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CD16 is indispensable for antibody-dependent cellular cytotoxicity by human monocytes

View Article: PubMed Central - PubMed

ABSTRACT

Antibody-dependent cellular cytotoxicity (ADCC) is exerted by immune cells expressing surface Fcγ receptors (FcγRs) against cells coated with antibody, such as virus-infected or transformed cells. CD16, the FcγRIIIA, is essential for ADCC by NK cells, and is also expressed by a subset of human blood monocytes. We found that human CD16− expressing monocytes have a broad spectrum of ADCC capacities and can kill cancer cell lines, primary leukemic cells and hepatitis B virus-infected cells in the presence of specific antibodies. Engagement of CD16 on monocytes by antibody bound to target cells activated β2-integrins and induced TNFα secretion. In turn, this induced TNFR expression on the target cells, making them susceptible to TNFα-mediated cell death. Treatment with TLR agonists, DAMPs or cytokines, such as IFNγ, further enhanced ADCC. Monocytes lacking CD16 did not exert ADCC but acquired this property after CD16 expression was induced by either cytokine stimulation or transient transfection. Notably, CD16+ monocytes from patients with leukemia also exerted potent ADCC. Hence, CD16+ monocytes are important effectors of ADCC, suggesting further developments of this property in the context of cellular therapies for cancer and infectious diseases.

No MeSH data available.


Related in: MedlinePlus

CD16+ monocytes perform ADCC through TNFα.(A) CD16+ monocytes were co-cultured with either uncoated (white bar) or KM966-coated A549 (grey bar) at E:T ratio of 10:1 in the absence or presence of blocking antibodies. Data are plotted as mean ± SD, n = 4. ****p ≤ 0.0001 with respect to KM966-coated A549 based on One-way ANOVA (****p ≤ 0.0001), ns = not significant. (B) SKBR3 cells were FACS sorted into TNFRlo and TNFRhi populations, coated with Trast and co-cultured with CD16+ monocytes. Data are plotted as mean ± SD, n = 3. **p ≤ 0.01 with respect to parental SKBR3 and based on One-way ANOVA (****p ≤ 0.0001). (C) Raji cells were either uncoated (white bar) or pre-coated with Rtx (grey bar) before fixing with 1% PFA and co-cultured with CD16+ monocytes for 4 hours. Supernatant was collected for TNFα ELISA. Data are plotted as mean ± SD, n = 3. ****p ≤ 0.0001 based on Student’s t test. (D) SKBR3 (top panel) and primary B-CLL cells (bottom panel) were pre-coated with Trast and Rtx respectively and treated with the indicated concentrations of recombinant human TNFα (rhTNFα) in the absence (No Fc) or presence (+ Fc) of anti-human IgG. Data shown are representative of 2 independent experiments for SKBR3 and 3 independent experiments for primary B-CLL cells and plotted as specific lysis with respect to untreated cells. **p ≤ 0.01 and ****p ≤ 0.0001 with respect to no Fc of the respective E:T ratios and based on Two-way ANOVA (****p ≤ 0.0001). (E) SKBR3 (top histograms) and primary B-CLL cells (bottom histograms) were pre-coated with Trast and Rtx respectively and treated with 5 µg/ml rhTNFα in the absence (No Fc) or presence (+ Fc) of anti-human IgG. Histogram plots of TNFR expression labelled with TNFR antibody (black solid line) versus isotype-matched control antibody (grey dashed line). The rMFI of TNFR was determined by subtracting MFI of isotype-matched control from the TNFR antibody labelling. Percentages indicate the proportion of positively stained cells. Data shown are representative of 1 experiment for SKBR3 and 3 independent experiments for primary B-CLL cells.
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f5: CD16+ monocytes perform ADCC through TNFα.(A) CD16+ monocytes were co-cultured with either uncoated (white bar) or KM966-coated A549 (grey bar) at E:T ratio of 10:1 in the absence or presence of blocking antibodies. Data are plotted as mean ± SD, n = 4. ****p ≤ 0.0001 with respect to KM966-coated A549 based on One-way ANOVA (****p ≤ 0.0001), ns = not significant. (B) SKBR3 cells were FACS sorted into TNFRlo and TNFRhi populations, coated with Trast and co-cultured with CD16+ monocytes. Data are plotted as mean ± SD, n = 3. **p ≤ 0.01 with respect to parental SKBR3 and based on One-way ANOVA (****p ≤ 0.0001). (C) Raji cells were either uncoated (white bar) or pre-coated with Rtx (grey bar) before fixing with 1% PFA and co-cultured with CD16+ monocytes for 4 hours. Supernatant was collected for TNFα ELISA. Data are plotted as mean ± SD, n = 3. ****p ≤ 0.0001 based on Student’s t test. (D) SKBR3 (top panel) and primary B-CLL cells (bottom panel) were pre-coated with Trast and Rtx respectively and treated with the indicated concentrations of recombinant human TNFα (rhTNFα) in the absence (No Fc) or presence (+ Fc) of anti-human IgG. Data shown are representative of 2 independent experiments for SKBR3 and 3 independent experiments for primary B-CLL cells and plotted as specific lysis with respect to untreated cells. **p ≤ 0.01 and ****p ≤ 0.0001 with respect to no Fc of the respective E:T ratios and based on Two-way ANOVA (****p ≤ 0.0001). (E) SKBR3 (top histograms) and primary B-CLL cells (bottom histograms) were pre-coated with Trast and Rtx respectively and treated with 5 µg/ml rhTNFα in the absence (No Fc) or presence (+ Fc) of anti-human IgG. Histogram plots of TNFR expression labelled with TNFR antibody (black solid line) versus isotype-matched control antibody (grey dashed line). The rMFI of TNFR was determined by subtracting MFI of isotype-matched control from the TNFR antibody labelling. Percentages indicate the proportion of positively stained cells. Data shown are representative of 1 experiment for SKBR3 and 3 independent experiments for primary B-CLL cells.

Mentions: Monocytes/macrophages can promote cytotoxicity through several mechanisms, such as the release of reactive oxygen species (ROS), reactive nitrogen species (RNS) and TNFα3334. To clarify the mechanism utilized by CD16+ monocytes to lyse antibody-coated targets, we added inhibitors and blocking antibodies to the ADCC assay. Blocking ROS with N-acetyl-cysteine (NAC) and NOS with NG-monomethyl-L-arginine (LMMA) did not inhibit the ability of CD16+ monocytes to exert ADCC (data not shown). However, the addition of blocking antibodies against both TNFα and TNF receptor (TNFR) significantly inhibited the lysis of antibody-coated A549 lung adenocarcinoma cells (Fig. 5A). Minimal inhibition of ADCC activity was observed when isotype control mouse IgG1 antibody was added. We further established that TNFR expression on the target cells is required in the ADCC by CD16+ monocytes through assessing the lysis of TNFR high-expressing (TNFRhi) and low-expressing (TNFRlo) SKBR3 cells sorted from the parental cell line. In comparison to the parental SKBR3 cells, a significantly greater proportion of the TNFRhi cells were lysed and a significant reduction in lysis was observed for the TNFRlo cells (Fig. 5B). Furthermore, CD16+ monocytes secreted significantly higher amount of TNFα when co-cultured with antibody-coated versus uncoated Raji cells (Fig. 5C). These data indicated that interaction of CD16+ monocytes with antibody-coated target cells induced their production of TNFα, which in turn binds to TNFR-expressing target cells to promote their cell death via a TNFα-mediated mechanism.


CD16 is indispensable for antibody-dependent cellular cytotoxicity by human monocytes
CD16+ monocytes perform ADCC through TNFα.(A) CD16+ monocytes were co-cultured with either uncoated (white bar) or KM966-coated A549 (grey bar) at E:T ratio of 10:1 in the absence or presence of blocking antibodies. Data are plotted as mean ± SD, n = 4. ****p ≤ 0.0001 with respect to KM966-coated A549 based on One-way ANOVA (****p ≤ 0.0001), ns = not significant. (B) SKBR3 cells were FACS sorted into TNFRlo and TNFRhi populations, coated with Trast and co-cultured with CD16+ monocytes. Data are plotted as mean ± SD, n = 3. **p ≤ 0.01 with respect to parental SKBR3 and based on One-way ANOVA (****p ≤ 0.0001). (C) Raji cells were either uncoated (white bar) or pre-coated with Rtx (grey bar) before fixing with 1% PFA and co-cultured with CD16+ monocytes for 4 hours. Supernatant was collected for TNFα ELISA. Data are plotted as mean ± SD, n = 3. ****p ≤ 0.0001 based on Student’s t test. (D) SKBR3 (top panel) and primary B-CLL cells (bottom panel) were pre-coated with Trast and Rtx respectively and treated with the indicated concentrations of recombinant human TNFα (rhTNFα) in the absence (No Fc) or presence (+ Fc) of anti-human IgG. Data shown are representative of 2 independent experiments for SKBR3 and 3 independent experiments for primary B-CLL cells and plotted as specific lysis with respect to untreated cells. **p ≤ 0.01 and ****p ≤ 0.0001 with respect to no Fc of the respective E:T ratios and based on Two-way ANOVA (****p ≤ 0.0001). (E) SKBR3 (top histograms) and primary B-CLL cells (bottom histograms) were pre-coated with Trast and Rtx respectively and treated with 5 µg/ml rhTNFα in the absence (No Fc) or presence (+ Fc) of anti-human IgG. Histogram plots of TNFR expression labelled with TNFR antibody (black solid line) versus isotype-matched control antibody (grey dashed line). The rMFI of TNFR was determined by subtracting MFI of isotype-matched control from the TNFR antibody labelling. Percentages indicate the proportion of positively stained cells. Data shown are representative of 1 experiment for SKBR3 and 3 independent experiments for primary B-CLL cells.
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f5: CD16+ monocytes perform ADCC through TNFα.(A) CD16+ monocytes were co-cultured with either uncoated (white bar) or KM966-coated A549 (grey bar) at E:T ratio of 10:1 in the absence or presence of blocking antibodies. Data are plotted as mean ± SD, n = 4. ****p ≤ 0.0001 with respect to KM966-coated A549 based on One-way ANOVA (****p ≤ 0.0001), ns = not significant. (B) SKBR3 cells were FACS sorted into TNFRlo and TNFRhi populations, coated with Trast and co-cultured with CD16+ monocytes. Data are plotted as mean ± SD, n = 3. **p ≤ 0.01 with respect to parental SKBR3 and based on One-way ANOVA (****p ≤ 0.0001). (C) Raji cells were either uncoated (white bar) or pre-coated with Rtx (grey bar) before fixing with 1% PFA and co-cultured with CD16+ monocytes for 4 hours. Supernatant was collected for TNFα ELISA. Data are plotted as mean ± SD, n = 3. ****p ≤ 0.0001 based on Student’s t test. (D) SKBR3 (top panel) and primary B-CLL cells (bottom panel) were pre-coated with Trast and Rtx respectively and treated with the indicated concentrations of recombinant human TNFα (rhTNFα) in the absence (No Fc) or presence (+ Fc) of anti-human IgG. Data shown are representative of 2 independent experiments for SKBR3 and 3 independent experiments for primary B-CLL cells and plotted as specific lysis with respect to untreated cells. **p ≤ 0.01 and ****p ≤ 0.0001 with respect to no Fc of the respective E:T ratios and based on Two-way ANOVA (****p ≤ 0.0001). (E) SKBR3 (top histograms) and primary B-CLL cells (bottom histograms) were pre-coated with Trast and Rtx respectively and treated with 5 µg/ml rhTNFα in the absence (No Fc) or presence (+ Fc) of anti-human IgG. Histogram plots of TNFR expression labelled with TNFR antibody (black solid line) versus isotype-matched control antibody (grey dashed line). The rMFI of TNFR was determined by subtracting MFI of isotype-matched control from the TNFR antibody labelling. Percentages indicate the proportion of positively stained cells. Data shown are representative of 1 experiment for SKBR3 and 3 independent experiments for primary B-CLL cells.
Mentions: Monocytes/macrophages can promote cytotoxicity through several mechanisms, such as the release of reactive oxygen species (ROS), reactive nitrogen species (RNS) and TNFα3334. To clarify the mechanism utilized by CD16+ monocytes to lyse antibody-coated targets, we added inhibitors and blocking antibodies to the ADCC assay. Blocking ROS with N-acetyl-cysteine (NAC) and NOS with NG-monomethyl-L-arginine (LMMA) did not inhibit the ability of CD16+ monocytes to exert ADCC (data not shown). However, the addition of blocking antibodies against both TNFα and TNF receptor (TNFR) significantly inhibited the lysis of antibody-coated A549 lung adenocarcinoma cells (Fig. 5A). Minimal inhibition of ADCC activity was observed when isotype control mouse IgG1 antibody was added. We further established that TNFR expression on the target cells is required in the ADCC by CD16+ monocytes through assessing the lysis of TNFR high-expressing (TNFRhi) and low-expressing (TNFRlo) SKBR3 cells sorted from the parental cell line. In comparison to the parental SKBR3 cells, a significantly greater proportion of the TNFRhi cells were lysed and a significant reduction in lysis was observed for the TNFRlo cells (Fig. 5B). Furthermore, CD16+ monocytes secreted significantly higher amount of TNFα when co-cultured with antibody-coated versus uncoated Raji cells (Fig. 5C). These data indicated that interaction of CD16+ monocytes with antibody-coated target cells induced their production of TNFα, which in turn binds to TNFR-expressing target cells to promote their cell death via a TNFα-mediated mechanism.

View Article: PubMed Central - PubMed

ABSTRACT

Antibody-dependent cellular cytotoxicity (ADCC) is exerted by immune cells expressing surface Fcγ receptors (FcγRs) against cells coated with antibody, such as virus-infected or transformed cells. CD16, the FcγRIIIA, is essential for ADCC by NK cells, and is also expressed by a subset of human blood monocytes. We found that human CD16− expressing monocytes have a broad spectrum of ADCC capacities and can kill cancer cell lines, primary leukemic cells and hepatitis B virus-infected cells in the presence of specific antibodies. Engagement of CD16 on monocytes by antibody bound to target cells activated β2-integrins and induced TNFα secretion. In turn, this induced TNFR expression on the target cells, making them susceptible to TNFα-mediated cell death. Treatment with TLR agonists, DAMPs or cytokines, such as IFNγ, further enhanced ADCC. Monocytes lacking CD16 did not exert ADCC but acquired this property after CD16 expression was induced by either cytokine stimulation or transient transfection. Notably, CD16+ monocytes from patients with leukemia also exerted potent ADCC. Hence, CD16+ monocytes are important effectors of ADCC, suggesting further developments of this property in the context of cellular therapies for cancer and infectious diseases.

No MeSH data available.


Related in: MedlinePlus