Immune response to a differentiation antigen induced by altered antigen: a study of tumor rejection and autoimmunity

Abstract

Recognition of self is emerging as a theme for the immune recognition of human cancer. One question is whether the immune system can actively respond to normal tissue autoantigens expressed by cancer cells. A second but related question is whether immune recognition of tissue autoantigens can actually induce tumor rejection. To address these issues, a mouse model was developed to investigate immune responses to a melanocyte differentiation antigen, tyrosinase-related protein 1 (or gp75), which is the product of the brown locus. In mice, immunization with purified syngeneic gp75 or syngeneic cells expressing gp75 failed to elicit antibody or cytotoxic T-cell responses to gp75, even when different immune adjuvants and cytokines were included. However, immunization with altered sources of gp75 antigen, in the form of either syngeneic gp75 expressed in insect cells or human gp75, elicited autoantibodies to gp75. Immunized mice rejected metastatic melanomas and developed patchy depigmentation in their coats. These studies support a model of tolerance maintained to a melanocyte differentiation antigen where tolerance can be broken by presenting sources of altered antigen (e.g., homologous xenogeneic protein or protein expressed in insect cells). Immune responses induced with these sources of altered antigen reacted with various processed forms of native, syngeneic protein and could induce both tumor rejection and autoimmunity.

Figure 1

Mice immunized with gp75/Sf9 produced antibodies against gp75 in syngeneic melanocytic cells. C57BL/6 mice were immunized with lysates of insect Sf9 cells infected with either recombinant baculovirus expressing syngeneic mouse gp75 (gp75/Sf9) or wt baculovirus (wt/Sf9). Mice were immunized four times subcutaneously with freeze–thawed Sf9 cells, 5 × 10⁶ cells per immunization. Sera from immunized mice were used to immunoprecipitate syngeneic gp75 from [³⁵S]methionine-labeled B16 melanoma lysates. The mAb TA99 was used as a positive control (right lane). Results for five mice in each group are shown (lanes 1–5).

Figure 2

Purified mouse gp75 mixed with lysates of wt/Sf9 cells does not induce autoantibodies to gp75. C57BL/6 mice were immunized with purified gp75 from B16 melanoma cells (purified gp75), purified gp75 from B16 melanoma cells mixed with lysates from 1 × 10⁶ Sf9 cells (purified gp75 + sf9), lysates of 1 × 10⁶ insect Sf9 cells infected with either recombinant baculovirus expressing syngeneic mouse gp75 (gp75/sf9 cells), or wt baculovirus (sf9 cells). Purified gp75 from B16 melanoma contained 10 μg of gp75, and gp75/Sf9 contained 14 μg of gp75. All immunizations included Freund’s adjuvant. Mice were immunized four times s.c. Sera from immunized mice were used to immunoprecipitate syngeneic gp75 from [³⁵S]methionine-labeled B16 melanoma lysates. The mAb TA99 was used as a positive control (left lane). Results for five mice in each group are shown (lanes 1–5).

Figure 3

Mice immunized with gp75 purified from Sf9 cells recognized an early processed form of gp75. Sera from C57BL/6 mice immunized with purified mouse gp75 (12 mg) produced in baculovirus were assessed for antibody responses to syngeneic gp75 by immunoprecipitation of lysates from [³⁵S]methionine-labeled syngeneic B16 melanoma cells or syngeneic gp75-negative TIB88 fibroblasts. The mAb TA99 was used as a positive control (right lane). Results for five mice are shown (lanes 1–5) after 3 immunizations (A), after 4 immunizations (B), and after 5 immunizations (C). All mice developed antibodies to syngeneic gp75 after four immunizations. These autoantibodies recognized an earlier processed form of gp75 (see Results), but mouse 2 developed autoantibodies that recognized mature, fully processed gp75 after four immunizations.

Figure 4

Mice immunized with human gp75 expressed in SK-MEL-19 melanoma cells produced antibodies against gp75 in syngeneic melanocytic cells. C57BL/6 mice were immunized with lysates of SK-MEL-19 melanoma cells with either Freund’s adjuvant or PBS. Mice were immunized four times s.c. with freeze–thawed cells, 5 × 10⁶ cells per immunization. Sera from immunized mice were used to immunoprecipitate syngeneic gp75 from [³⁵S]methionine-labeled B16 melanoma lysates. The mAb TA99 was used as a positive control (right lane). Results for three mice in each group are shown (lanes 1–3).

Figure 5

Immunization with gp75/Sf9 protects against melanoma lung metastases. C57BL/6 mice, five per group, were immunized with 5 × 10⁶ of gp75/Sf9 or control wt/Sf9 or were not immunized (Control). Lung metastases of B16F10 melanoma were assessed at day 14 following challenge with 100,000 melanoma cells intravenously. The mean number of lung colonies ± SD (error bars) is shown.

Figure 6

Alterations in coat color of C57BL/6 mice immunized with Sf9/gp75 but not Sf9/wt cell lysates. Black C57BL/6 mice, five per group, were immunized with lysates from 5 × 10⁶ Sf9/gp75 or Sf9/wt cells s.c. with Freund’s adjuvant every 10–14 days for five immunizations. Depigmentation was observed in coats of all five mice immunized with Sf9/gp75 but no mice immunized with Sf9/wt. Depigmentation appeared without dipilation of the coats. (Right) A representative mouse immunized with Sf9/gp75. (Left) A control mouse immunized with Sf9/wt.