Elevated Frequencies of Self-reactive CD8 + T Cells following Immunization with a Xenoantigen Are Due to the Presence of a Heteroclitic CD4 + T-Cell Helper Epitope Korosh Kianizad, 1 Laura A. Marshall, 1 Natalie Grinshtein, 1 Dannie Bernard, 1 Renate Margl, 1 Sheng Cheng, 1 Friedrich Beermann, 2 Yonghong Wan, 1 and Jonathan Bramson 1 1 Center for Gene Therapeutics, Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada and 2 Swiss Institute for Experimental Cancer Research, Lausanne, Switzerland Abstract Immunization of mice with human dopachrome tautomerase (hDCT) provides greater protection against melanoma than immunization with the murine homologue (mDCT). We mapped the CD8 + and CD4 + T-cell epitopes in both proteins to better understand the mechanisms of the enhanced protection. The dominant CD8 + T-cell epitopes were fully conserved between both proteins, yet immunization with hDCT produced frequencies of CD8 + T cells that were 5- to 10-fold higher than immunization with mDCT. This diffe- rence was not intrinsic to the two proteins because compa- rable frequencies of CD8 + T cells were elicited by both antigens in DCT-deficient mice. Strikingly, only hDCT elicited a significant level of specific CD4 + T cells in wild-type (WT) mice. The murine protein was not devoid of CD4 + T-cell epitopes because immunization of DCT-deficient mice with mDCT resulted in robust CD4 + T-cell immunity directed against two epitopes that were not identified in WT mice. These results suggested that the reduced immunogenicity of mDCT in WT mice may be a function of insufficient CD4 + T-cell help. To address this possibility, the dominant CD4 + T-cell epitope from hDCT was introduced into mDCT. Immunization with the mutated mDCT evoked CD8 + T-cell frequencies and protective immunity comparable with hDCT. These results reveal a novel mechanism by which xenoantigens overcome tolerance. Our data also suggest that immunologic tolerance is more stringent for CD4 + T cells than CD8 + T cells, providing a mechanism of peripheral tolerance where auto- reactive CD8 + T cells fail to be activated due to a lack of autoreactive CD4 + T cells specific for the same antigen. [Cancer Res 2007;67(13):6459–67] Introduction The identification of multiple tumor-associated antigens has greatly spurred interest in developing vaccination strategies for the treatment of cancer. However, most of these antigens are derived from naturally occurring proteins and, as such, immunization strategies must overcome immunologic tolerance, which limits the reactivity of T and B lymphocytes against self-antigen. One strategy that has been used successfully is ‘‘xenoimmunization,’’ where homologous proteins from different species are used as the basis for the vaccine (1–5). Although the mechanism of action of xeno- immunization remains to be fully understood, it is believed that subtle differences between the native and ‘‘xenoprotein’’ can overcome tolerance by eliciting T-cell and B-cell responses against the xenoantigen that cross-react with the native antigen, providing antitumor immunity. Previous investigations into the mechanism(s) of action of xenoimmunization showed that heteroclitic CD8 + T-cell epitopes present in xenoantigens could elicit CD8 + T-cell populations that are reactive to both the xenoantigen and the native antigen (2, 6, 7). Whether heteroclitic CD8 + T-cell epitopes underlie the activity of all xenoantigens is not known. Dopachrome tautomerase (DCT; also known as tyrosinase-related protein-2) is a nonmutated antigen that is widely expressed in melanoma. DCT is a promising candidate for the development of melanoma vaccines as it is naturally recognized by melanoma-specific CTL (8–10). Immuni- zation of mice with human DCT (hDCT) resulted in greater antitumor immunity than immunization with murine DCT (mDCT), providing a useful model for studying the mechanisms of xenoimmunization (11–14). Interestingly, the protective effect of hDCT can be recapitulated by immunization with only the dominant CD8 + T-cell epitope, SVYDFFVWL, which is 100% conserved within the murine sequence (15), showing that CD8 + T-cell immunity can be sufficient in this model. Linkage to strong CD4 + T-cell epitopes was required to produce protective immunity with SVYDFFVWL, suggesting that, rather than providing hetero- clitic CD8 + T-cell epitopes, hDCT may contain CD4 + T-cell epitopes that facilitate activation of the SVYDFFVWL-specific CD8 + T cells (15–17). However, it is also possible that the increased immuno- genicity of hDCT is not due to the SVYDFFVWL epitope. Our previous investigations have shown that both CD4 + and CD8 + T cells can provide protective immunity following immunization with hDCT (11). Therefore, the increased immunogenicity of hDCT could be due to heteroclitic variants of previously undefined epitopes that give rise to effector CD8 + and/or CD4 + T cells. Alternatively, immunization with mDCT may give rise to regulatory T cells as shown recently using a series of self-antigens identified by SEREX (18). In that regard, the increased immunogenicity of hDCT may be due to the absence of epitopes that elicit regulatory T cells. In the current article, we have characterized a series of T-cell epitopes elicited by recombinant adenovirus vaccines expressing DCT. Through combined studies in wild-type (WT) and DCT- deficient mice, we showed that the increased immunogenicity of hDCT relative to mDCT is due to the presence of heteroclitic CD4 + T helper epitopes. These studies also revealed a hierarchical Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Jonathan Bramson, Department of Pathology and Molecular Medicine, McMaster University, Room MDCL-5025, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5. Phone: 905-525-9140; Fax: 905-522-6750; E-mail: bramsonj@mcmaster.ca. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-4336 www.aacrjournals.org 6459 Cancer Res 2007; 67: (13). July 1, 2007 Research Article Research. on March 19, 2016. © 2007 American Association for Cancer cancerres.aacrjournals.org Downloaded from