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The pro-oxidative drug WF-10 inhibits serial killing by primary human cytotoxic T-cells

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ABSTRACT

Cytotoxic T-cells (CTLs) play an important role in many immune-mediated inflammatory diseases. Targeting cytotoxicity of CTLs would allow to interfere with immune-mediated tissue destruction. Here we demonstrate that WF-10, a pro-oxidative compound, inhibits CTL-mediated cytotoxicity. WF-10 did not influence early steps of target-cell killing, but impaired the ability of CTLs to detach from the initial target cell and to move to a second target cell. This reduced serial killing was accompanied by stronger enrichment of the adhesion molecule LFA-1 in the cytolytic immune synapse. LFA-1 clustering requires activation of the actin-bundling protein L-plastin and was accordingly diminished in L-plastin knockdown cells. Interestingly, WF-10 likely acts through regulating L-plastin: (I) It induced L-plastin activation through phosphorylation leading to enhanced LFA-1-mediated cell adhesion, and, importantly, (II) WF-10 lost its influence on target-cell killing in L-plastin knockdown cells. Finally, we demonstrate that WF-10 can improve immunosuppression by conventional drugs. Thus, while cyclosporine A alone had no significant effect on cytotoxicity of CTLs, a combination of cyclosporine A and WF-10 blocked target-cell killing synergistically. Together, our findings suggest that WF-10 – either alone or in combination with conventional immunosuppressive drugs – may be efficient to control progression of diseases, in which CTLs are crucially involved.

No MeSH data available.


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WF-10 induced L-plastin phosphorylation. (a) F-actin content in CTLs was analyzed by flow cytometry. CTLs were incubated with WF-10 for 2 h and stained with SiR actin (n=3; S.E.M.; *P<0.05). (b) CTLs were incubated with WF-10 for 2 h as indicated. After cell lysis, cytoplasmic fractions were prepared and proteins were subjected to western blot analysis using antibodies that are specific for phopsho-L-plastin (pLPL), L-plastin (LPL) or GAPDH. The blot is representative for three independent experiments. (c) Phosphorylation of L-plastin was analyzed in E/T couples using InFlow microscopy. The time point is 75 min after E/T couple formation (n=3; S.E.M.; *P<0.05).
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fig5: WF-10 induced L-plastin phosphorylation. (a) F-actin content in CTLs was analyzed by flow cytometry. CTLs were incubated with WF-10 for 2 h and stained with SiR actin (n=3; S.E.M.; *P<0.05). (b) CTLs were incubated with WF-10 for 2 h as indicated. After cell lysis, cytoplasmic fractions were prepared and proteins were subjected to western blot analysis using antibodies that are specific for phopsho-L-plastin (pLPL), L-plastin (LPL) or GAPDH. The blot is representative for three independent experiments. (c) Phosphorylation of L-plastin was analyzed in E/T couples using InFlow microscopy. The time point is 75 min after E/T couple formation (n=3; S.E.M.; *P<0.05).

Mentions: Enhancement of avidity of LFA-1 in the immune synapse between T-cells and APCs is dependent on the actin cytoskeleton.4 The higher LFA-1 enrichment in the cytolytic immune synapse could, therefore, be due to a missregulated actin cytoskeleton. In line with this assumption, we found a concentration-dependent increase of the F-actin content in CTLs that were treated with, respectively, 50, 100, 200 or 600 μM WF-10 for 2 h as measured by flow cytometry (Figure 5a).


The pro-oxidative drug WF-10 inhibits serial killing by primary human cytotoxic T-cells
WF-10 induced L-plastin phosphorylation. (a) F-actin content in CTLs was analyzed by flow cytometry. CTLs were incubated with WF-10 for 2 h and stained with SiR actin (n=3; S.E.M.; *P<0.05). (b) CTLs were incubated with WF-10 for 2 h as indicated. After cell lysis, cytoplasmic fractions were prepared and proteins were subjected to western blot analysis using antibodies that are specific for phopsho-L-plastin (pLPL), L-plastin (LPL) or GAPDH. The blot is representative for three independent experiments. (c) Phosphorylation of L-plastin was analyzed in E/T couples using InFlow microscopy. The time point is 75 min after E/T couple formation (n=3; S.E.M.; *P<0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4979520&req=5

fig5: WF-10 induced L-plastin phosphorylation. (a) F-actin content in CTLs was analyzed by flow cytometry. CTLs were incubated with WF-10 for 2 h and stained with SiR actin (n=3; S.E.M.; *P<0.05). (b) CTLs were incubated with WF-10 for 2 h as indicated. After cell lysis, cytoplasmic fractions were prepared and proteins were subjected to western blot analysis using antibodies that are specific for phopsho-L-plastin (pLPL), L-plastin (LPL) or GAPDH. The blot is representative for three independent experiments. (c) Phosphorylation of L-plastin was analyzed in E/T couples using InFlow microscopy. The time point is 75 min after E/T couple formation (n=3; S.E.M.; *P<0.05).
Mentions: Enhancement of avidity of LFA-1 in the immune synapse between T-cells and APCs is dependent on the actin cytoskeleton.4 The higher LFA-1 enrichment in the cytolytic immune synapse could, therefore, be due to a missregulated actin cytoskeleton. In line with this assumption, we found a concentration-dependent increase of the F-actin content in CTLs that were treated with, respectively, 50, 100, 200 or 600 μM WF-10 for 2 h as measured by flow cytometry (Figure 5a).

View Article: PubMed Central - PubMed

ABSTRACT

Cytotoxic T-cells (CTLs) play an important role in many immune-mediated inflammatory diseases. Targeting cytotoxicity of CTLs would allow to interfere with immune-mediated tissue destruction. Here we demonstrate that WF-10, a pro-oxidative compound, inhibits CTL-mediated cytotoxicity. WF-10 did not influence early steps of target-cell killing, but impaired the ability of CTLs to detach from the initial target cell and to move to a second target cell. This reduced serial killing was accompanied by stronger enrichment of the adhesion molecule LFA-1 in the cytolytic immune synapse. LFA-1 clustering requires activation of the actin-bundling protein L-plastin and was accordingly diminished in L-plastin knockdown cells. Interestingly, WF-10 likely acts through regulating L-plastin: (I) It induced L-plastin activation through phosphorylation leading to enhanced LFA-1-mediated cell adhesion, and, importantly, (II) WF-10 lost its influence on target-cell killing in L-plastin knockdown cells. Finally, we demonstrate that WF-10 can improve immunosuppression by conventional drugs. Thus, while cyclosporine A alone had no significant effect on cytotoxicity of CTLs, a combination of cyclosporine A and WF-10 blocked target-cell killing synergistically. Together, our findings suggest that WF-10 &ndash; either alone or in combination with conventional immunosuppressive drugs &ndash; may be efficient to control progression of diseases, in which CTLs are crucially involved.

No MeSH data available.


Related in: MedlinePlus