generation of cytotoxic T lymphocytes (CTL), few antibodies, secretion of interleukin-2 (IL-2), IL-12, and interferon-gamma and tumor protection. Immunotherapy with reduced M-FP or fusion protein (FP) alone leads to a T2 immune response characterized by the generation of MUC1 antibodies, few CTL, IL-4 secretion, and no tumor protection. In these studies, cytokine production from T cells was measured from cultures containing whole spleens. We now report the cytokine secretion patterns from spleen cells separated into CD4+ and CD8+ T cells obtained from mice immunized with either oxidized M-FP, reduced M-FP or FP, or the simultaneous administration of oxidized M-FP and FP. Immunization with oxidized M-FP led to the secretion of T1 cytokines from CD8+ T cells (IL-2, IFN-gamma, and tumor necrosis factor-alpha [TNF-alpha]) and from CD4+ T cells (IL-2 and IFN-gamma). IL-12 production, presumably from activated macrophages, was observed in CD8+ but not CD4+ cultures. Immunization with either reduced M-FP or FP led to the secretion of predominantly T2 cytokines from CD4+ T cells (IL-4 and IL-10) and IL-2 production in both CD4+ and CD8+ T cell cultures. The simultaneous immunization of both oxidized M-FP and FP led to the production of both T1 and T2 cytokines from CD8+ T cells (IL-2, IFN-gamma, and TNF-alpha) and CD4+ cells (IL-2, IFN-gamma, IL-4, and IL-10) and IL-12 production in CD8+ cultures that is, both types of immune responses could occur together. The results demonstrate that the cellular immune response observed in oxidized M-FP-immunized mice is indeed dependent on the T1 cytokine profile secreted by CD8+ T cells, and the simultaneous production of both T1 and T2 cytokines is not cross-inhibitory.

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