Limits...
Obinutuzumab-mediated high-affinity ligation of Fc γ RIIIA/CD16 primes NK cells for IFN γ production

View Article: PubMed Central - PubMed

ABSTRACT

Natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), based on the recognition of IgG-opsonized targets by the low-affinity receptor for IgG FcγRIIIA/CD16, represents one of the main mechanisms by which therapeutic antibodies (mAbs) mediate their antitumor effects. Besides ADCC, CD16 ligation also results in cytokine production, in particular, NK-derived IFNγ is endowed with a well-recognized role in the shaping of adaptive immune responses.

Obinutuzumab is a glycoengineered anti-CD20 mAb with a modified crystallizable fragment (Fc) domain designed to increase the affinity for CD16 and consequently the killing of mAb-opsonized targets. However, the impact of CD16 ligation in optimized affinity conditions on NK functional program is not completely understood.

Herein, we demonstrate that the interaction of NK cells with obinutuzumab-opsonized cells results in enhanced IFNγ production as compared with parental non-glycoengineered mAb or the reference molecule rituximab. We observed that affinity ligation conditions strictly correlate with the ability to induce CD16 down-modulation and lysosomal targeting of receptor-associated signaling elements. Indeed, a preferential degradation of FcεRIγ chain and Syk kinase was observed upon obinutuzumab stimulation independently from CD16-V158F polymorphism. Although the downregulation of FcεRIγ/Syk module leads to the impairment of cytotoxic function induced by NKp46 and NKp30 receptors, obinutuzumab-experienced cells exhibit an increased ability to produce IFNγ in response to different stimuli.

These data highlight a relationship between CD16 aggregation conditions and the ability to promote a degradative pathway of CD16-coupled signaling elements associated to the shift of NK functional program.

No MeSH data available.


Related in: MedlinePlus

CD16 engagement by obinutuzumab-opsonized targets enhances both CD107a mobilization and IFNγ production. PBMCs were left alone (baseline) or combined (2:1) with rituximab (Raji-RTX)-, obinutuzumab (Raji-GA101)-, wt-GA101 (Raji-wt-GA101)-opsonized or non-opsonized Raji (Raji-Ctrl) for 6 h. The percentage of IFNγ+ and CD107a+ cells among NK cells (CD3−CD56+) were analyzed by flow cytometry. (A) Plots from one representative donor are shown. (B) Data (mean ± SEM) from nine donors are shown. *p < 0.05, **p < 0.01, ***p < 0.0001. Compared to baseline or Raji-Ctrl samples, all the differences were statistically significant (p < 0.0001). (C) The median fluorescence intensity (FI) values of IFNγ NK cells from nine individuals are reported in the graph. Each line represents a single donor. **p < 0.01, ***p < 0.0001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5384385&req=5

f0001: CD16 engagement by obinutuzumab-opsonized targets enhances both CD107a mobilization and IFNγ production. PBMCs were left alone (baseline) or combined (2:1) with rituximab (Raji-RTX)-, obinutuzumab (Raji-GA101)-, wt-GA101 (Raji-wt-GA101)-opsonized or non-opsonized Raji (Raji-Ctrl) for 6 h. The percentage of IFNγ+ and CD107a+ cells among NK cells (CD3−CD56+) were analyzed by flow cytometry. (A) Plots from one representative donor are shown. (B) Data (mean ± SEM) from nine donors are shown. *p < 0.05, **p < 0.01, ***p < 0.0001. Compared to baseline or Raji-Ctrl samples, all the differences were statistically significant (p < 0.0001). (C) The median fluorescence intensity (FI) values of IFNγ NK cells from nine individuals are reported in the graph. Each line represents a single donor. **p < 0.01, ***p < 0.0001.

Mentions: To analyze at what extent individual NK cells can be induced to perform degranulation and/or cytokine production, the intracellular expression of IFNγ and surface expression of CD107a (marker of lytic granule exocytosis) were simultaneously assessed on peripheral blood CD3−CD56+ NK cells by multicolour cytofluorimetric analysis. Hereafter, in all our experimental settings, we obtained the stimulation of CD16 by co-culturing NK cells with CD20+ B cell lymphoma Raji cells that were opsonized with rituximab or obinutuzumab, in the defucosylated (GA101) or wild type version (wt-GA101), at saturating concentration as detailed in Materials and methods section and Fig. S1. Being Raji cells almost completely resistant to freshly isolated NK-mediated killing, the stimulation with non-opsonized Raji does not induce significant IFNγ production or CD107a expression (Fig. 1A and B). The stimulation of NK cells with rituximab-opsonized targets induces both degranulation and IFNγ production with a slightly greater frequency of CD107a+ than of IFNγ+ cells, confirming the hierarchy among NK cell responses described previously.31 Under such stimulation conditions, only 11% of the cells on average were able to perform both responses. Notably, when NK cells were stimulated with obinutuzumab-opsonized targets both degranulation and IFNγ production appeared significantly enhanced with respect to rituximab. Importantly, the percentage of NK cells that acquired the ability to degranulate and to produce IFNγ results almost twice in response to obinutuzumab than to rituximab. The enhanced activity of obinutuzumab is for large part attributable to defucosylation, in fact, wt-GA101 behaves similarly to rituximab in inducing NK functions. Besides the increased portion of IFNγ producing cells, the analysis of median values demonstrates that obinutuzumab stimulation increases the IFNγ quantity on a per cell basis (Fig. 1C).Figure 1.


Obinutuzumab-mediated high-affinity ligation of Fc γ RIIIA/CD16 primes NK cells for IFN γ production
CD16 engagement by obinutuzumab-opsonized targets enhances both CD107a mobilization and IFNγ production. PBMCs were left alone (baseline) or combined (2:1) with rituximab (Raji-RTX)-, obinutuzumab (Raji-GA101)-, wt-GA101 (Raji-wt-GA101)-opsonized or non-opsonized Raji (Raji-Ctrl) for 6 h. The percentage of IFNγ+ and CD107a+ cells among NK cells (CD3−CD56+) were analyzed by flow cytometry. (A) Plots from one representative donor are shown. (B) Data (mean ± SEM) from nine donors are shown. *p < 0.05, **p < 0.01, ***p < 0.0001. Compared to baseline or Raji-Ctrl samples, all the differences were statistically significant (p < 0.0001). (C) The median fluorescence intensity (FI) values of IFNγ NK cells from nine individuals are reported in the graph. Each line represents a single donor. **p < 0.01, ***p < 0.0001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384385&req=5

f0001: CD16 engagement by obinutuzumab-opsonized targets enhances both CD107a mobilization and IFNγ production. PBMCs were left alone (baseline) or combined (2:1) with rituximab (Raji-RTX)-, obinutuzumab (Raji-GA101)-, wt-GA101 (Raji-wt-GA101)-opsonized or non-opsonized Raji (Raji-Ctrl) for 6 h. The percentage of IFNγ+ and CD107a+ cells among NK cells (CD3−CD56+) were analyzed by flow cytometry. (A) Plots from one representative donor are shown. (B) Data (mean ± SEM) from nine donors are shown. *p < 0.05, **p < 0.01, ***p < 0.0001. Compared to baseline or Raji-Ctrl samples, all the differences were statistically significant (p < 0.0001). (C) The median fluorescence intensity (FI) values of IFNγ NK cells from nine individuals are reported in the graph. Each line represents a single donor. **p < 0.01, ***p < 0.0001.
Mentions: To analyze at what extent individual NK cells can be induced to perform degranulation and/or cytokine production, the intracellular expression of IFNγ and surface expression of CD107a (marker of lytic granule exocytosis) were simultaneously assessed on peripheral blood CD3−CD56+ NK cells by multicolour cytofluorimetric analysis. Hereafter, in all our experimental settings, we obtained the stimulation of CD16 by co-culturing NK cells with CD20+ B cell lymphoma Raji cells that were opsonized with rituximab or obinutuzumab, in the defucosylated (GA101) or wild type version (wt-GA101), at saturating concentration as detailed in Materials and methods section and Fig. S1. Being Raji cells almost completely resistant to freshly isolated NK-mediated killing, the stimulation with non-opsonized Raji does not induce significant IFNγ production or CD107a expression (Fig. 1A and B). The stimulation of NK cells with rituximab-opsonized targets induces both degranulation and IFNγ production with a slightly greater frequency of CD107a+ than of IFNγ+ cells, confirming the hierarchy among NK cell responses described previously.31 Under such stimulation conditions, only 11% of the cells on average were able to perform both responses. Notably, when NK cells were stimulated with obinutuzumab-opsonized targets both degranulation and IFNγ production appeared significantly enhanced with respect to rituximab. Importantly, the percentage of NK cells that acquired the ability to degranulate and to produce IFNγ results almost twice in response to obinutuzumab than to rituximab. The enhanced activity of obinutuzumab is for large part attributable to defucosylation, in fact, wt-GA101 behaves similarly to rituximab in inducing NK functions. Besides the increased portion of IFNγ producing cells, the analysis of median values demonstrates that obinutuzumab stimulation increases the IFNγ quantity on a per cell basis (Fig. 1C).Figure 1.

View Article: PubMed Central - PubMed

ABSTRACT

Natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), based on the recognition of IgG-opsonized targets by the low-affinity receptor for IgG Fc&gamma;RIIIA/CD16, represents one of the main mechanisms by which therapeutic antibodies (mAbs) mediate their antitumor effects. Besides ADCC, CD16 ligation also results in cytokine production, in particular, NK-derived IFN&gamma; is endowed with a well-recognized role in the shaping of adaptive immune responses.

Obinutuzumab is a glycoengineered anti-CD20 mAb with a modified crystallizable fragment (Fc) domain designed to increase the affinity for CD16 and consequently the killing of mAb-opsonized targets. However, the impact of CD16 ligation in optimized affinity conditions on NK functional program is not completely understood.

Herein, we demonstrate that the interaction of NK cells with obinutuzumab-opsonized cells results in enhanced IFN&gamma; production as compared with parental non-glycoengineered mAb or the reference molecule rituximab. We observed that affinity ligation conditions strictly correlate with the ability to induce CD16 down-modulation and lysosomal targeting of receptor-associated signaling elements. Indeed, a preferential degradation of Fc&epsilon;RI&gamma; chain and Syk kinase was observed upon obinutuzumab stimulation independently from CD16-V158F polymorphism. Although the downregulation of Fc&epsilon;RI&gamma;/Syk module leads to the impairment of cytotoxic function induced by NKp46 and NKp30 receptors, obinutuzumab-experienced cells exhibit an increased ability to produce IFN&gamma; in response to different stimuli.

These data highlight a relationship between CD16 aggregation conditions and the ability to promote a degradative pathway of CD16-coupled signaling elements associated to the shift of NK functional program.

No MeSH data available.


Related in: MedlinePlus