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Tumor-specific CD4+ T cells develop cytotoxic activity and eliminate virus-induced tumor cells in the absence of regulatory T cells.

Akhmetzyanova I, Zelinskyy G, Schimmer S, Brandau S, Altenhoff P, Sparwasser T, Dittmer U - Cancer Immunol. Immunother. (2012)

Bottom Line: We show here that there is an expansion of tumor-specific CD4(+) T cells producing cytokines and cytotoxic molecule granzyme B (GzmB) in the early phase of tumor growth.Importantly, we demonstrate that in vivo depletion of regulatory T cells (Tregs) and CD8(+) T cells in FBL-3-bearing DEREG transgenic mice augments IL-2 and GzmB production by CD4(+) T cells and increases FV-specific CD4(+) T-cell effector and cytotoxic responses leading to the complete tumor regression.Therefore, the capacity to reject tumor acquired by tumor-reactive CD4(+) T cells largely depends on the direct suppressive activity of Tregs.

View Article: PubMed Central - PubMed

Affiliation: Institute for Virology, University of Duisburg-Essen, Virchowstr 179, 45147, Essen, Germany. ilseyar.akhmetzyanova@uni-due.de

ABSTRACT
The important role of tumor-specific cytotoxic CD8(+) T cells is well defined in the immune control of the tumors, but the role of effector CD4(+) T cells is poorly understood. In the current research, we have used a murine retrovirus-induced tumor cell line of C57BL/6 mouse origin, namely FBL-3 cells, as a model to study basic mechanisms of immunological control and escape during tumor formation. This study shows that tumor-specific CD4(+) T cells are able to protect against virus-induced tumor cells. We show here that there is an expansion of tumor-specific CD4(+) T cells producing cytokines and cytotoxic molecule granzyme B (GzmB) in the early phase of tumor growth. Importantly, we demonstrate that in vivo depletion of regulatory T cells (Tregs) and CD8(+) T cells in FBL-3-bearing DEREG transgenic mice augments IL-2 and GzmB production by CD4(+) T cells and increases FV-specific CD4(+) T-cell effector and cytotoxic responses leading to the complete tumor regression. Therefore, the capacity to reject tumor acquired by tumor-reactive CD4(+) T cells largely depends on the direct suppressive activity of Tregs. We suggest that a cytotoxic CD4(+) T-cell immune response may be induced to enhance resistance against oncovirus-associated tumors.

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Influence of different cell populations on tumor formation: Effects of no depletion (injected with PBS) (a), depletion of CD8+ T cells (b), CD4+ T cells (c), Tregs (d), CD8+ and Tregs (e), CD8+, NK cells and Tregs (f) and CD4+ and CD8+ T cells (g) are shown. DEREG mice were injected s.c. with 1 × 107 FBL-3 cells (1 × 107) and tumor size was measured. Mice were depleted for their CD8+, CD4+ T cells, and Tregs as described in “Materials and methods.” Each line represents tumor progression in an individual mouse. h In vivo killing activity of CD8+ T cells in different treatment of mice. Mean percentages show killing of cells loaded with the FV DbgagL peptide in an in vivo CTL assay (described in “Materials and methods” section) at day 6 ptc in DEREG mice treated or not treated with DT. Data were pooled from four to six independent experiments with similar results. P values were determined by an unpaired t test (n.s., non-significant). Dragger symbol mice were euthanized due to progressive tumor growth
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Fig4: Influence of different cell populations on tumor formation: Effects of no depletion (injected with PBS) (a), depletion of CD8+ T cells (b), CD4+ T cells (c), Tregs (d), CD8+ and Tregs (e), CD8+, NK cells and Tregs (f) and CD4+ and CD8+ T cells (g) are shown. DEREG mice were injected s.c. with 1 × 107 FBL-3 cells (1 × 107) and tumor size was measured. Mice were depleted for their CD8+, CD4+ T cells, and Tregs as described in “Materials and methods.” Each line represents tumor progression in an individual mouse. h In vivo killing activity of CD8+ T cells in different treatment of mice. Mean percentages show killing of cells loaded with the FV DbgagL peptide in an in vivo CTL assay (described in “Materials and methods” section) at day 6 ptc in DEREG mice treated or not treated with DT. Data were pooled from four to six independent experiments with similar results. P values were determined by an unpaired t test (n.s., non-significant). Dragger symbol mice were euthanized due to progressive tumor growth

Mentions: It was previously reported that CD8+ T cells are essential in controlling FBL-3 progression, whereas CD4+ T cells did not affect the tumor growth [11, 13]. In agreement with these previous studies, tumor regression was completely abrogated when CD8+ T cells were ablated by monoclonal antibodies (Fig. 4b). In contrast, the depletion of CD4+ T cells did only temporary increase the tumor size at 6 days ptc but did not affect the subsequent rejection of FBL-3 tumor cells (Fig. 4c). The data demonstrate that mainly CD8+ T cell-mediated rejection of FBL-3 tumor cells but CD4+ T cells had only a minor effect. However, the influence of Tregs on the different effector T-cell populations during tumor rejection is poorly understood. To investigate this influence, we studied tumor regression and T-cell functions after selective depletion of Tregs. We used transgenic DEREG mice, which express a diphtheria toxin (DT) receptor under control of the Foxp3 promoter. An injection of DT selectively depleted more than 90 % of the Tregs (data not shown). No other cell population was depleted by this treatment. Interestingly, DT treatment of tumor-bearing DEREG mice did not significantly improve tumor elimination (Fig. 4d). Thus, in a tumor model in which immune surveillance and immune control are effectively mediated by tumor-specific CD8+ T cells, these cells do not seem to be functionally suppressed by Tregs. This is in line with our finding that depletion of Tregs did not enhance FBL-3-specific cytotoxicity of CD8+ T cells in an in vivo CTL assay with target cells loaded with a Friend virus immunodominant epitope peptide [9] (Fig. 4h). However, these results did not indicate whether or not tumor-specific CD4+ T-cell responses were suppressed by Tregs. To clarify this, we depleted Tregs and CD8+ T cells at the same time in tumor-bearing mice. Whereas CD8+ T-cell ablation alone resulted in progressive tumor growth (Fig. 4b), additional Treg depletion enabled the mice to again reject the tumor (Fig. 4e). The only difference to non-depleted wild-type mice was that the FBL-3 tumors increased in size for up to 4 days longer before they were rapidly rejected. To exclude a possible role of NK cells, the additional depletion of those cells in the group of mice lacking Tregs and CD8+ T cells was performed (Fig. 4f). Such mice could still reject the tumor, demonstrating that NK cells had no effect on tumor rejection in CD8+-depleted mice. In order to demonstrate that tumor rejection in mice depleted for CD8+ T cells and Tregs (Fig. 4e) was due to effector CD4+ T-cell responses, tumor growth was tested in mice lacking both CD4+ (including Tregs) and CD8+ T cells. In the absence of these T-cell compartments, no control of tumor growth was observed (Fig. 4g). This experiment suggests that CD4+ T cells can mediate potent anti-tumor effects when cytotoxic CD8+ T cells are absent but that they are tightly controlled in their activity by Foxp3+ Tregs.Fig. 4


Tumor-specific CD4+ T cells develop cytotoxic activity and eliminate virus-induced tumor cells in the absence of regulatory T cells.

Akhmetzyanova I, Zelinskyy G, Schimmer S, Brandau S, Altenhoff P, Sparwasser T, Dittmer U - Cancer Immunol. Immunother. (2012)

Influence of different cell populations on tumor formation: Effects of no depletion (injected with PBS) (a), depletion of CD8+ T cells (b), CD4+ T cells (c), Tregs (d), CD8+ and Tregs (e), CD8+, NK cells and Tregs (f) and CD4+ and CD8+ T cells (g) are shown. DEREG mice were injected s.c. with 1 × 107 FBL-3 cells (1 × 107) and tumor size was measured. Mice were depleted for their CD8+, CD4+ T cells, and Tregs as described in “Materials and methods.” Each line represents tumor progression in an individual mouse. h In vivo killing activity of CD8+ T cells in different treatment of mice. Mean percentages show killing of cells loaded with the FV DbgagL peptide in an in vivo CTL assay (described in “Materials and methods” section) at day 6 ptc in DEREG mice treated or not treated with DT. Data were pooled from four to six independent experiments with similar results. P values were determined by an unpaired t test (n.s., non-significant). Dragger symbol mice were euthanized due to progressive tumor growth
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Fig4: Influence of different cell populations on tumor formation: Effects of no depletion (injected with PBS) (a), depletion of CD8+ T cells (b), CD4+ T cells (c), Tregs (d), CD8+ and Tregs (e), CD8+, NK cells and Tregs (f) and CD4+ and CD8+ T cells (g) are shown. DEREG mice were injected s.c. with 1 × 107 FBL-3 cells (1 × 107) and tumor size was measured. Mice were depleted for their CD8+, CD4+ T cells, and Tregs as described in “Materials and methods.” Each line represents tumor progression in an individual mouse. h In vivo killing activity of CD8+ T cells in different treatment of mice. Mean percentages show killing of cells loaded with the FV DbgagL peptide in an in vivo CTL assay (described in “Materials and methods” section) at day 6 ptc in DEREG mice treated or not treated with DT. Data were pooled from four to six independent experiments with similar results. P values were determined by an unpaired t test (n.s., non-significant). Dragger symbol mice were euthanized due to progressive tumor growth
Mentions: It was previously reported that CD8+ T cells are essential in controlling FBL-3 progression, whereas CD4+ T cells did not affect the tumor growth [11, 13]. In agreement with these previous studies, tumor regression was completely abrogated when CD8+ T cells were ablated by monoclonal antibodies (Fig. 4b). In contrast, the depletion of CD4+ T cells did only temporary increase the tumor size at 6 days ptc but did not affect the subsequent rejection of FBL-3 tumor cells (Fig. 4c). The data demonstrate that mainly CD8+ T cell-mediated rejection of FBL-3 tumor cells but CD4+ T cells had only a minor effect. However, the influence of Tregs on the different effector T-cell populations during tumor rejection is poorly understood. To investigate this influence, we studied tumor regression and T-cell functions after selective depletion of Tregs. We used transgenic DEREG mice, which express a diphtheria toxin (DT) receptor under control of the Foxp3 promoter. An injection of DT selectively depleted more than 90 % of the Tregs (data not shown). No other cell population was depleted by this treatment. Interestingly, DT treatment of tumor-bearing DEREG mice did not significantly improve tumor elimination (Fig. 4d). Thus, in a tumor model in which immune surveillance and immune control are effectively mediated by tumor-specific CD8+ T cells, these cells do not seem to be functionally suppressed by Tregs. This is in line with our finding that depletion of Tregs did not enhance FBL-3-specific cytotoxicity of CD8+ T cells in an in vivo CTL assay with target cells loaded with a Friend virus immunodominant epitope peptide [9] (Fig. 4h). However, these results did not indicate whether or not tumor-specific CD4+ T-cell responses were suppressed by Tregs. To clarify this, we depleted Tregs and CD8+ T cells at the same time in tumor-bearing mice. Whereas CD8+ T-cell ablation alone resulted in progressive tumor growth (Fig. 4b), additional Treg depletion enabled the mice to again reject the tumor (Fig. 4e). The only difference to non-depleted wild-type mice was that the FBL-3 tumors increased in size for up to 4 days longer before they were rapidly rejected. To exclude a possible role of NK cells, the additional depletion of those cells in the group of mice lacking Tregs and CD8+ T cells was performed (Fig. 4f). Such mice could still reject the tumor, demonstrating that NK cells had no effect on tumor rejection in CD8+-depleted mice. In order to demonstrate that tumor rejection in mice depleted for CD8+ T cells and Tregs (Fig. 4e) was due to effector CD4+ T-cell responses, tumor growth was tested in mice lacking both CD4+ (including Tregs) and CD8+ T cells. In the absence of these T-cell compartments, no control of tumor growth was observed (Fig. 4g). This experiment suggests that CD4+ T cells can mediate potent anti-tumor effects when cytotoxic CD8+ T cells are absent but that they are tightly controlled in their activity by Foxp3+ Tregs.Fig. 4

Bottom Line: We show here that there is an expansion of tumor-specific CD4(+) T cells producing cytokines and cytotoxic molecule granzyme B (GzmB) in the early phase of tumor growth.Importantly, we demonstrate that in vivo depletion of regulatory T cells (Tregs) and CD8(+) T cells in FBL-3-bearing DEREG transgenic mice augments IL-2 and GzmB production by CD4(+) T cells and increases FV-specific CD4(+) T-cell effector and cytotoxic responses leading to the complete tumor regression.Therefore, the capacity to reject tumor acquired by tumor-reactive CD4(+) T cells largely depends on the direct suppressive activity of Tregs.

View Article: PubMed Central - PubMed

Affiliation: Institute for Virology, University of Duisburg-Essen, Virchowstr 179, 45147, Essen, Germany. ilseyar.akhmetzyanova@uni-due.de

ABSTRACT
The important role of tumor-specific cytotoxic CD8(+) T cells is well defined in the immune control of the tumors, but the role of effector CD4(+) T cells is poorly understood. In the current research, we have used a murine retrovirus-induced tumor cell line of C57BL/6 mouse origin, namely FBL-3 cells, as a model to study basic mechanisms of immunological control and escape during tumor formation. This study shows that tumor-specific CD4(+) T cells are able to protect against virus-induced tumor cells. We show here that there is an expansion of tumor-specific CD4(+) T cells producing cytokines and cytotoxic molecule granzyme B (GzmB) in the early phase of tumor growth. Importantly, we demonstrate that in vivo depletion of regulatory T cells (Tregs) and CD8(+) T cells in FBL-3-bearing DEREG transgenic mice augments IL-2 and GzmB production by CD4(+) T cells and increases FV-specific CD4(+) T-cell effector and cytotoxic responses leading to the complete tumor regression. Therefore, the capacity to reject tumor acquired by tumor-reactive CD4(+) T cells largely depends on the direct suppressive activity of Tregs. We suggest that a cytotoxic CD4(+) T-cell immune response may be induced to enhance resistance against oncovirus-associated tumors.

Show MeSH
Related in: MedlinePlus