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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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Application of F(ab) rATG and alemtuzumab fragments abolishes apoptosis and targeting of CD16 on CD56+CD16+ NK cells.NK cells were treated either with 1 µg/ml rATG or alemtuzumab or with 1 µg/ml F(ab) fragments of rATG or alemtuzumab for 1, 2, 3 and 6 hours. Controls remained untreated. Values of CD56+CD16+ NK cells are displayed as means±SD (n = 5); asterisks (*) indicate significant values compared to controls (***p<0.001, left panel). F(ab) fragments of rATG or alemtuzumab did not induce apoptosis and did not target CD16. Annexin V+/PI− CD56+CD3− cells were normalized to untreated cells. Values are displayed as means±SD of five independent experiments. P values are related to the rATG/alemtuzumab treatment: ***p<0.001.
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pone-0004709-g008: Application of F(ab) rATG and alemtuzumab fragments abolishes apoptosis and targeting of CD16 on CD56+CD16+ NK cells.NK cells were treated either with 1 µg/ml rATG or alemtuzumab or with 1 µg/ml F(ab) fragments of rATG or alemtuzumab for 1, 2, 3 and 6 hours. Controls remained untreated. Values of CD56+CD16+ NK cells are displayed as means±SD (n = 5); asterisks (*) indicate significant values compared to controls (***p<0.001, left panel). F(ab) fragments of rATG or alemtuzumab did not induce apoptosis and did not target CD16. Annexin V+/PI− CD56+CD3− cells were normalized to untreated cells. Values are displayed as means±SD of five independent experiments. P values are related to the rATG/alemtuzumab treatment: ***p<0.001.

Mentions: In order to investigate the influence of the unspecific binding of the Fc-part to FcγRIII, we generated F(ab) fragments and Fc-parts of a CD16 blocking antibody (clone 3G8), rATG and alemtuzumab. Blocking FcγRIII on NK cells by F(ab) fragments of an anti-CD16 resulted in a significant inhibition of FasL (p<0.001), TNFα (p<0.05) and IFNγ (p<0.001) mRNA induction. Additionally, the application of F(ab) fragments of either rATG or alemtuzumab further did not cause FasL, TNFα and IFNγ mRNA induction (Figure S2). Next, we tested generated Fc-parts and could illustrate that the application of rATG and alemtuzumab Fc-parts only is sufficient to induce a significant cytokine induction and apoptosis in NK cells compared with F(ab) fragments. This induction showed a similar expression level compared with intact IgG antibodies. In contrast, the application of control rIgG, a monoclonal anti-CD3 antibody (OKT3, IgG2a isotype) or daclizumab (Figure S1, 1 µg/ml for 1 hour) did not lead to induced cytokine levels and increased apoptosis (Figure 7A,B). Moreover, CD16 ligation by rATG and alemtuzumab Fc-parts resulted in degranulation of NK cells further emphasizing that antigen-specific crosslinking of antibodies is not necessary for the observed effector effects (Figure 7C). After the addition of rATG or alemtuzumab F(ab) fragments, targeting of CD16 on CD56+CD16+ cells and induction of NK cell apoptosis was abolished compared to the application of intact IgG antibodies (p<0.001, Figure 8). Thus, our results clearly illustrate that FcγRIII ligation with the Fc-part of rATG or alemtuzumab is sufficient to induce NK cell apoptosis and cytokine release and that this effect is independent of antibody specificity.


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)

Application of F(ab) rATG and alemtuzumab fragments abolishes apoptosis and targeting of CD16 on CD56+CD16+ NK cells.NK cells were treated either with 1 µg/ml rATG or alemtuzumab or with 1 µg/ml F(ab) fragments of rATG or alemtuzumab for 1, 2, 3 and 6 hours. Controls remained untreated. Values of CD56+CD16+ NK cells are displayed as means±SD (n = 5); asterisks (*) indicate significant values compared to controls (***p<0.001, left panel). F(ab) fragments of rATG or alemtuzumab did not induce apoptosis and did not target CD16. Annexin V+/PI− CD56+CD3− cells were normalized to untreated cells. Values are displayed as means±SD of five independent experiments. P values are related to the rATG/alemtuzumab treatment: ***p<0.001.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2651595&req=5

pone-0004709-g008: Application of F(ab) rATG and alemtuzumab fragments abolishes apoptosis and targeting of CD16 on CD56+CD16+ NK cells.NK cells were treated either with 1 µg/ml rATG or alemtuzumab or with 1 µg/ml F(ab) fragments of rATG or alemtuzumab for 1, 2, 3 and 6 hours. Controls remained untreated. Values of CD56+CD16+ NK cells are displayed as means±SD (n = 5); asterisks (*) indicate significant values compared to controls (***p<0.001, left panel). F(ab) fragments of rATG or alemtuzumab did not induce apoptosis and did not target CD16. Annexin V+/PI− CD56+CD3− cells were normalized to untreated cells. Values are displayed as means±SD of five independent experiments. P values are related to the rATG/alemtuzumab treatment: ***p<0.001.
Mentions: In order to investigate the influence of the unspecific binding of the Fc-part to FcγRIII, we generated F(ab) fragments and Fc-parts of a CD16 blocking antibody (clone 3G8), rATG and alemtuzumab. Blocking FcγRIII on NK cells by F(ab) fragments of an anti-CD16 resulted in a significant inhibition of FasL (p<0.001), TNFα (p<0.05) and IFNγ (p<0.001) mRNA induction. Additionally, the application of F(ab) fragments of either rATG or alemtuzumab further did not cause FasL, TNFα and IFNγ mRNA induction (Figure S2). Next, we tested generated Fc-parts and could illustrate that the application of rATG and alemtuzumab Fc-parts only is sufficient to induce a significant cytokine induction and apoptosis in NK cells compared with F(ab) fragments. This induction showed a similar expression level compared with intact IgG antibodies. In contrast, the application of control rIgG, a monoclonal anti-CD3 antibody (OKT3, IgG2a isotype) or daclizumab (Figure S1, 1 µg/ml for 1 hour) did not lead to induced cytokine levels and increased apoptosis (Figure 7A,B). Moreover, CD16 ligation by rATG and alemtuzumab Fc-parts resulted in degranulation of NK cells further emphasizing that antigen-specific crosslinking of antibodies is not necessary for the observed effector effects (Figure 7C). After the addition of rATG or alemtuzumab F(ab) fragments, targeting of CD16 on CD56+CD16+ cells and induction of NK cell apoptosis was abolished compared to the application of intact IgG antibodies (p<0.001, Figure 8). Thus, our results clearly illustrate that FcγRIII ligation with the Fc-part of rATG or alemtuzumab is sufficient to induce NK cell apoptosis and cytokine release and that this effect is independent of antibody specificity.

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