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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 results in enhanced degranulation as well as FcεRIγ lysosomal degradation irrespective of FCGR3A-V158F polymorphism. (A) PBMCs were left alone (baseline) or allowed to interact (2:1) with rituximab (Raji-RTX)-, obinutuzumab (Raji-GA101)-opsonized or non-opsonized Raji (Raji-Ctrl) for 6 h. The percentage of CD107a+ cells among CD3−CD56+ was evaluated by FACS analysis in individuals grouped by FCGR3A genotype (high-affinity allele V, low-affinity F; V/V, n = 6; V/F, n = 6; F/F, n = 6). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. For each group, compared with baseline or Raji-Ctrl samples, all the differences were statistically significant (p < 0.001). (B–F) Primary cultured NK cells (n = 5/genotype) were isolated upon 18 h co-culture (2:1) with biotinylated rituximab (RTX-exp)-, obinutuzumab (GA101-exp)-opsonized or non-opsonized Raji (Ctrl-exp) in the presence of medium alone (medium) or, when indicated, with 20 mM NH4Cl. (B) CD16 surface expression was evaluated by FACS analysis by anti-CD16 (Leu11c) mAb in individuals grouped by FCGR3A genotype. (Top) Histogram overlay of one representative donor/genotype .is shown and (bottom) normalized CD16 MFI calculated as described in Fig. 2A are depicted in bar graph (mean ± SEM). *p < 0.05. (C, E) An equal amount of proteins from whole cell lysates was immunoblotted as indicated. The same membrane was immunoblotted with anti-FcεRIγ and, after stripping, with anti-CD3ζ Abs followed by anti-β-tubulin for sample normalization. Membranes containing untreated- (medium) or NH4Cl-treated samples were developed in the same film. The black vertical lines indicate that intervening lanes were sliced out. The numbers represent the relative protein amount of the indicated proteins obtained by normalizing to the level of β-tubulin and expressed as fold change respect to Ctrl-exp samples (arbitrarily set to 1). One representative experiment is shown. (D, F) The relative values (mean ± SEM) of CD3ζ or FcεRIγ chains from five independent experiments are depicted in bar graphs.*p < 0.05, **p < 0.01.
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f0003: CD16 engagement by obinutuzumab-opsonized targets results in enhanced degranulation as well as FcεRIγ lysosomal degradation irrespective of FCGR3A-V158F polymorphism. (A) PBMCs were left alone (baseline) or allowed to interact (2:1) with rituximab (Raji-RTX)-, obinutuzumab (Raji-GA101)-opsonized or non-opsonized Raji (Raji-Ctrl) for 6 h. The percentage of CD107a+ cells among CD3−CD56+ was evaluated by FACS analysis in individuals grouped by FCGR3A genotype (high-affinity allele V, low-affinity F; V/V, n = 6; V/F, n = 6; F/F, n = 6). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. For each group, compared with baseline or Raji-Ctrl samples, all the differences were statistically significant (p < 0.001). (B–F) Primary cultured NK cells (n = 5/genotype) were isolated upon 18 h co-culture (2:1) with biotinylated rituximab (RTX-exp)-, obinutuzumab (GA101-exp)-opsonized or non-opsonized Raji (Ctrl-exp) in the presence of medium alone (medium) or, when indicated, with 20 mM NH4Cl. (B) CD16 surface expression was evaluated by FACS analysis by anti-CD16 (Leu11c) mAb in individuals grouped by FCGR3A genotype. (Top) Histogram overlay of one representative donor/genotype .is shown and (bottom) normalized CD16 MFI calculated as described in Fig. 2A are depicted in bar graph (mean ± SEM). *p < 0.05. (C, E) An equal amount of proteins from whole cell lysates was immunoblotted as indicated. The same membrane was immunoblotted with anti-FcεRIγ and, after stripping, with anti-CD3ζ Abs followed by anti-β-tubulin for sample normalization. Membranes containing untreated- (medium) or NH4Cl-treated samples were developed in the same film. The black vertical lines indicate that intervening lanes were sliced out. The numbers represent the relative protein amount of the indicated proteins obtained by normalizing to the level of β-tubulin and expressed as fold change respect to Ctrl-exp samples (arbitrarily set to 1). One representative experiment is shown. (D, F) The relative values (mean ± SEM) of CD3ζ or FcεRIγ chains from five independent experiments are depicted in bar graphs.*p < 0.05, **p < 0.01.

Mentions: We sought to analyze whether the FCGR3A c.559G>T polymorphism would impact on CD16 dynamics. V158F single nucleotide polymorphism was determined by cytofluorimetric approaches33 and confirmed by a PCR-based analysis.34,35 Of the 125 typed donors, 21 and 34 were V/V and F/F homozygous, respectively, while 70 were V/F heterozygous, testifying a large prevalence of individuals at intermediate–high affinity. In line with previous observations demonstrating that obinutuzumab triggers an enhanced ADCC irrespective of CD16 allotype,21,22 we observed that in F/F individuals surface CD107a levels in response to obinutuzumab were significantly higher with respect to rituximab, although lower than V/F or V/V donors (Fig. 3A).Figure 3.


Obinutuzumab-mediated high-affinity ligation of Fc γ RIIIA/CD16 primes NK cells for IFN γ production
CD16 engagement by obinutuzumab-opsonized targets results in enhanced degranulation as well as FcεRIγ lysosomal degradation irrespective of FCGR3A-V158F polymorphism. (A) PBMCs were left alone (baseline) or allowed to interact (2:1) with rituximab (Raji-RTX)-, obinutuzumab (Raji-GA101)-opsonized or non-opsonized Raji (Raji-Ctrl) for 6 h. The percentage of CD107a+ cells among CD3−CD56+ was evaluated by FACS analysis in individuals grouped by FCGR3A genotype (high-affinity allele V, low-affinity F; V/V, n = 6; V/F, n = 6; F/F, n = 6). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. For each group, compared with baseline or Raji-Ctrl samples, all the differences were statistically significant (p < 0.001). (B–F) Primary cultured NK cells (n = 5/genotype) were isolated upon 18 h co-culture (2:1) with biotinylated rituximab (RTX-exp)-, obinutuzumab (GA101-exp)-opsonized or non-opsonized Raji (Ctrl-exp) in the presence of medium alone (medium) or, when indicated, with 20 mM NH4Cl. (B) CD16 surface expression was evaluated by FACS analysis by anti-CD16 (Leu11c) mAb in individuals grouped by FCGR3A genotype. (Top) Histogram overlay of one representative donor/genotype .is shown and (bottom) normalized CD16 MFI calculated as described in Fig. 2A are depicted in bar graph (mean ± SEM). *p < 0.05. (C, E) An equal amount of proteins from whole cell lysates was immunoblotted as indicated. The same membrane was immunoblotted with anti-FcεRIγ and, after stripping, with anti-CD3ζ Abs followed by anti-β-tubulin for sample normalization. Membranes containing untreated- (medium) or NH4Cl-treated samples were developed in the same film. The black vertical lines indicate that intervening lanes were sliced out. The numbers represent the relative protein amount of the indicated proteins obtained by normalizing to the level of β-tubulin and expressed as fold change respect to Ctrl-exp samples (arbitrarily set to 1). One representative experiment is shown. (D, F) The relative values (mean ± SEM) of CD3ζ or FcεRIγ chains from five independent experiments are depicted in bar graphs.*p < 0.05, **p < 0.01.
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f0003: CD16 engagement by obinutuzumab-opsonized targets results in enhanced degranulation as well as FcεRIγ lysosomal degradation irrespective of FCGR3A-V158F polymorphism. (A) PBMCs were left alone (baseline) or allowed to interact (2:1) with rituximab (Raji-RTX)-, obinutuzumab (Raji-GA101)-opsonized or non-opsonized Raji (Raji-Ctrl) for 6 h. The percentage of CD107a+ cells among CD3−CD56+ was evaluated by FACS analysis in individuals grouped by FCGR3A genotype (high-affinity allele V, low-affinity F; V/V, n = 6; V/F, n = 6; F/F, n = 6). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. For each group, compared with baseline or Raji-Ctrl samples, all the differences were statistically significant (p < 0.001). (B–F) Primary cultured NK cells (n = 5/genotype) were isolated upon 18 h co-culture (2:1) with biotinylated rituximab (RTX-exp)-, obinutuzumab (GA101-exp)-opsonized or non-opsonized Raji (Ctrl-exp) in the presence of medium alone (medium) or, when indicated, with 20 mM NH4Cl. (B) CD16 surface expression was evaluated by FACS analysis by anti-CD16 (Leu11c) mAb in individuals grouped by FCGR3A genotype. (Top) Histogram overlay of one representative donor/genotype .is shown and (bottom) normalized CD16 MFI calculated as described in Fig. 2A are depicted in bar graph (mean ± SEM). *p < 0.05. (C, E) An equal amount of proteins from whole cell lysates was immunoblotted as indicated. The same membrane was immunoblotted with anti-FcεRIγ and, after stripping, with anti-CD3ζ Abs followed by anti-β-tubulin for sample normalization. Membranes containing untreated- (medium) or NH4Cl-treated samples were developed in the same film. The black vertical lines indicate that intervening lanes were sliced out. The numbers represent the relative protein amount of the indicated proteins obtained by normalizing to the level of β-tubulin and expressed as fold change respect to Ctrl-exp samples (arbitrarily set to 1). One representative experiment is shown. (D, F) The relative values (mean ± SEM) of CD3ζ or FcεRIγ chains from five independent experiments are depicted in bar graphs.*p < 0.05, **p < 0.01.
Mentions: We sought to analyze whether the FCGR3A c.559G>T polymorphism would impact on CD16 dynamics. V158F single nucleotide polymorphism was determined by cytofluorimetric approaches33 and confirmed by a PCR-based analysis.34,35 Of the 125 typed donors, 21 and 34 were V/V and F/F homozygous, respectively, while 70 were V/F heterozygous, testifying a large prevalence of individuals at intermediate–high affinity. In line with previous observations demonstrating that obinutuzumab triggers an enhanced ADCC irrespective of CD16 allotype,21,22 we observed that in F/F individuals surface CD107a levels in response to obinutuzumab were significantly higher with respect to rituximab, although lower than V/F or V/V donors (Fig. 3A).Figure 3.

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