Xenogeneic, but Not Syngeneic, TRP-2 DNA Induces Tumor Rejection.
hTRP-2 has 90% homology and 83% identity to the amino acid sequence of C57BL/6 mTRP-2. DNA immunization with xenogeneic hTRP-2 decreased B16F10/LM3 lung metastases by ≥90% (P < 0.0001) in tumor protection experiments (). There was no significant evidence of tumor immunity after immunization with syngeneic mTRP-2 DNA compared with untreated mice or mice injected with control null vector ( and ).
Figure 1 Protection and rejection of mouse melanoma B16F10/LM3 after immunization with human TRP-2 (hTRP-2), but not mouse TRP-2 (mTRP-2) DNA. C57BL/6 mice, 10–12 per group, were immunized cutaneously with hTRP-2 or mTRP-2 DNA by particle bombardment. (more ...)
To assess the potency of DNA immunization using xenogeneic hTRP-2 DNA, mice were immunized 4 d after tumor challenge or 10 d after tumor challenge, when lung metastases were numerous and macroscopic. Immunization at 4 d decreased metastases by >80% (P < 0.001; D). Therapeutic effects were observed 10 d after tumor challenge using immunization with hTRP-2 DNA plus recombinant mouse GM-CSF DNA as an immune adjuvant. Vaccination significantly decreased lung metastases by approximately half (P = 0.004; E). No significant decrease in lung metastases was observed after treatment with hTRP-2 or mTRP-2 DNA, or GM-CSF DNA alone ( E, see legend), although there was a trend towards decreased metastases with GM-CSF alone that did not reach significance (P > 0.05). These results showed a requirement for xenogeneic antigen and the adjuvant effect of GM-CSF in the treatment of established tumors.
Xenogeneic hTRP-2 DNA Vaccination Induces Autoantibodies and Autoreactive CTLs.
We next determined whether immunization with mTRP-2 or xenogeneic hTRP-2 generated antibody and CD8+ T cell responses against syngeneic mTRP-2. 6 of 12 mice immunized with hTRP-2 had detectable IgG antibodies (IgG1 and IgG2b isotype) against mTRP-2 (data not shown). No autoantibodies against syngeneic mouse TRP-2 were generated after immunization with mTRP-2 (0/12). Generation of autoantibodies after immunization with hTRP-2 required both CD4+ and CD8+ T cells, because no autoantibodies were detected in mice deficient in MHC class I (0/11) or II molecules (0/10).
CTL responses against TRP-2 were detected after immunization with xenogeneic hTRP-2, but not syngeneic mTRP-2 DNA. Specifically, CD8+ CTL from draining lymph nodes (supraclavicular nodes), stimulated in vitro for 5 d, recognized an MHC class I H-2Kb–restricted peptide of mTRP-2 after immunization with hTRP-2 DNA ( ). Interestingly, the H-2Kb–restricted peptide of mTRP-2, mTRP-2181–188, is identical between mouse and human TRP-2, including the immediate flanking amino acid residues. Thus, this self-peptide in the context of self–TRP-2 DNA does not induce CTL responses, but presentation of the same peptide in the context of xenogeneic hTRP-2 is immunogenic.
Figure 2 CTL response to TRP-2181–188 peptide in mice that were not immunized (A), or immunized with mTRP-2 (B) or hTRP-2 (C). C57BL/6 mice (2 or 3 per group) were immunized as described in the legend to . 7 d after the last immunization, draining (more ...)
Tumor Rejection Requires CD4+ and CD8+ Cells, but Not B Cells or NK Cells.
These results suggested that either antibody or CTL responses, or both, mediated tumor rejection. Roles for critical cell types were investigated by immunizing β2m−/−
mice deficient in MHC class I and CD8+
T cells, MHC II−/−
mice deficient in MHC class II and CD4+
T cells, Igμ2/−
mice deficient in mature B cells, and mice depleted of NK1.1+
cells, including NK cells (). Both MHC class I and II molecules were required for tumor rejection, supporting a central role for both CD8+
T cells. Neither NK cells nor B cells were necessary for tumor immunity. Noticeably, mice deficient in B cells developed fewer baseline metastases compared with wild-type C57BL/6 mice, and were completely free of any detectable tumor after treatment with hTRP-2 (12 of 12 mice). This phenomenon of enhanced T cell–dependent tumor rejection associated with B cell deficiency has been reported previously 10
. These results showed that T cell immunity, including both CD8+
T cells, was required for tumor rejection, but antibodies were not.
Figure 3 Tumor rejection in C57BL/6 mice deficient in immune molecules and cells. Mice, 10–12 per group, were immunized cutaneously with hTRP-2 DNA as described in the legend to . Lung metastases were evaluated after 18 d. Groups included mice deficient (more ...)
Xenogeneic Immunization Induces Autoimmunity That Also Requires T Cells.
Signs of autoimmunity, manifested as depigmentation, were observed in mice immunized with hTRP-2, but not generally in mice immunized with syngeneic mTRP-2 (). Depigmentation appeared 4–5 wk after starting immunization over depilated and shaved areas of the mouse coat, spreading to unshaved areas in most mice. Autoimmunity also required T cells, but not antibodies or NK cells, showing that tumor immunity and autoimmunity were coupled by a requirement for class I and II MHC expression leading to a requirement for T cells.
Figure 4 Depigmentation in mice treated with hTRP-2 or mTRP-2 DNA. Groups of mice (12–15 per group) were immunized with hTRP-2 or mTRP-2 DNA as described in the legend to , or remained untreated. Each dot represents a separate mouse. Groups include (more ...)
Requirement for Perforin in T Cell–dependent Autoimmunity, but Not Tumor Immunity.
CTLs have been proposed to be critical effector cells that mediate tumor rejection. Cytotoxicity of T cells can be mediated by exocytic granules involving perforin, or by cell membrane molecules that induce death of target cells. Tumor immunity proceeded in the complete absence of perforin (P = 0.0002) in pfp−/− mice (), whereas autoimmunity was mostly inhibited (). Fas ligand was not necessary for either autoimmunity () or tumor immunity (). These results are consistent with perforin-mediated killing of normal melanocytes in hair follicles playing a central role in autoimmunity. However, tumor immunity could proceed in a perforin-independent manner.
Figure 5 Tumor rejection in perforin-deficient (pfp−/−) mice and gld/gld mice (deficient in fas ligand) treated with hTRP-2, compared with mice not treated. Mice (9 or 10 per group) were immunized cutaneously with hTRP-2 DNA as described in the (more ...)