Genetic vaccination with "self" tyrosinase-related protein 2 causes melanoma eradication but not vitiligo

Abstract

"Self" melanocyte differentiation antigens are potential targets for specific melanoma immunotherapy. Vaccination against murine tyrosinase-related protein (TRP)-1/gp75 was shown recently to cause melanoma rejection, which was accompanied by autoimmune skin depigmentation (vitiligo). To further explore the linkage between immunotherapy and autoimmunity, we studied the response to vaccination with a related antigen, TRP-2. i.m. inoculation of plasmid DNA encoding murine trp-2 elicited antigen-specific CTLs that recognized the B16 mouse melanoma and protected the mice from challenge with tumor cells. Furthermore, mice bearing established s.c. B16 melanomas rejected the tumor upon vaccination with a recombinant vaccinia virus encoding trp-2. Depletion experiments showed that CD8+ lymphocytes and natural killer cells were crucial for the antitumor activity of the trp-2-encoding vaccines. Mice that rejected the tumor did not develop generalized vitiligo, indicating that protective immunity can be achieved in the absence of widespread autoimmune aggression.

Fig. 1

Plasmid DNA encoding TRP-2 induces Ag-specific CTL. A, 3 weeks after i.m. inoculation of plasmid DNA, spleens were removed, and splenocytes were stimulated in vitro in an MLPC with 1 μm of TRP-2_180–188 peptide. After 5 days of culture, cytotoxic activity of the splenocytes was tested in a ⁵¹Cr release assay carried out for 5 h against the following target cells: MBL-2, MBL-2 pulsed with TRP-2_180–188 peptide, and B16. Each panel shows the CTL response generated in a single mouse after inoculation of either cardiotoxin alone or cardiotoxin followed by either the empty pcDNA3 vector or pcDNA3-trp-2. Results are representative of five experiments. The SD of the triplicate determinations for each effector/target cell ratio (E:T ratio) was <10%, and spontaneous release never exceeded 20%. B, splenocytes (10⁵ cells) from MLPCs were restimulated for 24 h in triplicate wells with an equal amount of MBL-2 cells, MBL-2 cells pulsed for 1 h with 1 μm TRP-2_180–188 peptide, or B16 melanoma cells; the supernatant was then harvested and tested for released IFN-γ in a sandwich ELISA assay. The SD of the triplicate determinations for each effector/stimulator combination was <10%, and IFN-γ measured in control wells containing either effectors or stimulators alone did not exceed the lowest amount of IFN-γ detectable in our assay (i.e., 547 pg/ml, determined using serial dilutions of IFN-γ). Two-tailed Ps calculated by the Mann-Whitney test are reported in the figure for those groups showing significant differences over their respective controls (filled symbols).

Fig. 2

Immunization with a plasmid encoding TRP-2 prolongs survival of mice challenged with B16 melanoma cells. Three weeks after vaccination with pcDNA3-trp-2, mice were challenged s.c. in the right flank with 10⁵ B16 melanoma cells. Untreated mice or mice immunized with the empty pcDNA3 vector were used as negative controls. Results represent the sums of two independently performed experiments with 5–10 mice in each treatment group. Immunization with pcDNA3-trp-2 resulted in a significant prolongation of survival (P < 0.0001). Inoculation of the empty pcDNA3 vector also caused a significant prolongation of survival (P = 0.006) as compared with untreated mice.

Fig. 3

Protection against tumor challenge induced by DNA immunization depends on CD8⁺ lymphocytes and NK cells and is associated with local depigmentation and alopecia. A, to identify the effector population responsible for the antitumor effect, mice immunized with pcDNA3-trp-2 were depleted by i.p. injections of either anti-CD4, anti-CD8, or anti-NK1.1 mAbs as described in “Materials and Methods.” Mice not immunized with pcDNA3-trp-2 were used as controls (no treatment). Each treatment group contained six mice. The Mantel-Haenszel test gave P = 0.025 for the CD8⁺-depleted versus no depletion group and P = 0.09 for the NK-depleted versus no depletion group. Duplicate experiments confirmed these results. B, mice were routinely shaved on the right flank in preparation for the s.c. tumor challenge. Although the hair normally grew back in 3 weeks, some mice showed an area of persistent hair loss and depigmentation at the site of tumor injection. The photograph was taken 4 months after tumor challenge and shows mice (anesthetized for the photo) belonging to the “no depletion” and “CD4⁺ depletion” groups described in A.

Fig. 4

rVV encoding TRP-2 has a therapeutic effect on tumor-bearing mice. Mice were injected s.c. with 10⁵ B16 melanoma cells. Five days later, they received a single i.v. inoculation of 5 × 10⁶ PFU of an rVV encoding TRP-2 (rVV-trp-2). Untreated mice and mice injected with the same amount of mock virus (rVV) were used as negative controls. Results represent the sums of two independently performed experiments with five to six mice in each treatment group. The statistical analysis was performed according to the Mantel-Haenszel test (P = 0.04, no treatment versus rVV-trp-2; P = 0.001, rVV versus rVV-trp-2).