The effect of T1 and T2 cytokines on the cytotoxic T cell response to mannan-MUC1

Abstract

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.

MUC1-specific immune responses in human MUC1 transgenic mice immunized with various human MUC1 vaccines

“…As with the experiments described above, immunization of transgenic mice resulted in the inhibition of MUC1 + tumour growth. This inhibition could be augmented by the coadministration of cytokines with the vaccine preparation [28].…”

MUC1 immunotherapy against a metastatic mammary adenocarcinoma model: Importance of IFN-gamma

“…A MUC1-GST fusion protein containing 5 variable number of tandem repeat (VNTR) regions from the extracellular protein core of MUC1 [14] was produced in a bacterial expression system (pGEX-3X) and conjugated to oxidised mannan to form MFP as described previously [15][16][17][18][19][20][21][22][23]. BALB/c mice aged 6-10 weeks were given three intraperitoneal immunisations (on days 0, 7 and 14) with either MFP (containing 5μg of MUC1 fusion protein) or a control pH 9.0 phosphate buffer.…”

“…The following monoclonal antibodies were used; a) MUC1 (BC2: supernatant) [28], b) MHC class 1 H2 d (34.1.2s, 1/1000 dilution of ascites fluid) [29], c) MHC class II I-A8 (1/500 dilution of ascites fluid), d) B7.1 (4μg) (Pharmingen, San Diego, USA), e) ICAM-2 (1μg) (Pharmingen), f) CD28 (4μg) (Pharmingen), g) LFA-2 (1μg) (Pharmingen) and h) CTLA-4 (1μg) (Pharmingen). DA3-MUC1 tumour cells were prepared for FACS analysis by either a) culturing in growth media, b) culturing with 20 ng/ml vaccinia virus-IFN-γ [22] for 72 h, or c) culturing with 20 ng/ml IFN-γ for 72 h and then removing IFN-γ for subsequent culturing. In preparation for flow cytometry, tumour cells (2-5 x 10 5 cells/ml) were incubated with the specified antibodies for 45 min at 4 0 C, washed with phosphate buffer and incubated with either FITC-conjugated sheep (Fab')2 anti-mouse, anti-rat or anti-hamster immunoglobulin (Amersham, UK) (1/50 dilution) for a further 45 min at 4 0 C. Cells were washed and analysed by flow cytometry.…”

“…Prior to challenge, the tumour cells were cultured with 20 ng/ml vaccinia virus-IFNγ supernatant (UV inactivated) [22,23,25,26] for 72 h to increase cell surface MHC class I expression. Tumour growth was measured every 2-3 days for 2 weeks using electronic callipers.…”

Generation of cellular immune responses to antigenic tumor peptides

“…CTL responses were enhanced by using mannan-MUC1 with cyclophosphamide [130]. In addition, adjuvants such as GMDP, MDP and incomplete Freund's adjuvant can also increase the CTLp frequency [125], as did the cytokines IL-4 +k-IFN, IL-2+k-IFN and IL-12 [131,132]. However, one of the most potent modes of immunization is the in vitro targeting of mannose receptorbearing cells [133].…”