CD4+ and CD8+ T cells can act separately in tumour rejection after immunization with murine pneumotropic virus chimeric Her2/neu virus-like particles

Abstract

Background: Immunization with murine pneumotropic virus virus-like particles carrying Her2/neu (Her2MPtVLPs) prevents tumour outgrowth in mice when given prophylactically, and therapeutically if combined with the adjuvant CpG. We investigated which components of the immune system are involved in tumour rejection, and whether long-term immunological memory can be obtained.

Methodology and results: During the effector phase in BALB/c mice, only depletion of CD4+ and CD8+ in combination, with or without NK cells, completely abrogated tumour protection. Depletion of single CD4+, CD8+ or NK cell populations only had minor effects. During the immunization/induction phase, combined depletion of CD4+ and CD8+ cells abolished protection, while depletion of each individual subset had no or negligible effect. When tumour rejection was studied in knock-out mice with a C57Bl/6 background, protection was lost in CD4-/-CD8-/- and CD4-/-, but not in CD8-/- mice. In contrast, when normal C57Bl/6 mice were depleted of different cell types, protection was lost irrespective of whether only CD4+, only CD8+, or CD4+ and CD8+ cells in combination were eradicated. No anti-Her2/neu antibodies were detected but a Her2/neu-specific IFNgamma response was seen. Studies of long-term memory showed that BALB/c mice could be protected against tumour development when immunized together with CpG as long as ten weeks before challenge.

Conclusion: Her2MPtVLP immunization is efficient in stimulating several compartments of the immune system, and induces an efficient immune response including long-term memory. In addition, when depleting mice of isolated cellular compartments, tumour protection is not as efficiently abolished as when depleting several immune compartments together.

Figure 1. Tumour rejection following depletion of immune cells in the induction phase in BALB/c mice.

Mice immunized with Her2MPtVLPs were depleted of CD4⁺ cells (•), CD8⁺ cells (*), or both cell types (O), 4 days and 1 day before immunization, and 2 days after immunization, respectively. Unimmunized mice (x) and non-depleted Her2MPtVLP immunized mice (Δ) were included as negative and positive controls respectively. Mice were challenged with 5×10⁴ D2F2/E2 cells 2 weeks after immunization.

Figure 2. IFNγ responses following depletion of immune cells in BALB/c mice.

Mice were depleted of CD4⁺ and/or CD8⁺ cells 5 and 2 days before immunisation with Her2MPtVLPs. The IFNγ response was measured 7 days later by stimulating splenocytes with the immunodominant CD8⁺ peptide Her2_63–71 or the control peptide NP_118–126. Average values of triplicates from six animals are shown. Background values (stimulation in the absence of peptide) have been subtracted. Error bars represent S.E.

Figure 3. Surface expression of Her2/neu on EL4-Her2 cells.

Surface expression of human Her2/neu on EL4 (A), and EL4-Her2 cells (B), analysed by flow cytometry using the anti-Her2/neu-PE antibody (solid line) or relevant isotype control (dashed line).

Figure 4. Induction of long-term tumour rejection responses.

BALB/c mice were immunized with Her2MPtVLPs alone (*) or Her2MPtVLPs in combination with CpG (Δ) and challenged 6 weeks later with 5×10⁴ D2F2/E2 cells. Unimmunized mice (x) and mice immunized with MPtVLPs and CpG (•) were included as negative controls, while mice immunized with Her2MPtVLPs 2 weeks prior to challenge were used as positive controls (□).

Figure 5. Long-term IFNγ responses after Her2MPtVLP immunization.

BALB/c mice were immunized with Her2MPtVLPs with CpG either 10 weeks (a), 6 weeks (b) or 1 week (d) before ELISPOT analysis, or with Her2MPtVLPs without CpG 6 weeks (c) or 1 week (e) before ELISPOT. Unimmunized mice were included as negative controls (f). Splenocytes were stimulated with the immunodominant CD8⁺ peptide Her2_63–71 or the control peptide NP_118–126. Average values of triplicates from 4 animals are shown. Background values (stimulation in the absence of peptide) have been subtracted. Error bars represent S.E.