The most significant findings in the present study are that human malignant gliomas express EphA2 protein and that EphA2883–891 epitope can induce an antigen-specific, antiglioma CTL response in HLA-A2+ patient-derived PBMCs. These findings strongly support the rationale for targeting EphA2 in peptide-based vaccine trials for patients with AA and GBM.
Our immunohistochemical analyses demonstrated variable levels of EphA2 expression in all high-grade (grade III or IV) gliomas examined. However, unlike many other cancer types in which EphA2 expression levels predict malignant behaviors of cancers [10,15
], expression levels in gliomas did not correlate with the tumor grade, as both AA and GBM cases had variable expression levels and one of two grade II astrocytoma (diffuse fibrillary astrocytoma) cases expressed a high level of EphA2. This may be due to the fact that both AA and GBM are highly malignant tumors with aggressive invasiveness into the surrounding normal brain tissue. Further evaluation with additional low-grade astrocytoma cases and other gliomas, such as ependymoma, is warranted. Nevertheless, EphA2 appeared to be expressed widely in high-grade gliomas and is relatively restricted in tumor tissues as normal brain samples examined had little or no EphA2 expression. In our previous study with a mouse model [16
], EphA2 expression was not detected in the mouse brain based on Western blot analysis, supporting the finding that the EphA2 expression in the central nervous system is restricted to gliomas.
We also demonstrated that EphA2883–891 peptide induced a specific CTL response in HLA-A2+ donor-derived PBMCs in vitro. It was noteworthy that the levels of CTL activity observed in patient-derived PBMCs were as high as those observed in healthy donor-derived PBMCs in most cases, unless the donor patient suffered from clinical lymphopenia (defined as an absolute lymphocyte count < 800 µl-1) due to concurrent chemotherapy and/or radiotherapy. We are currently optimizing the conditions for the fluorescence-conjugated tetramer for EphA2883–891, which will allow us to evaluate the frequency of EphA2883–891, recognizing CD8+ T cells in glioma patients undergoing our perspective vaccine trials.
It has been well recognized that the CTL repertoire against high-affinity epitopes is often partially tolerized [13
], and this mechanism may explain the limited levels of CTL responses against a majority of tumor-associated autoantigens. However, our data employing HHD mice indicated that vaccinations with IFA containing the EphA2883–891
peptide effectively mounted an antigen-specific CTL response that was demonstrated not only against peptide-loaded EL-4-HHD cells but also against EL-4-HHD cells that endogenously express EphA2, suggesting that the EphA2883–891
peptide is capable of inducing a specific CTL response in vivo
with the assistance of effective adjuvants.
There is a theoretical possibility that vaccinations of patients with EphA2-derived peptides can induce autoimmune reactions because of their expression in the normal lung, spleen, and liver [16
]. However, EphA2 is not expressed in mouse brain [16
], and we have not observed any inflammatory pathology in normal organs in HHD mice following vaccinations with the EphA2883–891
(data not shown). This novel CTL epitope may therefore serve as an attractive component of peptide-based vaccines to treat patients with high-grade glioma [3,17
] and as a surrogate marker of T-cell immune responses in patients before and after immunotherapy.