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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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Cytokines and functional properties of T cells: B6 mice were inoculated s.c. with 1 × 107 FBL-3 cells. At different time points ptc, lymphocytes from lymph nodes were isolated and investigated. Kinetics of IFN-γ-, TNF-α-, and IL-2 expressing CD154+CD4+ (a) and CD43+CD8+ (c) T cells from lymph nodes are shown. b Numbers of cytokine producing CD4+CD154+ T cells at day 6 ptc are depicted. Each dot represents an individual mouse and the means are indicated by a line. d Intracellular expression of GzmB. Numbers of CD8+CD43+ (white box plots) and CD4+Foxp3− (grey box plots) T cells producing GzmB are shown in drLNs at different days ptc. Differences between two groups are indicated (*P < 0.05, **P < 0.005, ***P < 0.0005). All experiments were repeated three times with comparable results
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Fig2: Cytokines and functional properties of T cells: B6 mice were inoculated s.c. with 1 × 107 FBL-3 cells. At different time points ptc, lymphocytes from lymph nodes were isolated and investigated. Kinetics of IFN-γ-, TNF-α-, and IL-2 expressing CD154+CD4+ (a) and CD43+CD8+ (c) T cells from lymph nodes are shown. b Numbers of cytokine producing CD4+CD154+ T cells at day 6 ptc are depicted. Each dot represents an individual mouse and the means are indicated by a line. d Intracellular expression of GzmB. Numbers of CD8+CD43+ (white box plots) and CD4+Foxp3− (grey box plots) T cells producing GzmB are shown in drLNs at different days ptc. Differences between two groups are indicated (*P < 0.05, **P < 0.005, ***P < 0.0005). All experiments were repeated three times with comparable results

Mentions: Next, we wanted to analyze functional properties of CD4+ and CD8+ T cells during tumor rejection. To this end, we performed kinetic analysis of cytokine and granzyme B (GzmB) expression in T cells after tumor challenge. To analyze the total populations of CD4+ and CD8+ T cells that were activated during tumor rejection, we used the maker CD154 (CD40L) for CD4+ T cells [21] and the activation-associated glycoform of CD43 for CD8+ T cells [22]. In order to exclude Tregs from the effector CD4+ T-cell pool, we also stained for intracellular expression of Foxp3. In drLN nodes, the highest frequency of CD4+ T cells producing the three cytokines IFN-γ, TNF-α, and IL-2 was found between day 4 and 6 ptc (Fig. 2a). At day 8 ptc, the cytokine response already started to decrease, which was earlier than the decline in tetramer II-positive CD4+ T cells (Figs. 1a, 2b). The frequency of CD4+CD154+ T cells producing the three cytokines was significantly higher in drLN than in non-drLN (Fig. 2b and supplementary figure S1, available on-line), which correlated with the increased percentages of tetramer-positive CD4+ T cells in drLN (Fig. 1a). The peak cytokine production by CD8+ T cells in drLN was found at day 8 ptc again showing the delay in the CD8+ T-cell response compared to CD4+ T cells (Fig. 2a, c). In addition, the numbers of CD8+CD43+ T cells producing cytokines were much lower than those of activated CD4+ T cells. No cytokine production by CD8+ T cells was found in non-drLN during tumor rejection (data not shown). As expected, the cytotoxic molecule GzmB was also produced by CD8+CD43+ T cells after tumor challenge (Fig. 2d). The peak of this functional response was found between day 8 and 11 ptc. Remarkably, tumor-induced activation of Foxp3− CD4+ T cells in drLN also resulted in their differentiation into GzmB-producing cells, suggesting a potential cytotoxic role for these cells. Peak GzmB expression in CD4+CD43+ T cells was observed at day 6 ptc, which was again earlier as in the CD8+ T-cell compartment. Thus, both CD8+ and CD4+ T-cell populations in drLN expressed pro-inflammatory cytokines and GzmB in response to tumor challenge, but the CD4+ T-cell response initiated earlier and the magnitude of the response was higher than the CD8+ T-cell response.Fig. 2


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)

Cytokines and functional properties of T cells: B6 mice were inoculated s.c. with 1 × 107 FBL-3 cells. At different time points ptc, lymphocytes from lymph nodes were isolated and investigated. Kinetics of IFN-γ-, TNF-α-, and IL-2 expressing CD154+CD4+ (a) and CD43+CD8+ (c) T cells from lymph nodes are shown. b Numbers of cytokine producing CD4+CD154+ T cells at day 6 ptc are depicted. Each dot represents an individual mouse and the means are indicated by a line. d Intracellular expression of GzmB. Numbers of CD8+CD43+ (white box plots) and CD4+Foxp3− (grey box plots) T cells producing GzmB are shown in drLNs at different days ptc. Differences between two groups are indicated (*P < 0.05, **P < 0.005, ***P < 0.0005). All experiments were repeated three times with comparable results
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3569596&req=5

Fig2: Cytokines and functional properties of T cells: B6 mice were inoculated s.c. with 1 × 107 FBL-3 cells. At different time points ptc, lymphocytes from lymph nodes were isolated and investigated. Kinetics of IFN-γ-, TNF-α-, and IL-2 expressing CD154+CD4+ (a) and CD43+CD8+ (c) T cells from lymph nodes are shown. b Numbers of cytokine producing CD4+CD154+ T cells at day 6 ptc are depicted. Each dot represents an individual mouse and the means are indicated by a line. d Intracellular expression of GzmB. Numbers of CD8+CD43+ (white box plots) and CD4+Foxp3− (grey box plots) T cells producing GzmB are shown in drLNs at different days ptc. Differences between two groups are indicated (*P < 0.05, **P < 0.005, ***P < 0.0005). All experiments were repeated three times with comparable results
Mentions: Next, we wanted to analyze functional properties of CD4+ and CD8+ T cells during tumor rejection. To this end, we performed kinetic analysis of cytokine and granzyme B (GzmB) expression in T cells after tumor challenge. To analyze the total populations of CD4+ and CD8+ T cells that were activated during tumor rejection, we used the maker CD154 (CD40L) for CD4+ T cells [21] and the activation-associated glycoform of CD43 for CD8+ T cells [22]. In order to exclude Tregs from the effector CD4+ T-cell pool, we also stained for intracellular expression of Foxp3. In drLN nodes, the highest frequency of CD4+ T cells producing the three cytokines IFN-γ, TNF-α, and IL-2 was found between day 4 and 6 ptc (Fig. 2a). At day 8 ptc, the cytokine response already started to decrease, which was earlier than the decline in tetramer II-positive CD4+ T cells (Figs. 1a, 2b). The frequency of CD4+CD154+ T cells producing the three cytokines was significantly higher in drLN than in non-drLN (Fig. 2b and supplementary figure S1, available on-line), which correlated with the increased percentages of tetramer-positive CD4+ T cells in drLN (Fig. 1a). The peak cytokine production by CD8+ T cells in drLN was found at day 8 ptc again showing the delay in the CD8+ T-cell response compared to CD4+ T cells (Fig. 2a, c). In addition, the numbers of CD8+CD43+ T cells producing cytokines were much lower than those of activated CD4+ T cells. No cytokine production by CD8+ T cells was found in non-drLN during tumor rejection (data not shown). As expected, the cytotoxic molecule GzmB was also produced by CD8+CD43+ T cells after tumor challenge (Fig. 2d). The peak of this functional response was found between day 8 and 11 ptc. Remarkably, tumor-induced activation of Foxp3− CD4+ T cells in drLN also resulted in their differentiation into GzmB-producing cells, suggesting a potential cytotoxic role for these cells. Peak GzmB expression in CD4+CD43+ T cells was observed at day 6 ptc, which was again earlier as in the CD8+ T-cell compartment. Thus, both CD8+ and CD4+ T-cell populations in drLN expressed pro-inflammatory cytokines and GzmB in response to tumor challenge, but the CD4+ T-cell response initiated earlier and the magnitude of the response was higher than the CD8+ T-cell response.Fig. 2

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