Self antigens expressed by solid tumors Do not efficiently stimulate naive or activated T cells: implications for immunotherapy

Abstract

Induction and maintenance of cytotoxic T lymphocyte (CTL) activity specific for a primary endogenous tumor was investigated in vivo. The simian virus 40 T antigen (Tag) expressed under the control of the rat insulin promoter (RIP) induced pancreatic beta-cell tumors producing insulin, causing progressive hypoglycemia. As an endogenous tumor antigen, the lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) was introduced also under the control of the RIP. No significant spontaneous CTL activation against GP was observed. However, LCMV infection induced an antitumor CTL response which efficiently reduced the tumor mass, resulting in temporarily normalized blood glucose levels and prolonged survival of double transgenic RIP(GP x Tag2) mice (137 +/- 18 d) as opposed to control RIP-Tag2 mice (88 +/- 8 d). Surprisingly, the tumor-specific CTL response was not sustained despite the facts that the tumor cells continued to express MHC class I and LCMV-GP-specific CTLs were present and not tolerized. Subsequent adoptive transfer of virus activated spleen cells into RIP(GP x Tag2) mice further prolonged survival (168 +/- 11 d), demonstrating continued expression of the LCMV-GP tumor antigen and MHC class I. The data show that the tumor did not spontaneously induce or maintain an activated CTL response, revealing a profound lack of immunogenicity in vivo. Therefore, repetitive immunizations are necessary for prolonged antitumor immunotherapy. In addition, the data suggest that the risk for induction of chronic autoimmune diseases is limited, which may encourage immunotherapy against antigens selectively but not exclusively expressed by the tumor.

Figure 1

Blood glucose levels reflect the tumor burden and the effectiveness of tumor immunotherapy in vivo. (A) RIP(GP × Tag2) and RIP-Tag2 mice developed hypoglycemia indicative of β-cell hyperplasia and insulin overproduction, while control RIP-GP and C57BL/6 mice showed normal glucose levels. (B) Tag2 transgenic animals with two consecutive blood glucose readings <5 mM (at an age of 70–85 d) and age-matched control mice were infected with LCMV to induce tumor-specific CTLs. After infection, the GP-expressing mice (but not those without GP) increased the blood glucose levels. This effect was reversed in the RIP(GP × Tag2) mice and hypoglycemia relapsed within 4 wk. In 1 out of 10 RIP(GP × Tag2) mice the LCMV-induced hyperglycemia was very high and lethal. The figures show 1 mouse/group representative for 7–20 mice/group (4 mice for C57BL/6). ▪, RIP-GP; •, RIP(GP × Tag2); ○, RIP-Tag2; □, C57BL/6.

Figure 2

GP-specific T cells were neither spontaneously activated nor rendered unresponsive by the tumor. (A) LCMV-GP–specific proliferative responses. Spleen cells were taken from untreated mice (closed bars), 8 d after LCMV infection (open bars), or 30 d after LCMV infection (hatched bars) and incubated in vitro with stimulator macrophages treated with the LCMV glycoprotein peptide p33. Data are mean ± SD of triplicate cultures. Control cultures using macrophages without peptide had background levels <3,000 cpm (not shown). (B–E) Normal LCMV-specific cytotoxic responses were detected 8 d after LCMV infection (B and C), or 30 d after LCMV infection upon restimulation in vitro for 5 d (D and E). In the RIP(GP × Tag2) mice studied 30 d after LCMV infection the tumors had grown back and blood glucose was low (3–5 mM). No data were obtained for day 30 RIP-GP or RIP-Tag2 mice (n.d., not determined) because these animals were killed due to diabetes or tumor burden. Target cells were MC57G fibroblasts untreated (C and E) or treated with peptide p33 (B and D). The results shown are representative for three independent experiments and analysis of at least four mice per group. ▪, RIP-GP; •, RIP(GP × Tag2); ○, RIP-Tag2; □, C57BL/6.

Figure 2

GP-specific T cells were neither spontaneously activated nor rendered unresponsive by the tumor. (A) LCMV-GP–specific proliferative responses. Spleen cells were taken from untreated mice (closed bars), 8 d after LCMV infection (open bars), or 30 d after LCMV infection (hatched bars) and incubated in vitro with stimulator macrophages treated with the LCMV glycoprotein peptide p33. Data are mean ± SD of triplicate cultures. Control cultures using macrophages without peptide had background levels <3,000 cpm (not shown). (B–E) Normal LCMV-specific cytotoxic responses were detected 8 d after LCMV infection (B and C), or 30 d after LCMV infection upon restimulation in vitro for 5 d (D and E). In the RIP(GP × Tag2) mice studied 30 d after LCMV infection the tumors had grown back and blood glucose was low (3–5 mM). No data were obtained for day 30 RIP-GP or RIP-Tag2 mice (n.d., not determined) because these animals were killed due to diabetes or tumor burden. Target cells were MC57G fibroblasts untreated (C and E) or treated with peptide p33 (B and D). The results shown are representative for three independent experiments and analysis of at least four mice per group. ▪, RIP-GP; •, RIP(GP × Tag2); ○, RIP-Tag2; □, C57BL/6.

Figure 3

Intense lymphocytic infiltration after LCMV infection in double transgenic RIP(GP × Tag2) but not in RIP-Tag2 single transgenic mice. Mice were immunized with LCMV i.v. and killed on day 8 (A–F). Tag transgenic mice showed islet hyperplasia and tumor formation by proliferating β cells (C–H). Immunohistochemical analysis with CD8-specific (left) or CD4-specific (right) antibodies showed intense lymphocytic infiltration in the tumors of RIP-GP × Tag2 (E and F) but not RIP-Tag2 (C and D) mice or nontransgenic mice (A and B). In uninfected mice (G and H), the tumors showed minimal lymphocyte infiltration (only RIP-GP × Tag2 shown).