IFN-gamma- and TNF-dependent bystander eradication of antigen-loss variants in established mouse cancers

Abstract

Tumors elicit antitumor immune responses, but over time they evolve and can escape immune control through various mechanisms, including the loss of the antigen to which the response is directed. The escape of antigen-loss variants (ALVs) is a major obstacle to T cell-based immunotherapy for cancer. However, cancers can be cured if both the number of CTLs and the expression of antigen are high enough to allow targeting of not only tumor cells, but also the tumor stroma. Here, we showed that IFN-gamma and TNF produced by CTLs were crucial for the elimination of established mouse tumors, including ALVs. In addition, both BM- and non-BM-derived stromal cells were required to express TNF receptors and IFN-gamma receptors for the elimination of ALVs. Although IFN-gamma and TNF were not required by CTLs for perforin-mediated killing of antigen-expressing tumor cells, the strong inference is that tumor antigen-specific CTLs must secrete IFN-gamma and TNF for destruction of tumor stroma. Therefore, bystander killing of ALVs may result from IFN-gamma and TNF acting on tumor stroma.

Figure 1. IFN-γ and TNF produced by T cells are needed for rejection of established tumors.

OT-1 transgenic mice were injected s.c. with 2 × 10⁶ MC57-SIY-Hi cells; on day 14, the SIY-immune T cells from WT and Prf^–/– mice, as well as no T cells as controls (left), and the SIY-immune T cells from WT, TNF^–/–, and IFN-γ^–/– mice (middle) were adoptively transferred into the tumor-bearing mice. Results were pooled from 3 experiments, each controlled by tumor-bearing mice treated with WT T cells. Right: OT-1 transgenic mice were injected s.c. with 2 × 10⁶ MC57-SIY-Hi cells plus 2 × 10³ MC57 cells. At day 14, the SIY-immune T cells from WT, TNF^–/–, and IFN-γ^–/– mice were adoptively transferred into the tumor-bearing mice. The generation of SIY-immune T cells is described in Methods. Each curve represents an individual mouse.

Figure 2. Injection of cancer cells expressing SIY antigen induces a SIY-specific T cell response in mice deficient in IFN-γ or TNF.

(A) C57BL/6 WT, TNF^–/–, or IFN-γ^–/– mice were injected s.c. with 2 × 10⁶ MC57-SIY-Hi cells. After 8 d, anti-SIY–specific CD8⁺ T cells were detected in peripheral blood lymphocytes of mice using SIY:Ig dimers. (B) Stimulated T cells from TNF^–/– or IFN-γ^–/– mice produced the cytokine not knocked out. At 9 days after tumor challenge, splenocytes were restimulated with 1 μg/ml of the SIYRYYGL peptide. After 6 hours, intracellular IFN-γ and TNF were examined in CD8⁺ T cells obtained from these mice. Numbers within plots denote the percent of cells in the indicated quadrant.

Figure 3. TNF and IFN-γ are not required for SIY-specific T cell killing in vivo.

(A) Flow cytometric data showing representative examples of results. Left: SIY-pulsed (CFSE-high) or gp33-pulsed (CFSE-low) target cells from C57BL/6 WT, IFN-γR^–/–, TNFR^–/–, or lpr mice were transferred into C57BL/6 WT mice. Right: SIY-pulsed or gp33-pulsed target cells from C57BL/6 WT mice were transferred into IFN-γ^–/–, TNF^–/–, or Prf^–/– mice. Recipient mice were immunized with MC57-SIY-Hi cells 8 d prior to injection of target cells to generate host effector T cells. The unimmunized WT mice receiving target cells were used as controls (bottom right). Spleens were harvested 24 h later and analyzed for CFSE fluorescence. (B and C) Compiled data of percentage of killing. (B) n = 3 per group, pooled from 2 independent experiments. (C) n = 2 (WT and Ipr) or 4 (IFN-γR^–/– and TNFR^–/–), pooled from 2 independent experiments.

Figure 4. Expression of IFN-γR and TNFR on stromal cells is required for elimination of ALVs.

(A) Antigenic cancer escape in mice lacking the receptor for either TNF or IFN-γ. OT-1 WT, TNFR^–/–, or IFN-γR^–/– mice were injected s.c. with 2 × 10⁶ MC57-SIY-Hi cells or MC57-gp33-Hi cells as controls. At day 14, the SIY-specific 2C T cells were adoptively transferred into these tumor-bearing mice. Each curve represents an individual mouse. (B) Tumors relapsing in receptor-deficient mice were ALVs. The regrowing MC57-SIY-Hi tumor cells from OT-1 IFN-γR^–/– and OT-1 TNFR^–/– mice following T cell therapy were isolated at day 40. The parental MC57-Neo cells and MC57-SIY-Hi cells isolated from non–T cell–treated OT-1 IFN-γR^–/– or OT-1 TNFR^–/– mice were used as controls. The levels of SIY antigen expression on those cancer cells were examined by flow cytometry using the EGFP fluorescence of the SIY-EGFP fusion protein.

Figure 5. Tumor rejection requires BM- and non–BM-derived stromal cells expressing TNFR and IFN-γR.

(A) Tumor growth curves showing the escape in IFN-γR^–/– mice. IFN-γR^–/–→WT, WT→WT, and WT→IFN-γR^–/– mice were generated. (B) Tumor growth curves showing the escape in TNFR^–/– mice. TNFR^–/–→WT, WT→WT, and WT→TNFR^–/– mice were generated. Chimeric mice were injected s.c. with 2 × 10⁶ MC57-SIY-Hi cells. After 14 days, 5 × 10⁶ preactivated 2C T cells, or no T cells as a control, were transferred to these tumor-bearing mice, and tumor volume was monitored. Data are shown in Table 3, which shows the compiled results of further experiments.