Limits...
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.

Show MeSH

Related in: MedlinePlus

Rabbit ATG and alemtuzumab target surface expression of CD16 on NK cells.Human NK cells cultured with IL-2 (200 IU/ml) and different concentrations of rATG, alemtuzumab or control antibodies such as rIgG and daclizumab (18 hours) were harvested, washed and stained for CD3, CD56, CD8 and CD16. (A) The figure illustrates the average percentages of CD16 and CD8 staining on CD3−CD56+ NK cells after co-incubation with varying concentrations of rATG. Results are displayed as means±SD (n = 5); asterisks (*) denote significant differences compared to untreated controls: ***p<0.001. (B) The mean percentage of CD16 on CD3−CD56+ NK incubated with different concentrations of rATG, alemtuzumab, daclizumab and rIgG is shown. Results are displayed as means±SD of five independent experiments. Asterisks (*) indicate values that showed significantly less CD16 expression compared to untreated controls: ***p<0.001.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2651595&req=5

pone-0004709-g002: Rabbit ATG and alemtuzumab target surface expression of CD16 on NK cells.Human NK cells cultured with IL-2 (200 IU/ml) and different concentrations of rATG, alemtuzumab or control antibodies such as rIgG and daclizumab (18 hours) were harvested, washed and stained for CD3, CD56, CD8 and CD16. (A) The figure illustrates the average percentages of CD16 and CD8 staining on CD3−CD56+ NK cells after co-incubation with varying concentrations of rATG. Results are displayed as means±SD (n = 5); asterisks (*) denote significant differences compared to untreated controls: ***p<0.001. (B) The mean percentage of CD16 on CD3−CD56+ NK incubated with different concentrations of rATG, alemtuzumab, daclizumab and rIgG is shown. Results are displayed as means±SD of five independent experiments. Asterisks (*) indicate values that showed significantly less CD16 expression compared to untreated controls: ***p<0.001.

Mentions: NK cells derived from CD34+ hematopoietic stem cells undergo differentiation via NK cell precursors in the bone marrow through acquisition of functional surface receptors [34]. Therefore, certain NK cell specific receptors should not be targeted by rATG. As expected we could not detect any influence of rATG on the surface expression of the activating cytotoxicity receptors NKp30, NKp44, NKp46, NKG2D and killer-cell immunoglobulin-like receptors (KIRs) including KIR2DL1 and KIR3DL1 (data not shown). In contrast we confirmed previous data illustrating that rATG targets CD8 and CD16 and this effect was dose-dependent (Figure 2A) [35]. Targeting of CD16 was observed in the presence of 0.1 µg rATG (67%±10.8% versus 33.6%±4.1%, p<0.001), and CD16 was further decreased to 1.2%±0.6% at a concentration of 1 µg/ml rATG. In contrast, targeting of CD8 antigen required higher concentrations of rATG (e.g. 10 µg, 38.6±11.9% versus 9.6±4.3%, p<0.001). Whereas both rATG and alemtuzumab resulted in a significant and dose-dependent reduction of CD16 surface expression on CD56+ NK cells at a low dose concentration of 0.1 µg/ml, no CD16 downmodulation could be observed for daclizumab, an anti-IL-2Rα (CD25) humanized antibody similar to alemtuzumab (Figure 2B). In contrast, higher concentrations of rabbit IgG (rIgG) led to a decrease of CD16 (e.g. 10 µg, 80%±6.3% versus 48.4%±9.7% and 50 µg/ml 22.1%±4.3%, p<0.001).


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 target surface expression of CD16 on NK cells.Human NK cells cultured with IL-2 (200 IU/ml) and different concentrations of rATG, alemtuzumab or control antibodies such as rIgG and daclizumab (18 hours) were harvested, washed and stained for CD3, CD56, CD8 and CD16. (A) The figure illustrates the average percentages of CD16 and CD8 staining on CD3−CD56+ NK cells after co-incubation with varying concentrations of rATG. Results are displayed as means±SD (n = 5); asterisks (*) denote significant differences compared to untreated controls: ***p<0.001. (B) The mean percentage of CD16 on CD3−CD56+ NK incubated with different concentrations of rATG, alemtuzumab, daclizumab and rIgG is shown. Results are displayed as means±SD of five independent experiments. Asterisks (*) indicate values that showed significantly less CD16 expression compared to untreated controls: ***p<0.001.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2651595&req=5

pone-0004709-g002: Rabbit ATG and alemtuzumab target surface expression of CD16 on NK cells.Human NK cells cultured with IL-2 (200 IU/ml) and different concentrations of rATG, alemtuzumab or control antibodies such as rIgG and daclizumab (18 hours) were harvested, washed and stained for CD3, CD56, CD8 and CD16. (A) The figure illustrates the average percentages of CD16 and CD8 staining on CD3−CD56+ NK cells after co-incubation with varying concentrations of rATG. Results are displayed as means±SD (n = 5); asterisks (*) denote significant differences compared to untreated controls: ***p<0.001. (B) The mean percentage of CD16 on CD3−CD56+ NK incubated with different concentrations of rATG, alemtuzumab, daclizumab and rIgG is shown. Results are displayed as means±SD of five independent experiments. Asterisks (*) indicate values that showed significantly less CD16 expression compared to untreated controls: ***p<0.001.
Mentions: NK cells derived from CD34+ hematopoietic stem cells undergo differentiation via NK cell precursors in the bone marrow through acquisition of functional surface receptors [34]. Therefore, certain NK cell specific receptors should not be targeted by rATG. As expected we could not detect any influence of rATG on the surface expression of the activating cytotoxicity receptors NKp30, NKp44, NKp46, NKG2D and killer-cell immunoglobulin-like receptors (KIRs) including KIR2DL1 and KIR3DL1 (data not shown). In contrast we confirmed previous data illustrating that rATG targets CD8 and CD16 and this effect was dose-dependent (Figure 2A) [35]. Targeting of CD16 was observed in the presence of 0.1 µg rATG (67%±10.8% versus 33.6%±4.1%, p<0.001), and CD16 was further decreased to 1.2%±0.6% at a concentration of 1 µg/ml rATG. In contrast, targeting of CD8 antigen required higher concentrations of rATG (e.g. 10 µg, 38.6±11.9% versus 9.6±4.3%, p<0.001). Whereas both rATG and alemtuzumab resulted in a significant and dose-dependent reduction of CD16 surface expression on CD56+ NK cells at a low dose concentration of 0.1 µg/ml, no CD16 downmodulation could be observed for daclizumab, an anti-IL-2Rα (CD25) humanized antibody similar to alemtuzumab (Figure 2B). In contrast, higher concentrations of rabbit IgG (rIgG) led to a decrease of CD16 (e.g. 10 µg, 80%±6.3% versus 48.4%±9.7% and 50 µg/ml 22.1%±4.3%, p<0.001).

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