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Targeting of natural killer cells by rabbit antithymocyte globulin and campath-1H: similar effects independent of specificity.

Stauch D, Dernier A, Sarmiento Marchese E, Kunert K, Volk HD, Pratschke J, Kotsch K - PLoS ONE (2009)

Bottom Line: Here, we demonstrate that induction therapy with rATG following kidney/pancreas transplantation results in a rapid depletion of NK cells.By generating Fc-parts of rATG and alemtuzumab we illustrate that their ligation to FcgammaRIII (CD16) is sufficient for the significant induction of degranulation, apoptosis and inflammatory cytokine release (FasL, TNFalpha and IFNgamma) exclusively in CD3(-)CD56(dim) NK cells whereas application of rATG and alemtuzumab F(ab) fragments abolishes these effects.These findings are of general importance as our data suggest that NK cells are also mediators of the clinically relevant cytokine release syndrome and that their targeting by therapeutic antibodies should be considered as they are functionally relevant for the effective clearance of opportunistic viral infections and anti-tumor activity posttransplantation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Immunology, Charité Universitätsmedizin Berlin, Campus Mitte, Berlin, Germany.

ABSTRACT
T cell depleting strategies are an integral part of immunosuppressive regimens widely used in the hematological and solid organ transplant setting. Although it is known to induce lymphocytopenia, little is known about the effects of the polyclonal rabbit antithymocyte globulin (rATG) or the monoclonal anti-CD52 antibody alemtuzumab on Natural Killer (NK) cells in detail. Here, we demonstrate that induction therapy with rATG following kidney/pancreas transplantation results in a rapid depletion of NK cells. Treatment of NK cells with rATG and alemtuzumab in vitro leads to impairment of cytotoxicity and induction of apoptosis even at a 10-fold lower concentration (0.1 microg/ml) compared with T and B cells. By generating Fc-parts of rATG and alemtuzumab we illustrate that their ligation to FcgammaRIII (CD16) is sufficient for the significant induction of degranulation, apoptosis and inflammatory cytokine release (FasL, TNFalpha and IFNgamma) exclusively in CD3(-)CD56(dim) NK cells whereas application of rATG and alemtuzumab F(ab) fragments abolishes these effects. These findings are of general importance as our data suggest that NK cells are also mediators of the clinically relevant cytokine release syndrome and that their targeting by therapeutic antibodies should be considered as they are functionally relevant for the effective clearance of opportunistic viral infections and anti-tumor activity posttransplantation.

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Rabbit ATG and alemtuzumab increase FasL, TNFα and IFNγ mRNA in NK cells.(A) IL-2 (200 IU/ml) pre-activated NK cells cultured in the presence of rATG were analyzed for FasL, TNFα and IFNγ mRNA after 1, 2, 3 and 6 hours of co-culture. rATG induced a rapid and dose-dependent induction of FasL, TNFα and IFNγ mRNA in NK cells which decreased after 6 hours of co-incubation. (B) Similar to rATG, the application of alemtuzumab results in a rapid FasL, TNFα and IFNγ mRNA induction within the first hour of co-incubation. Values demonstrate the results relativized to untreated controls (2−ΔΔct) and are displayed as means of six independent experiments. Asterisks (*) indicate significant values compared to untreated controls: **p<0.01, ***p<0.001. (C) Induction of cytokines by rATG and alemtuzumab (10 µg/ml) was further confirmed for TNFα and IFNγ at the protein level, illustrating a significant induction over time. Asterisks (*) indicate significant values: ***p<0.001 compared to untreated controls.
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pone-0004709-g006: Rabbit ATG and alemtuzumab increase FasL, TNFα and IFNγ mRNA in NK cells.(A) IL-2 (200 IU/ml) pre-activated NK cells cultured in the presence of rATG were analyzed for FasL, TNFα and IFNγ mRNA after 1, 2, 3 and 6 hours of co-culture. rATG induced a rapid and dose-dependent induction of FasL, TNFα and IFNγ mRNA in NK cells which decreased after 6 hours of co-incubation. (B) Similar to rATG, the application of alemtuzumab results in a rapid FasL, TNFα and IFNγ mRNA induction within the first hour of co-incubation. Values demonstrate the results relativized to untreated controls (2−ΔΔct) and are displayed as means of six independent experiments. Asterisks (*) indicate significant values compared to untreated controls: **p<0.01, ***p<0.001. (C) Induction of cytokines by rATG and alemtuzumab (10 µg/ml) was further confirmed for TNFα and IFNγ at the protein level, illustrating a significant induction over time. Asterisks (*) indicate significant values: ***p<0.001 compared to untreated controls.

Mentions: The stimulation of CD16 (FcγRIII) on NK cells can initiate autocrine programmed cell death by the up-regulation of, for example, Fas ligand (FasL) [38]. Whereas freshly isolated NK cells did not constitutively express FasL mRNA, incubation with rATG resulted in a rapid and dose-dependent mRNA induction within the first hour (p<0.001) which decreased after 6 hours of co-culture (Figure 6A). Furthermore it has been demonstrated that anti-T-cell therapy results in the cytokine release syndrome early after administration [39], [40]. Similar to the induction of FasL mRNA, we detected a dose-dependent increase of TNFα and IFNγ mRNA in NK cells within the first hour of co-incubation with rATG (p<0.001, Figure 6A). Additionally, the same induction of FasL, TNFα and IFNγ mRNA was observed for alemtuzumab-treated NK cells (p<0.001, respectively), although the induction of IFNγ and TNFα mRNA was more intense compared to rATG-pretreated NK cells (Figure 6B). Analysis of the control antibodies rIgG and daclizumab showed that both induced a cytokine induction at higher concentrations (10–50 µg/ml), which is clearly below the induction profile of rATG and alemtuzumab (Figure S1). We confirmed the rapid and significant induction of TNFα and IFNγ at the protein level in cell culture supernatants (p<0.001, respectively, Figure 6C), further demonstrating that alemtuzumab resulted in higher cytokine expression levels compared to rATG.


Targeting of natural killer cells by rabbit antithymocyte globulin and campath-1H: similar effects independent of specificity.

Stauch D, Dernier A, Sarmiento Marchese E, Kunert K, Volk HD, Pratschke J, Kotsch K - PLoS ONE (2009)

Rabbit ATG and alemtuzumab increase FasL, TNFα and IFNγ mRNA in NK cells.(A) IL-2 (200 IU/ml) pre-activated NK cells cultured in the presence of rATG were analyzed for FasL, TNFα and IFNγ mRNA after 1, 2, 3 and 6 hours of co-culture. rATG induced a rapid and dose-dependent induction of FasL, TNFα and IFNγ mRNA in NK cells which decreased after 6 hours of co-incubation. (B) Similar to rATG, the application of alemtuzumab results in a rapid FasL, TNFα and IFNγ mRNA induction within the first hour of co-incubation. Values demonstrate the results relativized to untreated controls (2−ΔΔct) and are displayed as means of six independent experiments. Asterisks (*) indicate significant values compared to untreated controls: **p<0.01, ***p<0.001. (C) Induction of cytokines by rATG and alemtuzumab (10 µg/ml) was further confirmed for TNFα and IFNγ at the protein level, illustrating a significant induction over time. Asterisks (*) indicate significant values: ***p<0.001 compared to untreated controls.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2651595&req=5

pone-0004709-g006: Rabbit ATG and alemtuzumab increase FasL, TNFα and IFNγ mRNA in NK cells.(A) IL-2 (200 IU/ml) pre-activated NK cells cultured in the presence of rATG were analyzed for FasL, TNFα and IFNγ mRNA after 1, 2, 3 and 6 hours of co-culture. rATG induced a rapid and dose-dependent induction of FasL, TNFα and IFNγ mRNA in NK cells which decreased after 6 hours of co-incubation. (B) Similar to rATG, the application of alemtuzumab results in a rapid FasL, TNFα and IFNγ mRNA induction within the first hour of co-incubation. Values demonstrate the results relativized to untreated controls (2−ΔΔct) and are displayed as means of six independent experiments. Asterisks (*) indicate significant values compared to untreated controls: **p<0.01, ***p<0.001. (C) Induction of cytokines by rATG and alemtuzumab (10 µg/ml) was further confirmed for TNFα and IFNγ at the protein level, illustrating a significant induction over time. Asterisks (*) indicate significant values: ***p<0.001 compared to untreated controls.
Mentions: The stimulation of CD16 (FcγRIII) on NK cells can initiate autocrine programmed cell death by the up-regulation of, for example, Fas ligand (FasL) [38]. Whereas freshly isolated NK cells did not constitutively express FasL mRNA, incubation with rATG resulted in a rapid and dose-dependent mRNA induction within the first hour (p<0.001) which decreased after 6 hours of co-culture (Figure 6A). Furthermore it has been demonstrated that anti-T-cell therapy results in the cytokine release syndrome early after administration [39], [40]. Similar to the induction of FasL mRNA, we detected a dose-dependent increase of TNFα and IFNγ mRNA in NK cells within the first hour of co-incubation with rATG (p<0.001, Figure 6A). Additionally, the same induction of FasL, TNFα and IFNγ mRNA was observed for alemtuzumab-treated NK cells (p<0.001, respectively), although the induction of IFNγ and TNFα mRNA was more intense compared to rATG-pretreated NK cells (Figure 6B). Analysis of the control antibodies rIgG and daclizumab showed that both induced a cytokine induction at higher concentrations (10–50 µg/ml), which is clearly below the induction profile of rATG and alemtuzumab (Figure S1). We confirmed the rapid and significant induction of TNFα and IFNγ at the protein level in cell culture supernatants (p<0.001, respectively, Figure 6C), further demonstrating that alemtuzumab resulted in higher cytokine expression levels compared to rATG.

Bottom Line: Here, we demonstrate that induction therapy with rATG following kidney/pancreas transplantation results in a rapid depletion of NK cells.By generating Fc-parts of rATG and alemtuzumab we illustrate that their ligation to FcgammaRIII (CD16) is sufficient for the significant induction of degranulation, apoptosis and inflammatory cytokine release (FasL, TNFalpha and IFNgamma) exclusively in CD3(-)CD56(dim) NK cells whereas application of rATG and alemtuzumab F(ab) fragments abolishes these effects.These findings are of general importance as our data suggest that NK cells are also mediators of the clinically relevant cytokine release syndrome and that their targeting by therapeutic antibodies should be considered as they are functionally relevant for the effective clearance of opportunistic viral infections and anti-tumor activity posttransplantation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Immunology, Charité Universitätsmedizin Berlin, Campus Mitte, Berlin, Germany.

ABSTRACT
T cell depleting strategies are an integral part of immunosuppressive regimens widely used in the hematological and solid organ transplant setting. Although it is known to induce lymphocytopenia, little is known about the effects of the polyclonal rabbit antithymocyte globulin (rATG) or the monoclonal anti-CD52 antibody alemtuzumab on Natural Killer (NK) cells in detail. Here, we demonstrate that induction therapy with rATG following kidney/pancreas transplantation results in a rapid depletion of NK cells. Treatment of NK cells with rATG and alemtuzumab in vitro leads to impairment of cytotoxicity and induction of apoptosis even at a 10-fold lower concentration (0.1 microg/ml) compared with T and B cells. By generating Fc-parts of rATG and alemtuzumab we illustrate that their ligation to FcgammaRIII (CD16) is sufficient for the significant induction of degranulation, apoptosis and inflammatory cytokine release (FasL, TNFalpha and IFNgamma) exclusively in CD3(-)CD56(dim) NK cells whereas application of rATG and alemtuzumab F(ab) fragments abolishes these effects. These findings are of general importance as our data suggest that NK cells are also mediators of the clinically relevant cytokine release syndrome and that their targeting by therapeutic antibodies should be considered as they are functionally relevant for the effective clearance of opportunistic viral infections and anti-tumor activity posttransplantation.

Show MeSH
Related in: MedlinePlus