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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

Obinutuzumab-experienced NK cells exhibit an enhanced IFNγ production in response to cytokines, targets or obinutuzumab re-stimulation. Primary cultured NK cells were isolated upon 90 min of co-culture (2:1) with biotinylated rituximab (RTX-exp)-, obinutuzumab (GA101-exp)-opsonized or non-opsonized Raji (Ctrl-exp) and re-plated for 12 h. (A) NK cells were stained with anti-CD16 (Leu11c) mAb for FACS analysis. Graph depicts CD16 MFI and data are presented as median with the interquartile range. **p < 0.01, ***p < 0.0005. (B) NK cells were re-stimulated (2:1) with non-opsonized targets (Raji-Ctrl), rituximab-opsonized (Raji-RTX) or obinutuzumab-opsonized (Raji-GA101) target cells in the presence of IL-12 (10 ng/mL), or (C) left untreated (none) or treated with IL-12 (10 ng/mL) or IL-2 (100 U/mL). After 18 h, supernatants were collected and assessed for IFNγ levels. Data are presented as mean ± SEM of seven independent experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.0001. Compared to untreated (none), all the differences were statistically significant (p ≤ 0.001).
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f0005: Obinutuzumab-experienced NK cells exhibit an enhanced IFNγ production in response to cytokines, targets or obinutuzumab re-stimulation. Primary cultured NK cells were isolated upon 90 min of co-culture (2:1) with biotinylated rituximab (RTX-exp)-, obinutuzumab (GA101-exp)-opsonized or non-opsonized Raji (Ctrl-exp) and re-plated for 12 h. (A) NK cells were stained with anti-CD16 (Leu11c) mAb for FACS analysis. Graph depicts CD16 MFI and data are presented as median with the interquartile range. **p < 0.01, ***p < 0.0005. (B) NK cells were re-stimulated (2:1) with non-opsonized targets (Raji-Ctrl), rituximab-opsonized (Raji-RTX) or obinutuzumab-opsonized (Raji-GA101) target cells in the presence of IL-12 (10 ng/mL), or (C) left untreated (none) or treated with IL-12 (10 ng/mL) or IL-2 (100 U/mL). After 18 h, supernatants were collected and assessed for IFNγ levels. Data are presented as mean ± SEM of seven independent experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.0001. Compared to untreated (none), all the differences were statistically significant (p ≤ 0.001).

Mentions: As shown in Fig. 2A, CD16 levels were markedly down-modulated in the course of interaction with anti-CD20-opsonized targets. To obtain, albeit at partial levels, the re-expression of CD16, we cultured NK cells for 12 h after target detachment. Because of the stronger CD16 downregulation induced by obinutuzumab, the recovery of CD16 expression was significantly lower in obinutuzumab-experienced NK cells (Fig. 5A).Figure 5.


Obinutuzumab-mediated high-affinity ligation of Fc γ RIIIA/CD16 primes NK cells for IFN γ production
Obinutuzumab-experienced NK cells exhibit an enhanced IFNγ production in response to cytokines, targets or obinutuzumab re-stimulation. Primary cultured NK cells were isolated upon 90 min of co-culture (2:1) with biotinylated rituximab (RTX-exp)-, obinutuzumab (GA101-exp)-opsonized or non-opsonized Raji (Ctrl-exp) and re-plated for 12 h. (A) NK cells were stained with anti-CD16 (Leu11c) mAb for FACS analysis. Graph depicts CD16 MFI and data are presented as median with the interquartile range. **p < 0.01, ***p < 0.0005. (B) NK cells were re-stimulated (2:1) with non-opsonized targets (Raji-Ctrl), rituximab-opsonized (Raji-RTX) or obinutuzumab-opsonized (Raji-GA101) target cells in the presence of IL-12 (10 ng/mL), or (C) left untreated (none) or treated with IL-12 (10 ng/mL) or IL-2 (100 U/mL). After 18 h, supernatants were collected and assessed for IFNγ levels. Data are presented as mean ± SEM of seven independent experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.0001. Compared to untreated (none), all the differences were statistically significant (p ≤ 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f0005: Obinutuzumab-experienced NK cells exhibit an enhanced IFNγ production in response to cytokines, targets or obinutuzumab re-stimulation. Primary cultured NK cells were isolated upon 90 min of co-culture (2:1) with biotinylated rituximab (RTX-exp)-, obinutuzumab (GA101-exp)-opsonized or non-opsonized Raji (Ctrl-exp) and re-plated for 12 h. (A) NK cells were stained with anti-CD16 (Leu11c) mAb for FACS analysis. Graph depicts CD16 MFI and data are presented as median with the interquartile range. **p < 0.01, ***p < 0.0005. (B) NK cells were re-stimulated (2:1) with non-opsonized targets (Raji-Ctrl), rituximab-opsonized (Raji-RTX) or obinutuzumab-opsonized (Raji-GA101) target cells in the presence of IL-12 (10 ng/mL), or (C) left untreated (none) or treated with IL-12 (10 ng/mL) or IL-2 (100 U/mL). After 18 h, supernatants were collected and assessed for IFNγ levels. Data are presented as mean ± SEM of seven independent experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.0001. Compared to untreated (none), all the differences were statistically significant (p ≤ 0.001).
Mentions: As shown in Fig. 2A, CD16 levels were markedly down-modulated in the course of interaction with anti-CD20-opsonized targets. To obtain, albeit at partial levels, the re-expression of CD16, we cultured NK cells for 12 h after target detachment. Because of the stronger CD16 downregulation induced by obinutuzumab, the recovery of CD16 expression was significantly lower in obinutuzumab-experienced NK cells (Fig. 5A).Figure 5.

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