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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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The influence of regulatory T cells on tumor-specific CD4+ T-cell functions: DEREG mice were inoculated s.c. with 1 × 107 FBL-3 cells on day 0. One day before tumor inoculation, some mice also received DT to deplete Foxp3+ Tregs, and day later monoclonal antibody to deplete CD8+ T cells. At day 6 post-tumor transplantation, lymphocytes from draining lymph nodes were analyzed. a The percentages of CD4+ T cells reactive with I-Ab MHC class II tetramers are shown. Numbers of CD4+CD154+ T cells producing cytokines (IFN-γ, TNF-α, and IL-2) are shown (b). Numbers of activated (positive for the activation-induced isoform CD43) CD4+Foxp3− T cells producing GzmB (c) and representative dot plots of GzmB and tetramer II expression (d) in different treatment of mice are shown. Differences between two groups are indicated (*P < 0.05, **P < 0.005). Results were obtained from three experiments with comparable results
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Fig5: The influence of regulatory T cells on tumor-specific CD4+ T-cell functions: DEREG mice were inoculated s.c. with 1 × 107 FBL-3 cells on day 0. One day before tumor inoculation, some mice also received DT to deplete Foxp3+ Tregs, and day later monoclonal antibody to deplete CD8+ T cells. At day 6 post-tumor transplantation, lymphocytes from draining lymph nodes were analyzed. a The percentages of CD4+ T cells reactive with I-Ab MHC class II tetramers are shown. Numbers of CD4+CD154+ T cells producing cytokines (IFN-γ, TNF-α, and IL-2) are shown (b). Numbers of activated (positive for the activation-induced isoform CD43) CD4+Foxp3− T cells producing GzmB (c) and representative dot plots of GzmB and tetramer II expression (d) in different treatment of mice are shown. Differences between two groups are indicated (*P < 0.05, **P < 0.005). Results were obtained from three experiments with comparable results

Mentions: To prove that Tregs indeed control anti-tumor CD4+ T-cell functions in the FBL-3 model, we analyzed numbers of tumor-specific CD4+ T cells, their cytokine production and cytotoxic activity after Treg ablation with or without additional CD8+ T-cell depletion. In DT-treated DEREG mice challenged with tumor cells for 6 days, we observed a significant increase in the mean percentage of tumor-specific (tetramer II+) CD4+ T cells in comparison with mice that received only FBL-3 cells (Fig. 5a). Moreover, if depletion of Tregs was combined with CD8+ T-cell removal, the CD4+ T-cell response was further significantly enhanced. CD8+ T-cell depletion alone did not influence the mean percentage of tumor-specific CD4+ T cells, suggesting that their expansion was mainly controlled by Tregs (Fig. 5a). Tregs did not only influence CD4+ T-cell expansion but also modified their functional properties. In DT-treated mice, significantly more CD4+CD154+ T cells expressed the cytokines IFN-γ, TNF-α, and IL-2 than in mice receiving only tumor cells (Fig. 5b). Dual depletion of Tregs and CD8+ T cells resulted in slightly higher mean frequencies of cytokine producing CD4+ T cells than after Treg deletion alone but this difference was only significant for IL-2-producing cells (Fig. 5b). To determine possible cytotoxic effects against FBL-3 tumor cells, production of the cytolytic molecule GzmB by activated (CD43+) CD4+ T cells was analyzed. In mice lacking Tregs, the frequency of GzmB-positive cells was significantly higher compared to non-depleted tumor-bearing mice (Fig. 5c, d). Additional ablation of CD8+ T cells together with the Tregs resulted in a significant rise in the mean frequencies of GzmB+ CD4+ T cells in comparison with mice only depleted for Tregs (Fig. 5c). Again, CD8+ T-cell depletion alone did not influence the functional CD4+ T-cell response during tumor rejection (Fig. 5c, d).Fig. 5


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)

The influence of regulatory T cells on tumor-specific CD4+ T-cell functions: DEREG mice were inoculated s.c. with 1 × 107 FBL-3 cells on day 0. One day before tumor inoculation, some mice also received DT to deplete Foxp3+ Tregs, and day later monoclonal antibody to deplete CD8+ T cells. At day 6 post-tumor transplantation, lymphocytes from draining lymph nodes were analyzed. a The percentages of CD4+ T cells reactive with I-Ab MHC class II tetramers are shown. Numbers of CD4+CD154+ T cells producing cytokines (IFN-γ, TNF-α, and IL-2) are shown (b). Numbers of activated (positive for the activation-induced isoform CD43) CD4+Foxp3− T cells producing GzmB (c) and representative dot plots of GzmB and tetramer II expression (d) in different treatment of mice are shown. Differences between two groups are indicated (*P < 0.05, **P < 0.005). Results were obtained from three experiments with comparable results
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Related In: Results  -  Collection

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Fig5: The influence of regulatory T cells on tumor-specific CD4+ T-cell functions: DEREG mice were inoculated s.c. with 1 × 107 FBL-3 cells on day 0. One day before tumor inoculation, some mice also received DT to deplete Foxp3+ Tregs, and day later monoclonal antibody to deplete CD8+ T cells. At day 6 post-tumor transplantation, lymphocytes from draining lymph nodes were analyzed. a The percentages of CD4+ T cells reactive with I-Ab MHC class II tetramers are shown. Numbers of CD4+CD154+ T cells producing cytokines (IFN-γ, TNF-α, and IL-2) are shown (b). Numbers of activated (positive for the activation-induced isoform CD43) CD4+Foxp3− T cells producing GzmB (c) and representative dot plots of GzmB and tetramer II expression (d) in different treatment of mice are shown. Differences between two groups are indicated (*P < 0.05, **P < 0.005). Results were obtained from three experiments with comparable results
Mentions: To prove that Tregs indeed control anti-tumor CD4+ T-cell functions in the FBL-3 model, we analyzed numbers of tumor-specific CD4+ T cells, their cytokine production and cytotoxic activity after Treg ablation with or without additional CD8+ T-cell depletion. In DT-treated DEREG mice challenged with tumor cells for 6 days, we observed a significant increase in the mean percentage of tumor-specific (tetramer II+) CD4+ T cells in comparison with mice that received only FBL-3 cells (Fig. 5a). Moreover, if depletion of Tregs was combined with CD8+ T-cell removal, the CD4+ T-cell response was further significantly enhanced. CD8+ T-cell depletion alone did not influence the mean percentage of tumor-specific CD4+ T cells, suggesting that their expansion was mainly controlled by Tregs (Fig. 5a). Tregs did not only influence CD4+ T-cell expansion but also modified their functional properties. In DT-treated mice, significantly more CD4+CD154+ T cells expressed the cytokines IFN-γ, TNF-α, and IL-2 than in mice receiving only tumor cells (Fig. 5b). Dual depletion of Tregs and CD8+ T cells resulted in slightly higher mean frequencies of cytokine producing CD4+ T cells than after Treg deletion alone but this difference was only significant for IL-2-producing cells (Fig. 5b). To determine possible cytotoxic effects against FBL-3 tumor cells, production of the cytolytic molecule GzmB by activated (CD43+) CD4+ T cells was analyzed. In mice lacking Tregs, the frequency of GzmB-positive cells was significantly higher compared to non-depleted tumor-bearing mice (Fig. 5c, d). Additional ablation of CD8+ T cells together with the Tregs resulted in a significant rise in the mean frequencies of GzmB+ CD4+ T cells in comparison with mice only depleted for Tregs (Fig. 5c). Again, CD8+ T-cell depletion alone did not influence the functional CD4+ T-cell response during tumor rejection (Fig. 5c, d).Fig. 5

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