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

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.

Figure 1. CD16+ but not the CD16− monocytes are able to perform ADCC on therapeutic antibody–coated tumour and virus-infected cell lines.

ADCC by CD16+ (left panel) and CD16− (right panel) monocytes on A549 lung adenocarcinoma (A) Raji Burkitt’s lymphoma (B) and SKBR3 breast adenocarcinoma (C) at the indicated effector to target (E:T) cell ratios. Tumour cell lines were either uncoated (open symbols and dotted lines) or pre-coated with the respective therapeutic antibodies αGM2 (KM966), αCD20 (Rituximab) and αHER2 (Trastuzumab) (closed symbols and solid lines). Data shown are representative data of at least 5 independent experiments and plotted as mean ± SD of triplicate wells for each respective experiment. **p ≤ 0.01, ****p ≤ 0.0001 with respect to uncoated target cells at the respective E:T ratios based on Two-way ANOVA (****p ≤ 0.0001). (D) ADCC by CD16+ monocytes on Hepatitis B virus-infected cell line HepG2.2.15 (left) or parental HepG2 (right) cells at E:T ratio of 10:1. Both HepG2 cell lines were either uncoated (white bar) or coated with TCR-like antibodies recognising core or envelope peptides respective (grey bars). Data shown are plotted as mean ± SD; n = 4. ****p ≤ 0.0001 with respect to uncoated cells based on One-way ANOVA (****p ≤ 0.0001). (E) NK cells, CD16+ and CD16− monocytes isolated from the same individual were co-cultured with KM966-coated A549 at an E:T ratio of 10:1. Data plotted is mean ± SD, n = 2. **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001. One-way ANOVA (***p ≤ 0.001).

Figure 2. CD16+ monocytes from healthy individuals are able to perform ADCC on Rtx-coated primary B leukemic cells.

ADCC by CD16+ monocytes from three healthy individuals on primary B leukaemic cells isolated from three B-CLL patients (A–C) either uncoated (dotted lines) or coated with Rtx (solid lines) at the indicated E:T ratios. All data plotted are mean ± SD of triplicate wells for each experiment. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001 with respect to uncoated leukaemic cells at the respective E:T ratios and based on Two-way ANOVA (****p ≤ 0.0001).

Figure 3. 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.

Figure 4. Cell-cell contact mediated through β2-integrins is needed for ADCC to take place.

(A) CD16+ monocytes were co-cultured with SKBR3 at E:T ratio of 10:1. The x-axis depicts the conditions at which tumour targets were used. SKBR3 cells labelled with BATDA are depicted with black diamond inside and are either uncoated or trast-coated (antibody symbol). Data plotted are mean ± SD, n = 3. ****p ≤ 0.0001 with respect to trast-coated SKBR3 and based on One-way ANOVA (****p ≤ 0.0001), ns = not significant. (B) ADCC by CD16+ monocytes was assessed using time-lapsed imaging (see also Supplementary Movie). Interaction of CD16+ monocyte (black arrow) and trast-coated SKBR3 (dotted) was tracked at 20 secs intervals for up to 2 hrs and static images at various times as indicated are shown. Brightfield images were visualised under a FV-1000 confocal system with an inverted Olympus IX81 microscope at 200x magnification using Fluoview software FV10-ASW 2.0. Data shown is a representative of 3 independent experiments where >10 different cells were followed in each experiment. (C) CD16+ monocytes were either untreated or pre-treated with blocking antibodies for the indicated integrins prior to co-culturing with either uncoated (white bar) or trast-coated SKBR3 (grey bar) at E:T ratio of 10:1. Data plotted are mean ± SD, n = 3. ****p ≤ 0.0001 with respect to untreated trast-coated SKBR3 based on One-way ANOVA (****p ≤ 0.0001), ns = not significant.

Figure 5. 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.

Figure 6. Pre-treatment with various stimulus enhances the ability of CD16+ monocytes to perform ADCC.

(A–C) CD16+ monocytes, NK and CD16− monocytes respectively were either untreated (white bar) or pre-treated with various stimulus (grey bar). And then co-cultured with KM966-coated A549 at E:T ratio of 10:1. Data was plotted as fold difference in target cell lysis of treated to untreated effector cells. The percentage specific lysis of untreated CD16+, NK and CD16− cells were 10.1% (±4.6%), 20.6% (±10.5%) and 3.7% (±0.7%) respectively. Data are plotted as mean ± SD, n = 3. *p ≤ 0.05 and ****p ≤ 0.0001 with respect to untreated effector cells and based on One-way ANOVA (****p ≤ 0.0001 for CD16+ and NK), ns = not significant.

Figure 7. CD16+ monocytes from B-CLL patients exhibit ADCC activity similar to healthy individuals.

(A) CD16+ monocytes (left graph) or NK cells (right graph) isolated from the same leukemia patient (striped) or healthy donor (solid) were co-cultured with either uncoated (white bar) or Trast-coated SKBR3 (grey bar) at E:T ratio of 10:1. Data are plotted as mean ± SD, n = 2. ***p ≤ 0.001 based on One-way ANOVA (****p ≤ 0.0001). (B) CD16+ monocytes (left graph) or NK cells (right graph) isolated from the same patient (striped) or healthy donor (solid) were co-cultured with either uncoated (white bar) or Rtx-coated primary B-CLL cells (grey bar) at E:T ratio of 10:1. Data are plotted as mean ± SD, n = 4. **p ≤ 0.01 based on One-way ANOVA (****p ≤ 0.0001).