IFN-γ– and TNF-dependent bystander eradication of antigen-loss variants in established mouse cancers (original) (raw)
IFN-γ and TNF produced by transferred T cells are required for eradicating established tumors. To generate tumor-specific effector cells, spleen cells from SIY-immunized mice were activated in vitro with SIY peptide. These CTLs were infused i.v. into established MC57-SIY-Hi tumor–bearing OT-1 mice (see Methods). MC57-SIY-Hi tumors grew rapidly in untreated control mice or in mice receiving CTLs from Pfr–/– mice, whereas CTLs from WT mice caused complete tumor rejection (Figure 1, left, and Table 1), confirming our previous results (8). Adoptive transfer of CTLs from either IFN-γ–/– mice or TNF–/– mice caused MC57-SIY-Hi tumors to initially regress but then grow progressively, whereas CTLs from FasL–/– mice caused complete elimination of MC57-SIY-Hi tumors (Figure 1, middle, and Table 1). In addition, MC57-SIY-Hi tumors growing in IFN-γ–/– or TNF–/– mice were eradicated by SIY-specific 2C CTLs (data not shown), suggesting that host-derived IFN-γ and TNF were not required.
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.
TNF and IFN-γ produced by T cells are critical for complete elimination of established tumors
To confirm that ALVs preexistent in established tumors were eliminated by T cells secreting IFN-γ and TNF, 2,000 antigen-negative cancer cells (MC57 cells) as ALVs were mixed into 2 × 106 antigen-positive MC57-SIY-Hi cancer cells, inoculated s.c., and treated with adoptively transferred T cells on day 14, when the tumors were large. Transfer of CTLs generated either from IFN-γ–/– mice or from TNF–/– mice resulted in the temporary inhibition of tumor growth followed by relapse, whereas transfer of CTLs from WT mice eradicated these tumors completely (Figure 1, right). These data demonstrate that IFN-γ and TNF produced by CTLs are required for preventing relapse after T cell–mediated destruction of established tumors. As we previously observed (8), perforin secretion by the transferred T cells is essential to destroy the bulk of antigen-positive tumor cells, but we could not detect a significant role of Fas/FasL signaling in our system.
SIY-expressing cancer cells induce SIY-specific T cells in IFN-γ–/– and TNF–/– mice. To test whether CD8+ T cells are primed in IFN-γ–/– and TNF–/– mice, we challenged WT, IFN-γ–/–, and TNF–/– mice with MC57-SIY-Hi cancer cells. At 8 days after challenge, circulating anti-SIY CD8+ T cells were detected by peptide-MHC–dimer staining (Figure 2A); recovered T cells specifically responded to the SIY peptide but not to the irrelevant gp33 peptide (data not shown). IFN-γ–/– and TNF–/– mice produced the cytokine not knocked out at levels similar to those in WT mice; no IFN-γ was detected in IFN-γ–/– T cells and no TNF in TNF–/– T cells (Figure 2B). T cells from the WT host expressed both cytokines. IFN-γ– or TNF-expressing cells were not detected with an isotype control antibody (data not shown). These data suggest that antigen-specific T cells can be primed effectively in IFN-γ–/– or TNF–/– hosts.
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.
T cells require neither TNF nor IFN-γ to kill antigen-positive targets. We next investigated the roles of Fas, IFN-γ, TNF, and perforin pathways for antigen-specific killing by CD8+ T cells in vivo. As targets, spleen cells from WT control or IFN-γ receptor–deficient (IFN-γR–/–), TNF receptor–deficient (TNFR–/–), or lpr (i.e., Fas–/–) mice were pulsed with the gp33 or the SIY peptide and labeled with a low or a high concentration of CFSE. Cells of the 2 types of target populations were injected i.v. (2 × 107 cells) into Pfr–/–, IFN-γ–/–, TNF–/–, or WT mice that had been immunized 8 days earlier with MC57-SIY-Hi cells to generate effector T cells. Nonimmunized mice were used as controls. After 24 hours, spleen cells were harvested from immunized and control mice and analyzed for SIY-specific loss of the injected labeled peptide-pulsed target cells. Immunized Pfr–/– mice were severely compromised in their ability to kill SIY peptide–coated targets (52.9%; Figure 3). In contrast, immunized WT (94.7%), TNF–/– (94.9%), and IFN-γ–/– (95.3%) mice all displayed similarly effective antigen-specific killing in vivo. Furthermore, SIY peptide–pulsed target cells derived from WT, IFN-γR–/–, TNFR–/–, and lpr mice were similarly susceptible as targets in vivo. This indicated that SIY-specific CD8+ T cells do not require IFN-γ/IFN-γR or TNF/TNFR signals or Fas/FasL engagement for antigen-specific killing in vivo. The remaining level of killing detected in the absence of perforin presumably represents the collective contribution of perforin-independent killing and may be IFN-γR, TNFR, or Fas dependent, as has been previously observed (30). This contribution of Fas-, IFN-γ– and TNF-mediated killing is probably unmasked in the absence of the highly efficient perforin-mediated killing. However, our data indicate that neither of the TNF, IFN-γ, or FasL pathways are required for SIY-specific T cell killing in vivo and that killing by SIY-specific CD8+ T cells in vivo was largely mediated by the perforin-dependent granule exocytosis pathway.
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.
Expression of IFN-γR and TNFR on stromal cells is required for the successful elimination of cancer variants. In our model, targeting cancer cells as well as stromal cells was needed for perforin-mediated T cell rejection of tumors (ref. 8 and Figure 1, left). However, it remained unclear whether tumor rejection by T cells also required the action of IFN-γ or TNF on stromal cells. To address this question, OT-1 WT, OT-1 IFN-γR–/–, and OT-1 TNFR–/– mice were injected s.c. with MC57-SIY-Hi or MC57-gp33-Hi cancer cells, and 14 days later SIY-specific 2C T cells were adoptively transferred. As shown in Figure 4A, SIY tumors but not gp33 tumors were rejected by WT mice. Interestingly, SIY tumors in TNFR–/– or IFN-γR–/– mice regressed initially and then regrew (Figure 4A and Table 2). Cancer cells isolated from recurrent tumors of T cell–treated IFN-γR–/– (Figure 4B, top) or TNFR–/– mice (Figure 4B, bottom) were ALVs that had lost SIY-EGFP expression and were no longer recognized by 2C T cells (data not shown). Thus, for CD8+ T cell–mediated tumor rejection, IFN-γR or TNFR expression on cancer cells alone was not sufficient; stromal cells also had to express IFN-γR and TNFR to prevent relapse caused by ALVs.
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.
TNFR and IFN-γR expression on stromal cells is critical for complete elimination of established tumors
The stromal cell types necessary to prevent the outgrowth of ALVs in MC57-SIY-Hi tumors were subsequently determined by generating BM chimeras in which the respective BM-derived or non-BM–derived stromal cells expressed TNFR or IFN-γR (TNFR–/–→WT, OT-1 TNFR–/– BM to OT-1 WT recipient; WT→TNFR–/–, OT-1 WT BM to OT-1 TNFR–/– recipient; IFN-γR–/–→WT, OT-1 IFN-γR–/– BM to OT-1 WT recipient; WT→IFN-γR–/–, OT-1 WT BM to OT-1 IFN-γR–/– recipient). MC57-SIY-Hi tumors escaped rejection in IFN-γR–/–→WT and WT→IFN-γR–/– BM chimeric mice (Figure 5A). Similarly, MC57-SIY-Hi tumors also escaped rejection in TNFR–/–→WT and WT→TNFR–/– BM chimeric mice (Figure 5B), but were rejected in control WT→WT BM chimeric mice (Figure 5, A and B, and Table 3). Therefore, the elimination of ALVs by CTL most likely required both BM- and non-BM–derived stromal cells to express TNFR and IFN-γR during the effector phase of antitumor immune response.
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.
TNFR and IFN-γR expression on BM- and non-BM–derived stromal cells is required for complete elimination of established tumors