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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 anti-CD20-opsonized targets results in the impairment of FcεRIγ- and CD3ζ-dependent NKp46- and NKp30-mediated killing. (A) Primary cultured NK cells from V/F and V/V individuals (n = 5) were isolated upon 18 h co-culture (2:1) with biotinylated rituximab (RTX-exp)-, obinutuzumab (GA101-exp)-opsonized or non-opsonized Raji (Ctrl-exp). Cells were stained as indicated for FACS analysis and tested in 51Cr-release redirected killing assays toward P815 FcR+ cells in presence of Abs for the indicated receptors. (Top) The overlays of histograms from one representative experiment of five performed are shown. (Bottom) Lytic units (LU) from five independent experiments are shown. Bar graphs depict mean ± SEM. *p < 0.05, **p < 0.005, ***p < 0.0005. (B) NK cells as in A were stimulated for 4 h with the indicated plastic-immobilized mAbs. The percentage of CD107a+ cells was evaluated by FACS analysis gating on CD56+ cells. Data (mean ± SEM) from three independent experiments are shown. **p < 0.01, ***p < 0.001.
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f0004: CD16 engagement by anti-CD20-opsonized targets results in the impairment of FcεRIγ- and CD3ζ-dependent NKp46- and NKp30-mediated killing. (A) Primary cultured NK cells from V/F and V/V individuals (n = 5) were isolated upon 18 h co-culture (2:1) with biotinylated rituximab (RTX-exp)-, obinutuzumab (GA101-exp)-opsonized or non-opsonized Raji (Ctrl-exp). Cells were stained as indicated for FACS analysis and tested in 51Cr-release redirected killing assays toward P815 FcR+ cells in presence of Abs for the indicated receptors. (Top) The overlays of histograms from one representative experiment of five performed are shown. (Bottom) Lytic units (LU) from five independent experiments are shown. Bar graphs depict mean ± SEM. *p < 0.05, **p < 0.005, ***p < 0.0005. (B) NK cells as in A were stimulated for 4 h with the indicated plastic-immobilized mAbs. The percentage of CD107a+ cells was evaluated by FACS analysis gating on CD56+ cells. Data (mean ± SEM) from three independent experiments are shown. **p < 0.01, ***p < 0.001.

Mentions: NK cells were co-cultured for 18 h with anti-CD20-opsonized targets. Cytotoxic activity of experienced cells was assessed against FcR+ P815 target cells in a redirected killing assay in the presence of anti-CD16, anti-NKp46, anti-NKp30 or anti-NKG2D mAbs to explore the killing ability induced by ITAM-dependent- or ITAM-independent receptors. We also excluded that, by this time, residual anti-CD20 could be detected on the surface of NK cells (not shown). As expected, because of the reduction of CD16 expression levels, mAb experienced NK cells exhibited a marked reduction of CD16-dependent killing (reverse ADCC) (Fig. 4A). Moreover, being CD3ζ and FcεRIγ chains the molecular adaptors required for NKp46 and NKp30 signal transduction,29 we also observed a significant impairment of NKp46 and NKp30 cytotoxic response, both in rituximab- and obinutuzumab-experienced NK cells (Fig. 4A). We also noted a slight reduction of NKp46 expression levels in anti-CD20-experienced cells. On the opposite, NKG2D-dependent killing, relying on DAP10 adaptor,24 appeared more preserved.Figure 4.


Obinutuzumab-mediated high-affinity ligation of Fc γ RIIIA/CD16 primes NK cells for IFN γ production
CD16 engagement by anti-CD20-opsonized targets results in the impairment of FcεRIγ- and CD3ζ-dependent NKp46- and NKp30-mediated killing. (A) Primary cultured NK cells from V/F and V/V individuals (n = 5) were isolated upon 18 h co-culture (2:1) with biotinylated rituximab (RTX-exp)-, obinutuzumab (GA101-exp)-opsonized or non-opsonized Raji (Ctrl-exp). Cells were stained as indicated for FACS analysis and tested in 51Cr-release redirected killing assays toward P815 FcR+ cells in presence of Abs for the indicated receptors. (Top) The overlays of histograms from one representative experiment of five performed are shown. (Bottom) Lytic units (LU) from five independent experiments are shown. Bar graphs depict mean ± SEM. *p < 0.05, **p < 0.005, ***p < 0.0005. (B) NK cells as in A were stimulated for 4 h with the indicated plastic-immobilized mAbs. The percentage of CD107a+ cells was evaluated by FACS analysis gating on CD56+ cells. Data (mean ± SEM) from three independent experiments are shown. **p < 0.01, ***p < 0.001.
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getmorefigures.php?uid=PMC5384385&req=5

f0004: CD16 engagement by anti-CD20-opsonized targets results in the impairment of FcεRIγ- and CD3ζ-dependent NKp46- and NKp30-mediated killing. (A) Primary cultured NK cells from V/F and V/V individuals (n = 5) were isolated upon 18 h co-culture (2:1) with biotinylated rituximab (RTX-exp)-, obinutuzumab (GA101-exp)-opsonized or non-opsonized Raji (Ctrl-exp). Cells were stained as indicated for FACS analysis and tested in 51Cr-release redirected killing assays toward P815 FcR+ cells in presence of Abs for the indicated receptors. (Top) The overlays of histograms from one representative experiment of five performed are shown. (Bottom) Lytic units (LU) from five independent experiments are shown. Bar graphs depict mean ± SEM. *p < 0.05, **p < 0.005, ***p < 0.0005. (B) NK cells as in A were stimulated for 4 h with the indicated plastic-immobilized mAbs. The percentage of CD107a+ cells was evaluated by FACS analysis gating on CD56+ cells. Data (mean ± SEM) from three independent experiments are shown. **p < 0.01, ***p < 0.001.
Mentions: NK cells were co-cultured for 18 h with anti-CD20-opsonized targets. Cytotoxic activity of experienced cells was assessed against FcR+ P815 target cells in a redirected killing assay in the presence of anti-CD16, anti-NKp46, anti-NKp30 or anti-NKG2D mAbs to explore the killing ability induced by ITAM-dependent- or ITAM-independent receptors. We also excluded that, by this time, residual anti-CD20 could be detected on the surface of NK cells (not shown). As expected, because of the reduction of CD16 expression levels, mAb experienced NK cells exhibited a marked reduction of CD16-dependent killing (reverse ADCC) (Fig. 4A). Moreover, being CD3ζ and FcεRIγ chains the molecular adaptors required for NKp46 and NKp30 signal transduction,29 we also observed a significant impairment of NKp46 and NKp30 cytotoxic response, both in rituximab- and obinutuzumab-experienced NK cells (Fig. 4A). We also noted a slight reduction of NKp46 expression levels in anti-CD20-experienced cells. On the opposite, NKG2D-dependent killing, relying on DAP10 adaptor,24 appeared more preserved.Figure 4.

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