HER2 Peptide-Specific CD8 T Cells Are Proportionally Detectable Long After

Gene Therapy (2002) 9, 879–888 2002 Nature Publishing Group All rights reserved 0969-7128/02 $25.00 www.nature.com/gt RESEARCH ARTICLE HER2 peptide-specific CD8+ T cells are proportionally detectable long after multiple DNA vaccinations K Mukai1, Y Yasutomi2, M Watanabe1, A Kenjo3, T Aota1, L Wang1, H Nishikawa1, M Ishihara1, T Fujita3, K Kuribayashi2 and H Shiku1 1Second Department of Internal Medicine, Mie University School of Medicine, Mie, Japan; 2Department of Bioregulation, Mie University School of Medicine, Mie, Japan; and 3Department of Biochemistry, Fukushima Medical University, Fukushima, Japan We prepared a plasmid encoding 147 amino acid residues In hosts receiving more than three immunizations, surpris- from the N terminus of c-erbB-2/HER2/neu (HER2), which ingly high numbers of specific CD8+ T cells were persistently included both a cytotoxic T lymphocyte (CTL) epitope detectable. HER2 protein-specific antibodies of IgG class (HER2p63) and a helper epitope (HER2p1), using the mam- with dominance of IgG2a remain detectable 6 months after malian expression vector pCAGGS-New (pCAGGS147 single or multiple immunizations. The antibodies however, HER2). In a parallel analysis with a Tetramer assay and CTL were not reactive with cell surface HER2 antigens. Total assay, good specificity and sensitivity of a quantitative suppression of tumor growth was observed when syngeneic enzyme-linked immunospot (ELISPOT) assay to detect func- HER2+ tumor cells (2 × 106) were injected subcutaneously tional HER2p63-specific CD8+ T cells were demonstrated 14 days after a single immunization with pCAGGS147HER2. after intramuscular immunization of pCAGGS147HER2. In Furthermore, the number of pulmonary metastases an ELISPOT assay for HER2p63, spots of IFN␥-producing decreased significantly when DNA vaccination was initiated cells were first detected 10 days after the first immunization, on the day of, or 3 days after, intravenous injection (1 × and additional immunizations increased the number of 106 cells). spots. HER2p63-specific CD8+ T cells were detected over a Gene Therapy (2002) 9, 879–888. doi:10.1038/sj.gt.3301707 period of more than 10 months after the last immunization. Keywords: DNA vaccine; c-erbB-2/HER2/neu; CTL; CD8+ T cells Introduction for in vivo tumor-rejection immune responses.9–11 HLA- A2402, which is expressed by more than 60% of the DNA vaccines are capable of inducing potent cellular and Japanese population and also by a significant proportion humoral immunity in a variety of experimental systems of individuals in other countries, shares a similar peptide- (reviewed in Ref. 1). One of the characteristic features of binding motif with murine MHC class I, Kd. Indeed, we DNA vaccines is their ability to induce lasting immunity. have recently demonstrated that the human homolog of In many systems in mice, antibodies against molecules HER2p63 (TYLPTNASL; one amino acid difference to the encoded by cDNA in plasmids remain detectable for murine peptide) generated CTLs that could lyse HER2- 2–4 more than 1 year after the last immunization. This can expressing tumors in the context of HLA-A2402.12 be explained by persistent antigen expression by the HER2p63 peptides presented by two different MHC class DNA-transduced cells, including professional antigen- I molecules, murine Kd and HLA-A2402, provide us with presenting cells such as dendritic cells, and by persistent a unique system for developing experimental cancer vac- 5,6 memory cells. This is an extremely important feature cine models that could precede their clinical application. beneficial for constructing vaccines against a variety of + + It is becoming clear that CD4 T cells play essential infectious agents. While DNA vaccines induce CD8 cyto- roles in the efficient generation of CD8+ CTL.13–16 We toxic T lymphocytes (CTL) specific for peptides derived therefore prepared a plasmid construct encoding the N- from plasmid-encoded molecules, detailed analysis of the + terminal 147 amino acids of human HER2, which kinetics of CD8 T cells induced by vaccinations is still encompass a HER2p63 CTL epitope and a HER2 p1 7,8 limited. helper epitope (Wang et al, manuscript in preparation). We have recently demonstrated in a murine syngeneic We analyzed detailed kinetics of the induction and main- tumor system that 9mer peptide, murine HER2p63 + + tenance of HER2p63-specific CD8 T cells as a conse- (TYLPANASL), can induce CD8 CTLs with MHC class quence of single or multiple immunizations. I, Kd restriction and more importantly, can be a target Results Dose dependence of immune responses induced by Correspondence: H Shiku, Second Department of Internal Medicine, Mie plasmids encoding 147 amino acid residues from the N University School of Medicine, 2-174 Edobashi, Tsu, Mie, Japan 514-8507 terminus of HER2 (pCAGGS147HER2) Received 18 October 2001; accepted 22 February 2002 To determine the appropriate dose that could elicit the immune response to HER2p63 peptide by intramuscular Kinetics of CD8+ T cells induced by DNA vaccination K Mukai et al 880 Figure 1 Immune response specific for HER2p63 essentially correlates with the injection doses of pCAGGS147HER2 plasmids encoding 147 amino acid residues from the N terminus of HER2. Two BALB/c mice in each group were injected once with varied doses of pCAGGS147HER2. After vaccination, CD8+ T lymphocytes were quantitatively analyzed by ELISPOT assay. Pooled effector cells derived from two spleens of each group (1 × 105) were incubated with same number of peptide-pulsed P1.HTR for 20 h. HER2p63 and a control HER2p780 peptides were used. Data are mean ± s.d. of triplicate wells. Two additional experiments yielded similar results. injection of pCAGGS147HER2, we titrated the injection dose of pCAGGS147HER2 in mice and measured HER2- specific immune responses by ELISPOT assay to count interferon gamma (IFN␥) producing cells. BALB/c mice were immunized intramuscularly (i.m.) with pCAGGS147HER2 and their spleens were obtained 14days later, and CD8+ T cells were purified as described in Materials and methods. The number of HER2p63-specific CD8+ T cells in these spleen cells increased in a pCAGGS147HER2 dose-dependent manner, and reached a plateau with immunization of 100 ␮g DNA (Figure 1). The IFN␥ spots detected in the assay were antigen-specific, since only a marginal number of spots could be observed when target cells pulsed with a control Kd binding peptide (p780) were used. We therefore used the immunization dose of 100 ␮g in the following experiments. HER2p63-specific CD8+ T cells are quantitatively measurable with ELISPOT assay and Tetramer assay Mice were immunized i.m. twice at a 3-week interval, with pCAGGS147HER2. Spleen cells were obtained from animals 3 weeks after the second immunization and analyzed for HER2p63-specific CD8+ T cells with ELISPOT and Tetramer assays. A portion of splenocytes was sensitized in vitro with mitomycin C (MMC)-treated HER2- expressing CMS17HE cells shown in Figure 2 and they were similarly analyzed with the two assays. In the ELIS- POT assay, about 2 × 102 HER2p63-specific CD8+ T cells were detected in 105 CD8+ T cells (~0.2% of total CD8+ T cells; Figure 3a) before in vitro sensitization, and about 1 × 102 HER2 peptide-specific CD8+ T cells were detected in 103 CD8+ T cells (~10% of total CD8+ T cells; Figure 3d) after in vitro sensitization. A marginal number of spots could be observed when target cells pulsed with a control Kd binding peptide (9wt) were used. In the Tetramer assay, it was difficult to detect HER2p63-specific CD8+ T cells with significance before Figure 2 Three mouse fibrosarcoma cell lines transfected by HER2 cDNA in vitro sensitization (Figure 3b). However, their number express HER2 protein on their surface. Cell lines were described in Materials and methods. Shaded profiles, cells stained with secondary Ab; considerably increased after in vitro sensitization as open profiles, cells stained with anti HER2 monoclonal antibody, Trastu- d shown in Figure 3e. A control Tetramer with a control K zumab. In each panel, the ordinate represents the number of cells and the binding 9wt peptide did not respond to these splenocytes abscissa is the fluorescence intensity in logarithmic arbitrary units. Gene Therapy Kinetics of CD8+ T cells induced by DNA vaccination K Mukai et al 881 Figure 3 The ELISPOT and Tetramer assays provided comparative enumeration of HER2p63-specific CD8+ T cells induced by DNA vaccination. Mice were immunized i.m. twice at a 3-week interval with 100 ␮g pCAGGS147HER2. Spleen cells were sensitized in vitro with MMC-treated CMS17HE cells as described in Materials and methods. ELISPOT (a, d) and Tetramer (b, c, e, f) assays were performed before (a, b, c) or after (d, e, f) in vitro sensitization. In the ELISPOT assay, effector cells (a, 1 × 105, before sensitization; d, 1 × 103, after sensitization) were incubated with 1 × 105 peptide- pulsed P1.HTR cells for 20 h. HER2p63 and a control 9wt peptides were used. Data are for pooled effector cells of two spleens each and are mean ± s.d. of triplicate wells. In the Tetramer assay, CD8+ cells from the spleen cells were stained with Kd/HER2p63 TetramerPE and anti-CD8FITC (b, e) or Kd/9wt TetramerPE and anti-CD8FITC (c, f). Before in vitro sensitization, to improve the sensitivity of the experiment, CD8+ cells were positively enriched using a miniMACS device. One additional experiment yielded similar results. either before (Figure 3c) or after (Figure 3f) in vitro sensit- these animals were quantitatively analyzed by the ELIS- ization. Following in vitro sensitization, the frequencies POT assay. HER2p63-specific CD8+ T cells became detect- of HER2p63-specific CD8+ T cells detected with the ELIS- able 10 days after the first immunization (data not POT assay were essentially in the same range as those shown), and reached a level of 0.1% of the total CD8+ T determined by the Tetramer assay.

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