Coupling and uncoupling of tumor immunity and autoimmunity

Abstract

Self-antigens, in the form of differentiation antigens, are commonly recognized by the immune system on melanoma and other cancers. We have shown previously that active immunization of mice against the melanocyte differentiation antigen, a tyrosinase-related protein (TRP) gp75(TRP-1) (the brown locus protein) expressed by melanomas, could induce tumor immunity and autoimmunity manifested as depigmentation. In this system, tumor immunity and autoimmunity were mediated by autoantibodies. Here, we characterize immunity against another tyrosinase family glycoprotein TRP-2 (the slaty locus protein), using the same mouse model and method of immunization. As observed previously for gp75(TRP-1), immunity was induced by DNA immunization against a xenogeneic form of TRP-2, but not against the syngeneic gene, and depended on CD4(+) cells. Immunization against TRP-2 induced autoantibodies and autoreactive cytotoxic T cells. In contrast to immunization against gp75(TRP-1), both tumor immunity and autoimmunity required CD8(+) T cells, but not antibodies. Only autoimmunity required perforin, whereas tumor immunity proceeded in the absence of perforin. Thus, immunity induced against two closely related autoantigens that are highly conserved throughout vertebrate evolution involved qualitatively different mechanisms, i.e., antibody versus CD8(+) T cell. However, both pathways led to tumor immunity and identical phenotypic manifestations of autoimmunity.

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. Mice were challenged with B16 melanoma cells intravenously and scored for surface lung metastases after 14–17 d. (A and B) Mice were immunized three times at weekly intervals with hTRP-2, mTRP-2, or PCR3 control vector (vector), and compared to untreated mice. Mice were challenged with B16F10/LM3 melanoma 5 d after the last immunization. B is a repeat of the experiment in A, without the vector control and mTRP-2 groups. Significant tumor protection was observed in both experiments (P < 0.0001). (C) Mice were immunized five times at weekly intervals with hTRP-2, or mTRP-2, or were untreated. Significant protection was observed with hTRP-2 compared with no treatment (P = 0.001) or with mTRP-2 (P = 0.001). The difference between mice treated with mTRP-2 and untreated mice was not significant (P = 0.16). (D) Immunization with hTRP-2 DNA started 4 d after tumor challenge, or mice remained untreated. A significant therapeutic effect was observed (P < 0.001). (E) Immunization with hTRP-2 plus GM-CSF DNA started 10 d after tumor challenge. Significant therapeutic effect was observed compared with other treatment and control groups (P < 0.01). GM-CSF treatment alone yielded 692 ± 69 metastases, mTRP-2 gave 902 ± 65 metastases, hTRP-2 gave 783 ± 75 metastases, and control null vector gave 705 ± 61 metastases (not shown in figure). Results are shown as mean number of lung metastases ± SEM.

Figure 2

CTL response to TRP-2_181–188 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 Fig. 1. 7 d after the last immunization, draining lymph nodes were pooled and stimulated as described in Materials and Methods for 5 d and tested for cytotoxicity against EL-4 target cells, either pulsed with TRP-2 peptide or unpulsed. Results are representative of two experiments.

Figure 2

CTL response to TRP-2_181–188 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 Fig. 1. 7 d after the last immunization, draining lymph nodes were pooled and stimulated as described in Materials and Methods for 5 d and tested for cytotoxicity against EL-4 target cells, either pulsed with TRP-2 peptide or unpulsed. Results are representative of two experiments.

Figure 2

CTL response to TRP-2_181–188 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 Fig. 1. 7 d after the last immunization, draining lymph nodes were pooled and stimulated as described in Materials and Methods for 5 d and tested for cytotoxicity against EL-4 target cells, either pulsed with TRP-2 peptide or unpulsed. Results are representative of two experiments.

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 legend to Fig. 1. Significant tumor protection was observed in pfp ^−/− (P = 0.0002) and gld/gld (P < 0.0001) mice.

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 Fig. 1. Lung metastases were evaluated after 18 d. Groups included mice deficient in β2 microglobulin (β2^−/−), MHC II, immunoglobulins (μ^2/−), and depleted of NK1.1⁺ cells (NK deplete). All mice were treated in the same experiment, and differences in lung metastases in the no treatment groups reflect tumorigenicity in different mouse strains or under different conditions (e.g., NK deplete). Significant protection was observed in NK depleted (P < 0.0001) and μ^2/− (P = 0.03), but not β2^−/− or MHC II^−/− (P > 0.10) mice. This experiment is representative of two experiments. Results are shown as mean ± SEM.

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 Fig. 1, or remained untreated. Each dot represents a separate mouse. Groups include C57BL/6 (wt) mice treated with hTRP-2 (h) or mTRP-2 (m) DNA. In addition, immunoglobulin-deficient (μ^2/−), β2 microglobulin-deficient (β2^−/−), MHC II–deficient (MHC II^−/−), NK-depleted, and perforin-deficient (perf^−/−) mice were immunized with hTRP-2.