Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Feb 1;111(3):1456-63.
doi: 10.1182/blood-2007-02-074716. Epub 2007 Nov 16.

NK-cell activation and antibody-dependent cellular cytotoxicity induced by rituximab-coated target cells is inhibited by the C3b component of complement

Siao-Yi Wang  1 , Emilian Racila, Ronald P Taylor, George J Weiner
Affiliations

NK-cell activation and antibody-dependent cellular cytotoxicity induced by rituximab-coated target cells is inhibited by the C3b component of complement

Siao-Yi Wang et al. Blood. .

Abstract

Antibody-dependent cellular cytotoxicity (ADCC) and complement fixation both appear to play a role in mediating antitumor effects of monoclonal antibodies (mAbs), including rituximab. We evaluated the relationship between rituximab-induced complement fixation, natural killer (NK)-cell activation, and NK cell-mediated ADCC. Down-modulation of NK- cell CD16 and NK-cell activation induced by rituximab-coated target cells was blocked by human serum but not heat-inactivated serum. This inhibition was also observed in the absence of viable target cells. C1q and C3 in the serum were required for these inhibitory effects, while C5 was not. An antibody that stabilizes C3b on the target cell surface enhanced the inhibition of NK-cell activation induced by rituximab-coated target cells. Binding of NK cells to rituximab-coated plates through CD16 was inhibited by the fixation of complement. C5-depleted serum blocked NK cell-mediated ADCC. These data suggest that C3b deposition induced by rituximab-coated target cells inhibits the interaction between the rituximab Fc and NK-cell CD16, thereby limiting the ability of rituximab-coated target cells to induce NK activation and ADCC. Further studies are needed to define in more detail the impact of complement fixation on ADCC, and whether mAbs that fail to fix complement will be more effective at mediating ADCC.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Serum inhibits rituximab-induced NK-cell CD16 down-modulation and CD54 up-regulation. PBMCs and Raji, Daudi, or FL cells were mixed at a 1:1 ratio for 20 hours in the presence or absence of 50% serum with varying concentrations of rituximab. Surface marker expression was determined using flow cytometry with gating on CD3−, CD56+ lymphocytes. (A) NK-cell CD16, expressed as median fluorescence, in the absence and presence of serum (n = 3). (B) NK-cell CD54, expressed as a percentage of CD54 bright, in the absence and presence of serum (n = 3). Error bars represent the standard deviation (SD) of the mean.
Figure 2
Figure 2
Inhibitory effect of serum is dose dependent and abrogated by heat inactivation. PBMCs and Raji cells were mixed at a 1:1 ratio for 20 hours in varying concentrations of serum or heat-inactivated serum in the presence of 0.2 μg/mL rituximab. Surface marker expression was determined using flow cytometry with gating on CD3−, CD56+ lymphocytes. (A) NK-cell CD16, expressed as median fluorescence, after incubation with varying concentrations of serum and heat-inactivated serum (n = 3). (B) NK-cell CD54, expressed as a percentage of CD54 bright, after incubation with varying concentrations of serum and heat inactivated serum (n = 3). Error bars represent SD of the mean.
Figure 3
Figure 3
Target cells are lysed in the presence of serum and rituximab irrespective of presence of effector cells. Raji cells were incubated for 20 hours in media, 50% serum, or 50% serum plus PBMCs with varying concentrations of rituximab. The percent of viable target cells were determined using flow cytometry by counting annexin V and propidium iodide–negative target cells (n = 3). Error bars represent SD of the mean.
Figure 4
Figure 4
Serum inhibits changes in CD16 expression in the absence of viable target cells. (A) Raji cells were fixed in 1% formaldehyde and washed extensively. Fixed cells were mixed with PBMCs at a 1:1 ratio for 20 hours with 5 μg/mL rituximab (n = 3). (B) Flat-bottom plates were coated with 10 μg/mL of rituximab. Plates were washed, and PBMCs were added and cultured for 20 hours. Incubations with fixed Raji cells and rituximab or rituximab-coated plates were preformed in the presence or absence of 50% serum and heat-inactivated serum (n = 5). Surface marker expression was determined using flow cytometry with gating on CD3−, CD56+ lymphocytes. NK-cell CD16, expressed as median fluorescence, was measured after incubation with media, serum, and heat-inactivated serum in the presence of fixed Raji cells (A) or flat-bottom plates coated with rituximab (B). Error bars represent SD of the mean.
Figure 5
Figure 5
Serum-depleted C5 inhibits NK-cell CD54 up-regulation, while serum-depleted C1q or C3 does not. PBMCs were mixed with Raji cells at a 1:1 ratio for 20 hours with 5 μg/mL of rituximab in the presence or absence of C1q-, C3-, or C5-depleted serum. Surface marker expression was determined using flow cytometry with gating on CD3−, CD56+ lymphocytes. NK-cell CD54, expressed as a percentage of CD54 bright, was determined. (A) Evaluation in C1q-depleted serum, C1q-depleted serum with purified C1q added back, or purified C1q alone (n = 3). (B) Evaluation in C3-depleted serum, C3-depleted serum with purified C3 added back, or purified C3 alone (n = 3). (C) Evaluation in various concentrations of C5-depleted serum (n = 3). Error bars represent SD of the mean.
Figure 6
Figure 6
C3b-stabilizing antibody (3E7) enhances the inhibition of NK-cell CD16 down-modulation and CD54 up-regulation. PBMCs and Raji cells were mixed at a 1:1 ratio for 20 hours in the presence or absence of 50% serum with varying concentrations of rituximab and 10 μg/mL of 3E7. Surface marker expression was determined using flow cytometry with gating on CD3−, CD56+ lymphocytes. NK-cell CD54, expressed as a percentage of CD54 bright, was cultured in media, serum, or serum plus 3E7 (n = 3). Error bars represent SD of the mean.
Figure 7
Figure 7
NK cell CD16 binding to rituximab is blocked by complement fixation. U-bottomed 96-well microtiter plates were coated with varying concentrations of rituximab. Wells were incubated with media, 50% serum, or 50% heat-inactivated serum. After washing, NK cells were allowed to sit on plate for 30 minutes at room temperature. High absorbance resulted from NK-cell pelleting that occurred in the absence of binding to rituximab. All samples were run in triplicate (n = 3). Data are representative of 3 independent experiments. Error bars represent SD of the mean.
Figure 8
Figure 8
C5-depleted serum inhibits rituximab-mediated ADCC. Raji cells were labeled with 51Cr and incubated with purified NK cells in various E/T ratios, 5 μg/mL rituximab, and either C5-depleted serum, heat-inactivated C5-depleted serum, or media. Percentage of specific lysis was measured based on 51Cr release. All samples were run in triplicate (n = 3). Data are representative of 3 independent experiments. Error bars represent SD of the mean.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Deans JP, Li H, Polyak MJ. CD20-mediated apoptosis: signalling through lipid rafts. Immunology. 2002;107:176–182. - PMC - PubMed
    1. van Meerten T, van Rijn RS, Hol S, Hagenbeek A, Ebeling SB. Complement-induced cell death by rituximab depends on CD20 expression level and acts complementary to antibody-dependent cellular cytotoxicity. Clin Cancer Res. 2006;12:4027–4035. - PubMed
    1. Golay J, Zaffaroni L, Vaccari T, et al. Biologic response of B lymphoma cells to anti-CD20 monoclonal antibody rituximab in vitro: CD55 and CD59 regulate complement-mediated cell lysis. Blood. 2000;95:3900–3908. - PubMed
    1. Harjunpaa A, Junnikkala S, Meri S. Rituximab (anti-CD20) therapy of B-cell lymphomas: direct complement killing is superior to cellular effector mechanisms. Scand J Immunol. 2000;51:634–641. - PubMed
    1. Weng WK, Levy R. Expression of complement inhibitors CD46, CD55, and CD59 on tumor cells does not predict clinical outcome after rituximab treatment in follicular non-Hodgkin lymphoma. Blood. 2001;98:1352–1357. - PubMed

Publication types

MeSH terms