The biological effects of syngeneic and allogeneic cytokine-expressing prophylactic whole cell vaccines and the influence of irradiation in a murine melanoma model

Abstract

Allogeneic whole tumour cell vaccines are inherently practical compared with autologous vaccines. Cell lines are derived from allogeneic tumour, grown in bulk and then administered as a vaccine to the patient, following irradiation, which not only prevents any replication but also enhances antigen presentation. Protection is believed to occur through the presentation of antigens shared between the syngeneic and allogeneic tumours. Although cytokine-transfected tumour whole cell vaccines have been used clinically, little data is available comparing the effects of immunomodulatory cytokine-transfection directly on the same cells when used as both an allogeneic and autologous vaccine. To address this, weakly immunogenic B16-F10 (H-2b) murine melanoma was transfected to secrete either GM-CSF, IL-4 or IL-7. Prophylactic vaccination of both syngeneic C57/BL6 (H-2b) (B6) and allogeneic C3H/Hej (H-2k) (C3H) mice showed the effects of transfected cytokine varied between models. Both GM-CSF and IL-7 significantly (P<0.05) increased the levels of protection within syngeneic B6 mice, but had a diminished effect (P>0.05) within C3H allogeneic mice. Allogeneic B16-F10 cells and syngeneic K1735 cells generated CTL against K1735 suggesting cross-reactive immunity. Using cells labeled with fluorescent dye we demonstrate that irradiated vaccines, of either syngeneic or allogeneic origin, appear to generate potent immune responses and fragments of either vaccine remain at the injection site for up to 9 days. This study shows that protection can be enhanced in vivo by using transfected cytokine, but suggests that irradiated whole cell vaccines, of either tissue-type, are rapidly processed. This leads to the conclusion that the cytokine effects are transient and thus transfection with cytokine may be of limited long-term use in situ.

Fig. 1

Kaplan-Meyer survival curves showing cytokine transfection of B16-F10 cells (H-2^b) significantly increases the levels of protection in syngeneic B6 (H-2^b) mice (a) but not in allogeneic mice C3H (H-2^k) (b). Mice (five per group) were vaccinated with 2 s.c. doses of 100 Gy irradiated B16-F10 cells transfected with various cytokine (shown in legend), and challenged with a tumourigenic dose of live syngeneic tumour 7 days post-boost. Data is representative of three independent experiments. Results are summarised in the table below the graphs. **P <0.05 when compared to B16-F10 wild type immunised group

Fig. 2

Kaplan-Meyer survival curves showing K1735 allogeneic vaccine inducing melanoma-specific cross reactive immunity in female B6 mice. Animals, five per group, were vaccinated with three 1×10⁶ s.c. doses of either K1735, B16-F10 or PMC-1 prostate cells (all irradiated, shown in legend), and challenged with 5×10⁴ live B16-F10 cells 7 days after final immunisation. A control group immunised with saline alone is also shown

Fig. 3

Kinetics of elimination of vaccine cells. B6 mice were vaccinated with PKH2 labelled cells subcutaneously in the flank. The injection sites were excised, cell suspensions prepared and the fraction of green fluorescent cells determined by flow cytometry at different time points after injection. Samples were tested prior to injection (pre), and at three time points after administration (0, 2 and 7 days). At the same time, cells obtained from the excised injection sites were stained with monoclonal antibodies specific for different cell surface markers and analysed at different time points after injection. Cells carrying the following surface markers were determined: H-2K^b (open square), I-A^b (filled square), CD80 (open circle), CD86 (filled circle). Values are representative of two experiments. a Unirradiated B16-F10 (syngeneic), b Unirradiated K1735 (allogeneic), c Irradiated B16-F10 (syngeneic), d Irradiated K1735 (allogeneic)

Fig. 4

In situ demonstration of the kinetics of elimination of syngeneic or allogeneic vaccine cells from the injection site. B6 mice were inoculated with PKH2-labelled irradiated B16-F10 or K1735 cells subcutaneously behind the right forelimb. The injection sites were excised at different time points after injection, and the tissue samples were embedded in paraffin before freezing. Frozen tissues were cut with a cryomicrotome to 5 μm slices. Consecutive slices were examined directly for PKH-2 fluorescence on day 1, 3 and 9 (Magnification×40)

Fig. 5

In situ demonstration of fragments of the injected tumour cells in the draining lymph nodes. B6 mice were inoculated with PKH2-labelled irradiated B16-F10 or K1735 cells subcutaneously behind the right forelimb. The axillary lymph nodes were excised at different time points after injection, and the tissues samples were embedded in paraffin before freezing. Frozen tissues were cut with a cryomicrotome to 5 μm slices. Slices were examined directly for PKH-2 fluorescence on day 1, 3 and 9. (Magnification×40)

Fig. 6

Vaccination with cytokine-transfected B16-F10 induces anti-tumour CTL activity. Splenocytes were taken 8 days after the second vaccination from either syngeneic B6 (a, b) or allogeneic C3H (c, d) mice and stimulated for 5 days with irradiated (50 Gy) syngeneic tumour. Stimulated splenocytes were then analysed for either CTL activity against tumour cells syngeneic for the host mouse strain (a, c), or for NK-like activity against P815 murine mastocytoma cells (b, d) using a standard 4 h ⁵¹Cr release assay. The mean specific-percentage lysis is shown for triplicate samples, except for wild type vaccinated C3H which was tested in duplicate