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

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 – 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.


Enhanced LFA-1 enrichment in the cytolytic immune synapse in WF-10-treated CTLs. (a) InFlow microscopy images show representative E/T couples from redirected killing assays. The nuclei were stained with Dapi (blue), LFA-1 with anti-CD18-FITC antibodies (green) and CD3 with anti-CD3-PeTxR antibodies (red). The merge shows the overlay of all colors. The images are representative for four independent experiments. Yellow indicates the overlay of red and green. (b) The fluorescence intensity of LFA-1 was calculated in the E/T contact zone (=cytolytic immune synapse) (n=3; S.E.M.; ***P<0.001). (c) CTLs of E/T couples that had a strong LFA-1 enrichment, that is, at least 50% of the cellular LFA-1, were quantified for control or WF-10-treated CTLs and for two time points (15 and 75 min) (n=3; S.E.M.; *P<0.05).
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fig4: Enhanced LFA-1 enrichment in the cytolytic immune synapse in WF-10-treated CTLs. (a) InFlow microscopy images show representative E/T couples from redirected killing assays. The nuclei were stained with Dapi (blue), LFA-1 with anti-CD18-FITC antibodies (green) and CD3 with anti-CD3-PeTxR antibodies (red). The merge shows the overlay of all colors. The images are representative for four independent experiments. Yellow indicates the overlay of red and green. (b) The fluorescence intensity of LFA-1 was calculated in the E/T contact zone (=cytolytic immune synapse) (n=3; S.E.M.; ***P<0.001). (c) CTLs of E/T couples that had a strong LFA-1 enrichment, that is, at least 50% of the cellular LFA-1, were quantified for control or WF-10-treated CTLs and for two time points (15 and 75 min) (n=3; S.E.M.; *P<0.05).

Mentions: We next sought to investigate the underlying reason for sustained cytolytic synapse formation and eventual inhibition of serial killing. One possible reason for the inhibition of serial killing could be an increased adhesion of CTLs to their (initial) target cells. An important adhesion molecule that mediates such cell/cell interactions is LFA-1.30 We therefore measured LFA-1 clustering at the E/T immune synapse using InFlow microscopy (Figure 4a). We found that LFA-1 was equally distributed on the CTL surface in the absence of OKT-3. LFA-1 enriched in the cytolytic immune synapse in the presence of OKT-3. This strong LFA-1 enrichment was transient, as the number of cells with a strong LFA-1 enrichment in the cytolytic immune synapse reduced over time (Figure 4c). In contrast to control conditions, WF-10 pretreatment of CTLs led to a profound (Figure 4b) and sustained (Figure 4c) increment of LFA-1 enrichment in the cytolytic immune synapse. Such an increase in LFA-1 concentration in the cytolytic immune synapse provides a molecular explanation for the inhibition of serial killing by WF-10.


The pro-oxidative drug WF-10 inhibits serial killing by primary human cytotoxic T-cells
Enhanced LFA-1 enrichment in the cytolytic immune synapse in WF-10-treated CTLs. (a) InFlow microscopy images show representative E/T couples from redirected killing assays. The nuclei were stained with Dapi (blue), LFA-1 with anti-CD18-FITC antibodies (green) and CD3 with anti-CD3-PeTxR antibodies (red). The merge shows the overlay of all colors. The images are representative for four independent experiments. Yellow indicates the overlay of red and green. (b) The fluorescence intensity of LFA-1 was calculated in the E/T contact zone (=cytolytic immune synapse) (n=3; S.E.M.; ***P<0.001). (c) CTLs of E/T couples that had a strong LFA-1 enrichment, that is, at least 50% of the cellular LFA-1, were quantified for control or WF-10-treated CTLs and for two time points (15 and 75 min) (n=3; S.E.M.; *P<0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig4: Enhanced LFA-1 enrichment in the cytolytic immune synapse in WF-10-treated CTLs. (a) InFlow microscopy images show representative E/T couples from redirected killing assays. The nuclei were stained with Dapi (blue), LFA-1 with anti-CD18-FITC antibodies (green) and CD3 with anti-CD3-PeTxR antibodies (red). The merge shows the overlay of all colors. The images are representative for four independent experiments. Yellow indicates the overlay of red and green. (b) The fluorescence intensity of LFA-1 was calculated in the E/T contact zone (=cytolytic immune synapse) (n=3; S.E.M.; ***P<0.001). (c) CTLs of E/T couples that had a strong LFA-1 enrichment, that is, at least 50% of the cellular LFA-1, were quantified for control or WF-10-treated CTLs and for two time points (15 and 75 min) (n=3; S.E.M.; *P<0.05).
Mentions: We next sought to investigate the underlying reason for sustained cytolytic synapse formation and eventual inhibition of serial killing. One possible reason for the inhibition of serial killing could be an increased adhesion of CTLs to their (initial) target cells. An important adhesion molecule that mediates such cell/cell interactions is LFA-1.30 We therefore measured LFA-1 clustering at the E/T immune synapse using InFlow microscopy (Figure 4a). We found that LFA-1 was equally distributed on the CTL surface in the absence of OKT-3. LFA-1 enriched in the cytolytic immune synapse in the presence of OKT-3. This strong LFA-1 enrichment was transient, as the number of cells with a strong LFA-1 enrichment in the cytolytic immune synapse reduced over time (Figure 4c). In contrast to control conditions, WF-10 pretreatment of CTLs led to a profound (Figure 4b) and sustained (Figure 4c) increment of LFA-1 enrichment in the cytolytic immune synapse. Such an increase in LFA-1 concentration in the cytolytic immune synapse provides a molecular explanation for the inhibition of serial killing by WF-10.

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.