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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

FcγRIII is involved in CD16+ monocyte mediated ADCC.(A) CD16+ monocytes were untreated or pre-treated with FcγR blocking antibodies or isotype control and co-cultured with uncoated (white bar) or trast-coated SKBR3 (grey bar), n = 3. **p ≤ 0.01, ***p ≤ 0.001. ****p ≤ 0.0001 is compared to untreated trast-coated SKBR3. One-way ANOVA (****p ≤ 0.0001), ns = not significant. (B) CD16+ monocytes were FACS-sorted to intermediate and non-classical subsets (FACS plot). Non-classical monocytes (triangle symbol) exhibit higher ADCC than intermediate monocytes (square symbol) on A549 (left graph) and SKBR3 (right graph) pre-coated with respective antibody (closed symbols) at various E:T ratios. Uncoated target cells are represented by open symbols. Data shown are representative of 2 independent experiments for each tumour cell lines. **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001 compared to intermediate monocytes at the respective E:T ratios. Two-way ANOVA (****p ≤ 0.0001). (C) Histogram plot showing SLAN expression on non-classical monocytes (left). SLAN+ and SLAN− monocytes were co-cultured with uncoated (white bar) or trast-coated SKBR3 (grey bar), n = 3. One-way ANOVA (***p ≤ 0.001) (right). (D) CD16− monocytes were untreated or pre-treated with M-CSF, TGF-β or IL-10. The rMFI of CD16 was determined by subtracting mean fluorescence intensity (MFI) of isotype-matched control (dashed line) from the CD16 labelling (solid line). Percentages indicate positively-stained cells. Bar graph depicts ADCC assay performed using untreated and treated CD16− monocytes with trast-coated SKBR3. Data plotted as fold difference with respect to specific target lysis of freshly isolated CD16− monocytes, which was 5%, n = 7. *p ≤ 0.05, ***p ≤ 0.001 compared to untreated CD16− monocytes and One-way ANOVA (****p ≤ 0.0001). (E) CD16− monocytes were either mock or CD16 mRNA transfected. Histogram plots showing CD16 expression either labelled with CD16 antibody (solid line) versus isotype-matched control (dashed line). Percentages indicate positively stained cells. ADCC assay was performed using mock and CD16 mRNA-transfected CD16− monocytes co-cultured with trast-coated SKBR3 cells (bar graph), n = 3. **p ≤ 0.01 based on Student’s t test. All the ADCC assays are based on E:T ratio of 10:1 and all data are plotted as mean ± SD.
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f3: FcγRIII is involved in CD16+ monocyte mediated ADCC.(A) CD16+ monocytes were untreated or pre-treated with FcγR blocking antibodies or isotype control and co-cultured with uncoated (white bar) or trast-coated SKBR3 (grey bar), n = 3. **p ≤ 0.01, ***p ≤ 0.001. ****p ≤ 0.0001 is compared to untreated trast-coated SKBR3. One-way ANOVA (****p ≤ 0.0001), ns = not significant. (B) CD16+ monocytes were FACS-sorted to intermediate and non-classical subsets (FACS plot). Non-classical monocytes (triangle symbol) exhibit higher ADCC than intermediate monocytes (square symbol) on A549 (left graph) and SKBR3 (right graph) pre-coated with respective antibody (closed symbols) at various E:T ratios. Uncoated target cells are represented by open symbols. Data shown are representative of 2 independent experiments for each tumour cell lines. **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001 compared to intermediate monocytes at the respective E:T ratios. Two-way ANOVA (****p ≤ 0.0001). (C) Histogram plot showing SLAN expression on non-classical monocytes (left). SLAN+ and SLAN− monocytes were co-cultured with uncoated (white bar) or trast-coated SKBR3 (grey bar), n = 3. One-way ANOVA (***p ≤ 0.001) (right). (D) CD16− monocytes were untreated or pre-treated with M-CSF, TGF-β or IL-10. The rMFI of CD16 was determined by subtracting mean fluorescence intensity (MFI) of isotype-matched control (dashed line) from the CD16 labelling (solid line). Percentages indicate positively-stained cells. Bar graph depicts ADCC assay performed using untreated and treated CD16− monocytes with trast-coated SKBR3. Data plotted as fold difference with respect to specific target lysis of freshly isolated CD16− monocytes, which was 5%, n = 7. *p ≤ 0.05, ***p ≤ 0.001 compared to untreated CD16− monocytes and One-way ANOVA (****p ≤ 0.0001). (E) CD16− monocytes were either mock or CD16 mRNA transfected. Histogram plots showing CD16 expression either labelled with CD16 antibody (solid line) versus isotype-matched control (dashed line). Percentages indicate positively stained cells. ADCC assay was performed using mock and CD16 mRNA-transfected CD16− monocytes co-cultured with trast-coated SKBR3 cells (bar graph), n = 3. **p ≤ 0.01 based on Student’s t test. All the ADCC assays are based on E:T ratio of 10:1 and all data are plotted as mean ± SD.

Mentions: As CD16+ monocytes also express high levels of CD32 and low levels of CD64 (two other Fcγ receptors), we determined their relative role in ADCC exerted by CD16+ monocytes. Minimal inhibition of ADCC activity was observed when isotype control mouse IgG1 antibody or CD64 blocking antibody was added (Fig. 3A; left panel). A 2-fold inhibition in lysis was observed when 10 μg/ml of CD32 blocking antibody was added. However, the addition of 10 μg/ml of CD16 blocking antibody inhibited target cell lysis to levels similar to basal cytotoxicity (Fig. 3A; left panel). Even when a lower concentration of CD32 or CD16 blocking antibodies (5 μg/ml) was used, the extent of inhibition was consistently higher when CD16 was blocked (Fig. 3A; right panel). However, ADCC was further inhibited when 5 μg/ml of CD32 and CD16 blocking antibodies were added together (Fig. 3A; right panel).


CD16 is indispensable for antibody-dependent cellular cytotoxicity by human monocytes
FcγRIII is involved in CD16+ monocyte mediated ADCC.(A) CD16+ monocytes were untreated or pre-treated with FcγR blocking antibodies or isotype control and co-cultured with uncoated (white bar) or trast-coated SKBR3 (grey bar), n = 3. **p ≤ 0.01, ***p ≤ 0.001. ****p ≤ 0.0001 is compared to untreated trast-coated SKBR3. One-way ANOVA (****p ≤ 0.0001), ns = not significant. (B) CD16+ monocytes were FACS-sorted to intermediate and non-classical subsets (FACS plot). Non-classical monocytes (triangle symbol) exhibit higher ADCC than intermediate monocytes (square symbol) on A549 (left graph) and SKBR3 (right graph) pre-coated with respective antibody (closed symbols) at various E:T ratios. Uncoated target cells are represented by open symbols. Data shown are representative of 2 independent experiments for each tumour cell lines. **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001 compared to intermediate monocytes at the respective E:T ratios. Two-way ANOVA (****p ≤ 0.0001). (C) Histogram plot showing SLAN expression on non-classical monocytes (left). SLAN+ and SLAN− monocytes were co-cultured with uncoated (white bar) or trast-coated SKBR3 (grey bar), n = 3. One-way ANOVA (***p ≤ 0.001) (right). (D) CD16− monocytes were untreated or pre-treated with M-CSF, TGF-β or IL-10. The rMFI of CD16 was determined by subtracting mean fluorescence intensity (MFI) of isotype-matched control (dashed line) from the CD16 labelling (solid line). Percentages indicate positively-stained cells. Bar graph depicts ADCC assay performed using untreated and treated CD16− monocytes with trast-coated SKBR3. Data plotted as fold difference with respect to specific target lysis of freshly isolated CD16− monocytes, which was 5%, n = 7. *p ≤ 0.05, ***p ≤ 0.001 compared to untreated CD16− monocytes and One-way ANOVA (****p ≤ 0.0001). (E) CD16− monocytes were either mock or CD16 mRNA transfected. Histogram plots showing CD16 expression either labelled with CD16 antibody (solid line) versus isotype-matched control (dashed line). Percentages indicate positively stained cells. ADCC assay was performed using mock and CD16 mRNA-transfected CD16− monocytes co-cultured with trast-coated SKBR3 cells (bar graph), n = 3. **p ≤ 0.01 based on Student’s t test. All the ADCC assays are based on E:T ratio of 10:1 and all data are plotted as mean ± SD.
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f3: FcγRIII is involved in CD16+ monocyte mediated ADCC.(A) CD16+ monocytes were untreated or pre-treated with FcγR blocking antibodies or isotype control and co-cultured with uncoated (white bar) or trast-coated SKBR3 (grey bar), n = 3. **p ≤ 0.01, ***p ≤ 0.001. ****p ≤ 0.0001 is compared to untreated trast-coated SKBR3. One-way ANOVA (****p ≤ 0.0001), ns = not significant. (B) CD16+ monocytes were FACS-sorted to intermediate and non-classical subsets (FACS plot). Non-classical monocytes (triangle symbol) exhibit higher ADCC than intermediate monocytes (square symbol) on A549 (left graph) and SKBR3 (right graph) pre-coated with respective antibody (closed symbols) at various E:T ratios. Uncoated target cells are represented by open symbols. Data shown are representative of 2 independent experiments for each tumour cell lines. **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001 compared to intermediate monocytes at the respective E:T ratios. Two-way ANOVA (****p ≤ 0.0001). (C) Histogram plot showing SLAN expression on non-classical monocytes (left). SLAN+ and SLAN− monocytes were co-cultured with uncoated (white bar) or trast-coated SKBR3 (grey bar), n = 3. One-way ANOVA (***p ≤ 0.001) (right). (D) CD16− monocytes were untreated or pre-treated with M-CSF, TGF-β or IL-10. The rMFI of CD16 was determined by subtracting mean fluorescence intensity (MFI) of isotype-matched control (dashed line) from the CD16 labelling (solid line). Percentages indicate positively-stained cells. Bar graph depicts ADCC assay performed using untreated and treated CD16− monocytes with trast-coated SKBR3. Data plotted as fold difference with respect to specific target lysis of freshly isolated CD16− monocytes, which was 5%, n = 7. *p ≤ 0.05, ***p ≤ 0.001 compared to untreated CD16− monocytes and One-way ANOVA (****p ≤ 0.0001). (E) CD16− monocytes were either mock or CD16 mRNA transfected. Histogram plots showing CD16 expression either labelled with CD16 antibody (solid line) versus isotype-matched control (dashed line). Percentages indicate positively stained cells. ADCC assay was performed using mock and CD16 mRNA-transfected CD16− monocytes co-cultured with trast-coated SKBR3 cells (bar graph), n = 3. **p ≤ 0.01 based on Student’s t test. All the ADCC assays are based on E:T ratio of 10:1 and all data are plotted as mean ± SD.
Mentions: As CD16+ monocytes also express high levels of CD32 and low levels of CD64 (two other Fcγ receptors), we determined their relative role in ADCC exerted by CD16+ monocytes. Minimal inhibition of ADCC activity was observed when isotype control mouse IgG1 antibody or CD64 blocking antibody was added (Fig. 3A; left panel). A 2-fold inhibition in lysis was observed when 10 μg/ml of CD32 blocking antibody was added. However, the addition of 10 μg/ml of CD16 blocking antibody inhibited target cell lysis to levels similar to basal cytotoxicity (Fig. 3A; left panel). Even when a lower concentration of CD32 or CD16 blocking antibodies (5 μg/ml) was used, the extent of inhibition was consistently higher when CD16 was blocked (Fig. 3A; right panel). However, ADCC was further inhibited when 5 μg/ml of CD32 and CD16 blocking antibodies were added together (Fig. 3A; right panel).

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