Antigenic cancer cells grow progressively in immune hosts without evidence for T cell exhaustion or systemic anergy

Abstract

One enigma in tumor immunology is why animals bearing malignant grafts can reject normal grafts that express the same nonself-antigen. An explanation for this phenomenon could be that different T cell clones react to the normal graft and the malignant cells, respectively, and only the tumor-reactive clonotypes may be affected by the growing tumor. To test this hypothesis, we used a T cell receptor transgenic mouse in which essentially all CD8(+) T cells are specific for a closely related set of self-peptides presented on the MHC class I molecule Ld. We find that the tumor expressed Ld in the T cell receptor transgenic mice but grew, while the Ld-positive skin was rejected. Thus, despite an abundance of antigen-specific T cells, the malignant tissue grew while normal tissue expressing the same epitopes was rejected. Therefore, systemic T cell exhaustion or anergy was not responsible for the growth of the antigenic cancer cells. Expression of costimulatory molecules on the tumor cells after transfection and preimmunization by full-thickness skin grafts was required for rejection of a subsequent tumor challenge, but there was no detectable effect of active immunization once the tumor was established. Thus, the failure of established tumors to attract and activate tumor-specific T cells at the tumor site may be a major obstacle for preventive or therapeutic vaccination against antigenic cancer.

Figure 1

Homogenous expression of the L^d molecule on the transfected tumor cells and of the anti-L^d TCR 2C on the transgenic CD8⁺ T cells. (A) Stable transfection of AG104A tumor cells with an L^d cDNA expression vector resulted in L^d expression 40-fold above background as shown by FACS^® analysis. Shaded curve, anti-L^d staining; unshaded curve, staining with goat anti–mouse FITC secondary antibody alone. (B) Two-color staining of peripheral blood cells from the C3H × 2C transgenic mice with biotinylated anti-CD8a, and 1B2–FITC (anti–2C) mAbs showed that all CD8⁺ T cells express the 2C TCR.

Figure 1

Homogenous expression of the L^d molecule on the transfected tumor cells and of the anti-L^d TCR 2C on the transgenic CD8⁺ T cells. (A) Stable transfection of AG104A tumor cells with an L^d cDNA expression vector resulted in L^d expression 40-fold above background as shown by FACS^® analysis. Shaded curve, anti-L^d staining; unshaded curve, staining with goat anti–mouse FITC secondary antibody alone. (B) Two-color staining of peripheral blood cells from the C3H × 2C transgenic mice with biotinylated anti-CD8a, and 1B2–FITC (anti–2C) mAbs showed that all CD8⁺ T cells express the 2C TCR.

Figure 2

AG104A–L^d tumor cells grow progressively and retain L^d expression in normal C3H × C57BL/6 mice and anti-L^d TCR transgenic mice. AG104A–L^d cells were injected subcutaneously into the flanks of four normal C3H × C57BL/6 mice and five anti-L^d C3H × 2C TCR transgenic mice (A) or two nude mice and three anti-L^d C3H × 2C TCR transgenic mice (B). Tumors grew out in all types of mice. Bars indicate the SEM. FACS^® analysis of AG104A–L^d tumor cells reisolated at day 27 from a nontransgenic mouse (C) or at day 33 from a TCR transgenic mouse (D) showed that antigen expression was retained in the course of tumor growth. Shaded curve, anti-L^d staining; unshaded curve, staining with goat anti–mouse FITC secondary antibody alone.

Figure 3

AG104A–L^d tumor cells do not express Fas ligand. Total RNA was isolated from AG104A–L^d tumor cells grown in culture or as tumors in vivo, P815 tumor cells, and spleen cells. Total RNA from the T cell clone pGL10 (37), activated for 7 d on syngeneic feeder cells in the presence of 0.2 mg/ml chicken ovalbumin and 12 IU/ml rhuIL-2, was provided by Dr. U. Korthäuer (University of Chicago). Expression of Fas ligand and β-actin was analyzed by RT-PCR. To test for genomic DNA contamination, RT-PCR was also performed without the addition of reverse transcriptase for the cDNA synthesis (no RT).

Figure 4

An anti-L^d–specific CD8⁺ T cell clone from TCR transgenic mice has cytolytic activity against AG104A–L^d and AG104ABC–L^d tumor cells in vitro. The CD8⁺ T cell clone 900-2 derived from the 2C TCR transgenic mice specifically recognizes and lyses AG104A–L^d and AG104ABC–L^d tumor cells, but not AG104A–wt tumor cells in a 4.5-h ⁵¹Cr release assay in vitro.

Figure 5

Anti-L^d TCR transgenic mice reject L^d-positive skin, but do not reject a simultaneous challenge with L^d-expressing tumor cells. Two TCR transgenic mice were transplanted with full-thickness L^d-positive skin from a BALB/c mouse and concurrently received subcutaneous injections of AG104A–wt cells and AG104A–L^d cells on opposite flanks. Whereas both mice rejected the skin graft at day 13 after transplantation (see Table 1), both wt and L^d-expressing tumors grew progressively (A and C). Bars indicate the SEM. The outgrowth of AG104A–L^d was not due to antigen loss, because tumor cells reisolated on day 24 still stained positive for L^d in a FACS^® analysis (B and D). Shaded curve, anti-L^d staining, unshaded curve, staining with goat anti–mouse FITC secondary antibody alone.

Figure 6

Expression of the costimulatory molecules B7-1 and CD48 by the L^d-positive AG104A tumor cells leads to slower tumor outgrowth in naive TCR transgenic mice. AG104A cells expressing the costimulatory molecules B7-1 and CD48 (18) were transfected with an L^d cDNA expression vector. The expression level of L^d, B7-1, and CD48 in these triple transfected cells, named AG104ABC–L^d, is shown in A. To determine the effect of expression of B7-1 and CD48 on tumor cell outgrowth (B), naive TCR transgenic mice were injected with either AG104A–L^d cells (3 mice) or AG104ABC–L^d cells (11 mice). AG104ABC–L^d tumor cells grew slower than AG104A–L^d tumor cells. Bars indicate the SEM.

Figure 7

Antigen-positive tumor cells providing costimulation are effectively rejected after preimmunization of TCR transgenic mice with antigen-positive skin. Six TCR transgenic mice were challenged subcutaneously with AG104ABC–L^d tumor cells and AG104ABC tumor cells on opposite flanks 3–5 d after rejection of a full-thickness BALB/c skin graft. All six mice rejected the AG104ABC–L^d tumor cells, but not the simultaneous AG104ABC tumor cell challenge, showing that the protective effect of L^d-positive skin was antigen specific (A). Four mice, each of which rejected the skin graft, were treated with anti-CD8 antibody (B) or anti-CD4 antibody (C) 3 d before challenge with AG104ABC–L^d tumor cells. The tumors grew in the anti-CD8–treated mice, but not in the anti-CD4–treated mice, showing that tumor rejection is mediated by CD8⁺ T cells. Spleen cells from the six mice that rejected the AG104ABC–L^d tumor cells (D) and six naive TCR transgenic mice (E) were stimulated in vitro with AG104A–L^d tumor cells in a MLTC. Specific cytolytic T cells were detected only in the culture from mice that had rejected the AG104ABC–L^d tumor challenge. Bars indicate the SEM.