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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 influence the degranulation of peripheral blood CD3−CD56dim NK cells.(A) K562 cells (E/T ratio 2∶1) and CD107a antibody were added to NK cells cultured with IL-2 (200 IU/ml) and varying concentrations of rATG, alemtuzumab, daclizumab and rIgG. NK cells were harvested and stained for CD3 and CD56. For analysis the percentage of CD107a+ NK cells of controls (without K562) was subtracted from the CD107a+ NK cells co-incubated with K562 cells. Values demonstrate CD107a expression on CD56dim NK cells normalized to untreated cells, which were set at 100%. Results are displayed as means±SD for 5 independent experiments. Asterisks (*) indicate values that showed significantly less degranulation compared to untreated controls: *p<0.05, ** p<0.01, *** p<0.001. (B) A degranulation assay was performed (A) without the addition of K562 cells. Results are displayed as the mean of CD107a+ cells on CD56dim NK cells±SD (n = 5). Asterisks (*) indicate values that showed significantly higher degranulation compared to untreated controls: *p<0.05, ** p<0.01, *** p<0.001. (C) Representative FACS dot plots of CD56+CD3− NK cells stained for CD107a. Treatment with 0.1 µg/ml rATG and alemtuzumab produced a higher induction of CD107a in CD56dim NK cells compared to 0.1 µg/ml daclizumab and rIgG.
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pone-0004709-g004: Rabbit ATG and alemtuzumab influence the degranulation of peripheral blood CD3−CD56dim NK cells.(A) K562 cells (E/T ratio 2∶1) and CD107a antibody were added to NK cells cultured with IL-2 (200 IU/ml) and varying concentrations of rATG, alemtuzumab, daclizumab and rIgG. NK cells were harvested and stained for CD3 and CD56. For analysis the percentage of CD107a+ NK cells of controls (without K562) was subtracted from the CD107a+ NK cells co-incubated with K562 cells. Values demonstrate CD107a expression on CD56dim NK cells normalized to untreated cells, which were set at 100%. Results are displayed as means±SD for 5 independent experiments. Asterisks (*) indicate values that showed significantly less degranulation compared to untreated controls: *p<0.05, ** p<0.01, *** p<0.001. (B) A degranulation assay was performed (A) without the addition of K562 cells. Results are displayed as the mean of CD107a+ cells on CD56dim NK cells±SD (n = 5). Asterisks (*) indicate values that showed significantly higher degranulation compared to untreated controls: *p<0.05, ** p<0.01, *** p<0.001. (C) Representative FACS dot plots of CD56+CD3− NK cells stained for CD107a. Treatment with 0.1 µg/ml rATG and alemtuzumab produced a higher induction of CD107a in CD56dim NK cells compared to 0.1 µg/ml daclizumab and rIgG.

Mentions: When we analyzed the degranulation capacity of NK cells preincubated with rATG by the measurement of CD107a, a marker significantly upregulated on the surface of NK cells following stimulation with MHC-devoid targets, we detected a significant impairment even at a low concentration of 0.1 µg/ml rATG, and this was observed exclusively in CD56dim NK cells (56.8%±11.6% versus 100%, p<0.05, Figure 4A). As was seen with rATG, a significant decrease of degranulation capacity of CD56dim NK cells after pre-incubation with alemtuzumab was detected, even at a concentration of 0.1 µg/ml (52.5%±7.8% versus 100%, p<0.001, Figure 4A), in contrast to control antibodies (rIgG, daclizumab), which exhibit induction of degranulation at higher concentrations (50 µg/ml–100 µg/ml). In contrast to rATG, no dose-dependent effect was observed for alemtuzumab (Figure 4A). Interestingly, both rATG and alemtuzumab induced degranulation of NK cells without additional sensitization by adding K562 cells. In comparison with rATG a stronger CD107a induction in NK cells with alemtuzumab was detected (e.g. 0.1 µg/ml alemtuzumab 18.3%±6.6% versus 0.1 µg/ml rATG 7.3%±3.0%, p<0.001, Figure 4B,C). In contrast, induction of degranulation by rIgG and daclizumab was dose-dependent, while the latter antibody showed only moderate degranulation induction on NK cells (5.3%±2.8% degranulation, 100 µg/ml).


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 influence the degranulation of peripheral blood CD3−CD56dim NK cells.(A) K562 cells (E/T ratio 2∶1) and CD107a antibody were added to NK cells cultured with IL-2 (200 IU/ml) and varying concentrations of rATG, alemtuzumab, daclizumab and rIgG. NK cells were harvested and stained for CD3 and CD56. For analysis the percentage of CD107a+ NK cells of controls (without K562) was subtracted from the CD107a+ NK cells co-incubated with K562 cells. Values demonstrate CD107a expression on CD56dim NK cells normalized to untreated cells, which were set at 100%. Results are displayed as means±SD for 5 independent experiments. Asterisks (*) indicate values that showed significantly less degranulation compared to untreated controls: *p<0.05, ** p<0.01, *** p<0.001. (B) A degranulation assay was performed (A) without the addition of K562 cells. Results are displayed as the mean of CD107a+ cells on CD56dim NK cells±SD (n = 5). Asterisks (*) indicate values that showed significantly higher degranulation compared to untreated controls: *p<0.05, ** p<0.01, *** p<0.001. (C) Representative FACS dot plots of CD56+CD3− NK cells stained for CD107a. Treatment with 0.1 µg/ml rATG and alemtuzumab produced a higher induction of CD107a in CD56dim NK cells compared to 0.1 µg/ml daclizumab and rIgG.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2651595&req=5

pone-0004709-g004: Rabbit ATG and alemtuzumab influence the degranulation of peripheral blood CD3−CD56dim NK cells.(A) K562 cells (E/T ratio 2∶1) and CD107a antibody were added to NK cells cultured with IL-2 (200 IU/ml) and varying concentrations of rATG, alemtuzumab, daclizumab and rIgG. NK cells were harvested and stained for CD3 and CD56. For analysis the percentage of CD107a+ NK cells of controls (without K562) was subtracted from the CD107a+ NK cells co-incubated with K562 cells. Values demonstrate CD107a expression on CD56dim NK cells normalized to untreated cells, which were set at 100%. Results are displayed as means±SD for 5 independent experiments. Asterisks (*) indicate values that showed significantly less degranulation compared to untreated controls: *p<0.05, ** p<0.01, *** p<0.001. (B) A degranulation assay was performed (A) without the addition of K562 cells. Results are displayed as the mean of CD107a+ cells on CD56dim NK cells±SD (n = 5). Asterisks (*) indicate values that showed significantly higher degranulation compared to untreated controls: *p<0.05, ** p<0.01, *** p<0.001. (C) Representative FACS dot plots of CD56+CD3− NK cells stained for CD107a. Treatment with 0.1 µg/ml rATG and alemtuzumab produced a higher induction of CD107a in CD56dim NK cells compared to 0.1 µg/ml daclizumab and rIgG.
Mentions: When we analyzed the degranulation capacity of NK cells preincubated with rATG by the measurement of CD107a, a marker significantly upregulated on the surface of NK cells following stimulation with MHC-devoid targets, we detected a significant impairment even at a low concentration of 0.1 µg/ml rATG, and this was observed exclusively in CD56dim NK cells (56.8%±11.6% versus 100%, p<0.05, Figure 4A). As was seen with rATG, a significant decrease of degranulation capacity of CD56dim NK cells after pre-incubation with alemtuzumab was detected, even at a concentration of 0.1 µg/ml (52.5%±7.8% versus 100%, p<0.001, Figure 4A), in contrast to control antibodies (rIgG, daclizumab), which exhibit induction of degranulation at higher concentrations (50 µg/ml–100 µg/ml). In contrast to rATG, no dose-dependent effect was observed for alemtuzumab (Figure 4A). Interestingly, both rATG and alemtuzumab induced degranulation of NK cells without additional sensitization by adding K562 cells. In comparison with rATG a stronger CD107a induction in NK cells with alemtuzumab was detected (e.g. 0.1 µg/ml alemtuzumab 18.3%±6.6% versus 0.1 µg/ml rATG 7.3%±3.0%, p<0.001, Figure 4B,C). In contrast, induction of degranulation by rIgG and daclizumab was dose-dependent, while the latter antibody showed only moderate degranulation induction on NK cells (5.3%±2.8% degranulation, 100 µg/ml).

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