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Superantigens increase the survival of mice bearing T cell lymphomas by inducing apoptosis of neoplastic cells.

Mundiñano J, Berguer PM, Cabrera G, Lorenzo D, Nepomnaschy I, Piazzon I - PLoS ONE (2010)

Bottom Line: Superantigens bind to major histocompatibility complex class II molecules and interact with T cells expressing a particular T cell receptor Vβ inducing a strong proliferation/deletion response of the superantigen-reactive T cells.However, there have been no attempts to investigate the ability of Sags to induce apoptosis in neoplastic T cells by signaling through the Vβ region of their TCR.The possibility of a therapeutic use of superantigens in lymphoma/leukemia T cell malignancies is discussed.

View Article: PubMed Central - PubMed

Affiliation: ILEX-CONICET, División Medicina Experimental, Instituto de Investigaciones Hematológicas, Academia Nacional de Medicina, Buenos Aires, Argentina.

ABSTRACT
Superantigens bind to major histocompatibility complex class II molecules and interact with T cells expressing a particular T cell receptor Vβ inducing a strong proliferation/deletion response of the superantigen-reactive T cells. However, there have been no attempts to investigate the ability of Sags to induce apoptosis in neoplastic T cells by signaling through the Vβ region of their TCR. In the present study we show that bacterial and MMTV-encoded superantigens induce the apoptosis of AKR/J cognate lymphoma T cells both in vitro and in vivo. The Fas-Fas-L pathway was shown to be involved in the apoptosis of lymphoma T cells induced by bacterial superantigens. In vivo exposure to bacterial superantigens was able to improve the survival of lymphoma bearing mice. Moreover, the permanent expression of a retroviral encoded superantigen induced the complete remission of an aggressive lymphoma in a high percentage of mice. The possibility of a therapeutic use of superantigens in lymphoma/leukemia T cell malignancies is discussed.

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Mechanisms involved in Sag-induced apoptosis.A) Inhibition of Sag-induced apoptosis of lymphoma cells by caspase-8 and caspase-9 inhibitors. T8 cells (2.5×105) co-cultured with peritoneal macrophages (0.7×105) were treated during two hours with 25 µM of Z-IETD-FMK (caspase-8 inhibitor), Z-LEHD-FMK (caspase-9 inhibitor) or DMSO prior to the addition of 10 µg/ml of SEB or PBS. At day 3, cells were recovered, stained with Annexin/7-AAD and analyzed by FACS. The percentage of specific apoptosis was calculated as follows: 100× (% Sag induced apoptosis - % spontaneous apoptosis with PBS)/(100 - % spontaneous apoptosis with PBS). Inhibition of apoptosis by caspase inhibitors was calculated as reduction of specific apoptosis compared to specific apoptosis in the presence of DMSO set as 100%.(B) Inhibition of apoptosis by Fas-Fc fusion protein. T5 or T8 cells (2.5×105) co-cultured with peritoneal macrophages (0.7×105) were treated with SEI, SEB (10 µg/ml) or PBS. Ten µg/ml of Fas-Fc, HuIgG or PBS were added at day 2. At day 4, cells were harvested and stained with Annexin V-7AAD. Specific apoptosis was calculated as described in (A). Inhibition was calculated as reduction of apoptosis compared to apoptosis in the presence of HuIgG set as 100%. (C–D) Sags cause mitochondrial depolarization in cognate lymphoma T cells. T8 lymphoma cells (2.5×105) were cultured in the presence of 10 µg/ml of SEE, SEB or PBS as described in Material and Methods. Cells were harvested 72 hours after Sag exposure and the mitochondrial membrane potential (Δψm) was measured by FACS using DiOC2(3) staining. Representative overlayed histograms for DiOC2(3) fluorescence are depicted in (C).(D) Show the percentage of DiOC2(3)low cells. Data are expressed as the mean ± SD. *p<0.05. (E) Caspase-8 inhibitor decreases the loss of Δψm induced by SEB. T8 lymphoma cells (0.25×106) were incubated with 25 µM Z-IETD-FMK or DMSO for 2 hours before exposure to 10 µg/ml of SEE, SEB or PBS. Cells were harvested 72 hours later and Δψm was measured by FACS using DiOC2(3) staining. Apoptotic cells were identified by their decrease in Δψm (DiOC2(3)low). Data show the percentage of specific apoptosis calculated as described in (A). All the experiments were performed at least three times with similar results.
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pone-0015694-g006: Mechanisms involved in Sag-induced apoptosis.A) Inhibition of Sag-induced apoptosis of lymphoma cells by caspase-8 and caspase-9 inhibitors. T8 cells (2.5×105) co-cultured with peritoneal macrophages (0.7×105) were treated during two hours with 25 µM of Z-IETD-FMK (caspase-8 inhibitor), Z-LEHD-FMK (caspase-9 inhibitor) or DMSO prior to the addition of 10 µg/ml of SEB or PBS. At day 3, cells were recovered, stained with Annexin/7-AAD and analyzed by FACS. The percentage of specific apoptosis was calculated as follows: 100× (% Sag induced apoptosis - % spontaneous apoptosis with PBS)/(100 - % spontaneous apoptosis with PBS). Inhibition of apoptosis by caspase inhibitors was calculated as reduction of specific apoptosis compared to specific apoptosis in the presence of DMSO set as 100%.(B) Inhibition of apoptosis by Fas-Fc fusion protein. T5 or T8 cells (2.5×105) co-cultured with peritoneal macrophages (0.7×105) were treated with SEI, SEB (10 µg/ml) or PBS. Ten µg/ml of Fas-Fc, HuIgG or PBS were added at day 2. At day 4, cells were harvested and stained with Annexin V-7AAD. Specific apoptosis was calculated as described in (A). Inhibition was calculated as reduction of apoptosis compared to apoptosis in the presence of HuIgG set as 100%. (C–D) Sags cause mitochondrial depolarization in cognate lymphoma T cells. T8 lymphoma cells (2.5×105) were cultured in the presence of 10 µg/ml of SEE, SEB or PBS as described in Material and Methods. Cells were harvested 72 hours after Sag exposure and the mitochondrial membrane potential (Δψm) was measured by FACS using DiOC2(3) staining. Representative overlayed histograms for DiOC2(3) fluorescence are depicted in (C).(D) Show the percentage of DiOC2(3)low cells. Data are expressed as the mean ± SD. *p<0.05. (E) Caspase-8 inhibitor decreases the loss of Δψm induced by SEB. T8 lymphoma cells (0.25×106) were incubated with 25 µM Z-IETD-FMK or DMSO for 2 hours before exposure to 10 µg/ml of SEE, SEB or PBS. Cells were harvested 72 hours later and Δψm was measured by FACS using DiOC2(3) staining. Apoptotic cells were identified by their decrease in Δψm (DiOC2(3)low). Data show the percentage of specific apoptosis calculated as described in (A). All the experiments were performed at least three times with similar results.

Mentions: An important inhibition of specific apoptosis could be observed when T8 cells were treated with SEB in the presence of caspase-8 inhibitor (Figure 6A). Similar results were obtained when T5 cells were cultured with SEI in the presence of caspase-8 inhibitor (data not shown). Finally, mouse Fas-Fc protein was used as a competitive inhibitor of Fas/Fas-L interactions [36]. We found that Fas-Fc effectively inhibited apoptosis (Figure 6B) confirming the involvement of the Fas-Fas-L pathway in apoptosis induced by bacterial Sags.


Superantigens increase the survival of mice bearing T cell lymphomas by inducing apoptosis of neoplastic cells.

Mundiñano J, Berguer PM, Cabrera G, Lorenzo D, Nepomnaschy I, Piazzon I - PLoS ONE (2010)

Mechanisms involved in Sag-induced apoptosis.A) Inhibition of Sag-induced apoptosis of lymphoma cells by caspase-8 and caspase-9 inhibitors. T8 cells (2.5×105) co-cultured with peritoneal macrophages (0.7×105) were treated during two hours with 25 µM of Z-IETD-FMK (caspase-8 inhibitor), Z-LEHD-FMK (caspase-9 inhibitor) or DMSO prior to the addition of 10 µg/ml of SEB or PBS. At day 3, cells were recovered, stained with Annexin/7-AAD and analyzed by FACS. The percentage of specific apoptosis was calculated as follows: 100× (% Sag induced apoptosis - % spontaneous apoptosis with PBS)/(100 - % spontaneous apoptosis with PBS). Inhibition of apoptosis by caspase inhibitors was calculated as reduction of specific apoptosis compared to specific apoptosis in the presence of DMSO set as 100%.(B) Inhibition of apoptosis by Fas-Fc fusion protein. T5 or T8 cells (2.5×105) co-cultured with peritoneal macrophages (0.7×105) were treated with SEI, SEB (10 µg/ml) or PBS. Ten µg/ml of Fas-Fc, HuIgG or PBS were added at day 2. At day 4, cells were harvested and stained with Annexin V-7AAD. Specific apoptosis was calculated as described in (A). Inhibition was calculated as reduction of apoptosis compared to apoptosis in the presence of HuIgG set as 100%. (C–D) Sags cause mitochondrial depolarization in cognate lymphoma T cells. T8 lymphoma cells (2.5×105) were cultured in the presence of 10 µg/ml of SEE, SEB or PBS as described in Material and Methods. Cells were harvested 72 hours after Sag exposure and the mitochondrial membrane potential (Δψm) was measured by FACS using DiOC2(3) staining. Representative overlayed histograms for DiOC2(3) fluorescence are depicted in (C).(D) Show the percentage of DiOC2(3)low cells. Data are expressed as the mean ± SD. *p<0.05. (E) Caspase-8 inhibitor decreases the loss of Δψm induced by SEB. T8 lymphoma cells (0.25×106) were incubated with 25 µM Z-IETD-FMK or DMSO for 2 hours before exposure to 10 µg/ml of SEE, SEB or PBS. Cells were harvested 72 hours later and Δψm was measured by FACS using DiOC2(3) staining. Apoptotic cells were identified by their decrease in Δψm (DiOC2(3)low). Data show the percentage of specific apoptosis calculated as described in (A). All the experiments were performed at least three times with similar results.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3008744&req=5

pone-0015694-g006: Mechanisms involved in Sag-induced apoptosis.A) Inhibition of Sag-induced apoptosis of lymphoma cells by caspase-8 and caspase-9 inhibitors. T8 cells (2.5×105) co-cultured with peritoneal macrophages (0.7×105) were treated during two hours with 25 µM of Z-IETD-FMK (caspase-8 inhibitor), Z-LEHD-FMK (caspase-9 inhibitor) or DMSO prior to the addition of 10 µg/ml of SEB or PBS. At day 3, cells were recovered, stained with Annexin/7-AAD and analyzed by FACS. The percentage of specific apoptosis was calculated as follows: 100× (% Sag induced apoptosis - % spontaneous apoptosis with PBS)/(100 - % spontaneous apoptosis with PBS). Inhibition of apoptosis by caspase inhibitors was calculated as reduction of specific apoptosis compared to specific apoptosis in the presence of DMSO set as 100%.(B) Inhibition of apoptosis by Fas-Fc fusion protein. T5 or T8 cells (2.5×105) co-cultured with peritoneal macrophages (0.7×105) were treated with SEI, SEB (10 µg/ml) or PBS. Ten µg/ml of Fas-Fc, HuIgG or PBS were added at day 2. At day 4, cells were harvested and stained with Annexin V-7AAD. Specific apoptosis was calculated as described in (A). Inhibition was calculated as reduction of apoptosis compared to apoptosis in the presence of HuIgG set as 100%. (C–D) Sags cause mitochondrial depolarization in cognate lymphoma T cells. T8 lymphoma cells (2.5×105) were cultured in the presence of 10 µg/ml of SEE, SEB or PBS as described in Material and Methods. Cells were harvested 72 hours after Sag exposure and the mitochondrial membrane potential (Δψm) was measured by FACS using DiOC2(3) staining. Representative overlayed histograms for DiOC2(3) fluorescence are depicted in (C).(D) Show the percentage of DiOC2(3)low cells. Data are expressed as the mean ± SD. *p<0.05. (E) Caspase-8 inhibitor decreases the loss of Δψm induced by SEB. T8 lymphoma cells (0.25×106) were incubated with 25 µM Z-IETD-FMK or DMSO for 2 hours before exposure to 10 µg/ml of SEE, SEB or PBS. Cells were harvested 72 hours later and Δψm was measured by FACS using DiOC2(3) staining. Apoptotic cells were identified by their decrease in Δψm (DiOC2(3)low). Data show the percentage of specific apoptosis calculated as described in (A). All the experiments were performed at least three times with similar results.
Mentions: An important inhibition of specific apoptosis could be observed when T8 cells were treated with SEB in the presence of caspase-8 inhibitor (Figure 6A). Similar results were obtained when T5 cells were cultured with SEI in the presence of caspase-8 inhibitor (data not shown). Finally, mouse Fas-Fc protein was used as a competitive inhibitor of Fas/Fas-L interactions [36]. We found that Fas-Fc effectively inhibited apoptosis (Figure 6B) confirming the involvement of the Fas-Fas-L pathway in apoptosis induced by bacterial Sags.

Bottom Line: Superantigens bind to major histocompatibility complex class II molecules and interact with T cells expressing a particular T cell receptor Vβ inducing a strong proliferation/deletion response of the superantigen-reactive T cells.However, there have been no attempts to investigate the ability of Sags to induce apoptosis in neoplastic T cells by signaling through the Vβ region of their TCR.The possibility of a therapeutic use of superantigens in lymphoma/leukemia T cell malignancies is discussed.

View Article: PubMed Central - PubMed

Affiliation: ILEX-CONICET, División Medicina Experimental, Instituto de Investigaciones Hematológicas, Academia Nacional de Medicina, Buenos Aires, Argentina.

ABSTRACT
Superantigens bind to major histocompatibility complex class II molecules and interact with T cells expressing a particular T cell receptor Vβ inducing a strong proliferation/deletion response of the superantigen-reactive T cells. However, there have been no attempts to investigate the ability of Sags to induce apoptosis in neoplastic T cells by signaling through the Vβ region of their TCR. In the present study we show that bacterial and MMTV-encoded superantigens induce the apoptosis of AKR/J cognate lymphoma T cells both in vitro and in vivo. The Fas-Fas-L pathway was shown to be involved in the apoptosis of lymphoma T cells induced by bacterial superantigens. In vivo exposure to bacterial superantigens was able to improve the survival of lymphoma bearing mice. Moreover, the permanent expression of a retroviral encoded superantigen induced the complete remission of an aggressive lymphoma in a high percentage of mice. The possibility of a therapeutic use of superantigens in lymphoma/leukemia T cell malignancies is discussed.

Show MeSH
Related in: MedlinePlus