Immunoglobulin G (IgG) antibody subclasses play major roles in the control of bacterial and viral infections, the killing of tumour cells during antibody therapy and the pathological destruction of healthy tissues in autoimmune diseases. As a result of their potency and range of actions, antibodies have become one of the most rapidly growing classes of human therapeutics in recent years, particularly in cancer treatments.

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.

Introduction

Antibodies mediate their anti-tumour effects directly, by interfering with tumor cell growth, or indirectly by activating immune-mediated complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC). A growing body of evidence suggests that ADCC may be the dominant mechanism operating in vivo^1,2,3,4,5. The process of ADCC begins with recognition of an antigen expressed on the target cell surface by specific immunoglobulins. The Fc domain of these antibodies is then bound by Fcγ receptors (FcγRs) expressed on immune effector cells, which triggers the release of cytotoxic granules towards the target cell or upregulates death receptors expression on the cell surface. In murine cancer models, both rituximab and trastuzumab efficacy has been shown to completely depend on activating FcγRs^6,7, in particular FcγRI and/or FcγRIV^8,9. This appears to be similar in humans where polymorphisms in either FcγRIIa or FcγRIII that affect their affinity for IgG influence the clinical success of rituximab^10,11, trastuzumab^{1,2,3,12,13,14} and cetuximab^4,5,6 treatment for B-cell lymphoma, breast cancer and colorectal cancer, respectively.

NK cells are considered to be the main mediators of ADCC both in physiological and therapeutic settings. However, NK cells are present only in low numbers in the microenvironment of colorectal^7,8, renal^9,10, liver, skin, breast and lung carcinomas^11,12,13,14. Defects in their cytotoxic function due to changes in activating and inhibiting receptor expression, upregulation in MHC class I expression, decreased expression in the signal transducing ζ chain, CD16 and cytotoxic machinery has been reported in numerous studies^15,16,17. A recent study further showed that cross-linking of CD16 on NK cells promoted a phenomenon known as NK cell abnormalities (NKCA), which not only included CD16 down-regulation, but also an increased in the frequency of apoptotic NK cells as well as enhanced depletion of NK cells in the presence of leukemic cells¹⁸. Apparently, this NKCA can be overcome by inhibiting MMP activation with TIMP3¹⁸. Notably, trastuzumab treatment was more effective in mice lacking the inhibitory FcγRIIb⁷, which is not expressed by NK cells, implying the involvement of other immune cell populations. Using target cell depletion approaches in mice, several studies have demonstrated that monocytes and macrophages to be the principal mediators of ADCC against α-CD20-coated B cells in vivo^4,19.

In both mice and humans, subsets of blood monocytes exhibit differential surface expression of the various FcγRs²⁰. FcγRIIIA (CD16) distinguishes human monocytes into two major subsets (i.e. CD16+ and CD16−) and they can be further subdivided using additional surface markers such as CD56^21,22. The minor subset that expresses low level of CD56 is mostly CD16− and is expanded in numbers under inflammatory conditions like Crohn’s disease and rheumatoid arthritis^23,24. While both the CD16+ and CD16− monocyte populations express similar levels of FcγRIIA (CD32), FcγRI (CD64) is preferentially expressed on the CD16− subset²⁰. The infiltration of monocytes into tumours has been widely observed. Interestingly, a recent study showed that tumour infiltration of CD16+ myeloid was associated with improved survival of colorectal cancer patients⁸. Whether CD16 expression on monocytes could promote cytotoxicity like it does in NK cells is unknown. In this study, we determine the capacity of human monocyte subsets to perform ADCC, and specifically assessed the role of CD16 in this function.

Discussion

Our data show that the human blood monocyte subsets that express CD16 possess the capacity to exert ADCC on cell lines, primary tumor cells and virally infected cells. ADCC by CD16+ monocytes was as efficient as that of NK cells. The CD16− subset when acquired CD16 expression could promote ADCC, revealing that this subset intrinsically possessed the machinery required to promote cytolysis of antibody-coated targets. ADCC activity could be further enhanced upon stimulation of CD16+ monocytes with TLR agonists, cytokines such as IFNγ and DAMPs. Cell-cell contact was essential and target cells lysis occurred through a TNFα-mediated mechanism. CD16+ monocytes from B-CLL patients did not exhibit discernible dysfunctions and showed ADCC activity similar to that of CD16+ monocytes from healthy individuals.

The involvement of other immune cell types in mediating ADCC has been clearly evident in numerous preclinical studies. These mouse studies demonstrated that monocytes and/or macrophages and not NK cells are the principal mediators of ADCC against α-CD20-coated B cells in vivo^4,19 further supporting the importance of monocyte in eradicating antibody-coated cells in vivo.

Our observation that the capacity for ADCC is unique to the CD16+ subset of human monocytes, is in line with recent findings both in humans³⁶ and in mice¹⁹. Mice deficient in FcγRIV, the murine homolog of human CD16³⁷ exhibit defects in several models of ADCC³⁸. In our study, the engagement of CD16 and potentially CD32, but not CD64, was necessary to trigger ADCC, which was similar to that reported for SLAN+ DC²⁹. However, unlike the data by Schmitz et al.²⁹ showing equal contribution of both CD32 and CD16 to ADCC by DC, our study showed that blocking CD16 inhibited lysis to a greater extent than CD32. CD16− monocytes were unable to exert ADCC despite the expression of CD64 and CD32 unless CD16 expression was enforced. Their differential ADCC could not be explained by the expression of CD32 isoforms, i.e. CD32a (activating) and CD32b (inhibiting) since CD32a expression is similar on both monocyte subsets and CD32b expression is higher on CD16+ monocytes^20,39 and unpublished data. Furthermore, the fact that the level of ADCC activity of these cells positively correlated with the level of CD16 expression further confirms the essential role played by CD16. Functional polymorphisms in the coding regions of the different FcγR s are known to impact their affinity for IgG. In fact, many studies correlating FcγR polymorphisms, particularly for CD32 and CD16 with clinical response, suggest a role for FcγR-mediated effector functions in antibody therapy. In both rituximab and trastuzumab treatments for follicular lymphoma and metastatic breast cancer respectively, polymorphism in both CD16 (i.e. FcγRIIIa-158V/F) and CD32 (i.e. FcγRIIa -131H/R) were shown to correlate with clinical responses^1,11. Another study in metastatic breast cancer found homozygosity for FcγRIIa-131H alone to be significantly associated with a stronger anti-tumour response and progression free survival when patients are treated with trastuzumab⁴⁰. These further support a predominant role of myeloid cells including monocytes in antibody therapy.

Panitumumab, an EGF receptor antibody, currently the only approved human IgG2 antibody, has been shown to promote ADCC by myeloid cells including monocytes as effectively as the IgG1 antibody at low doses⁴¹. Unlike IgG1, they bind CD32 with higher affinity⁴². With our study demonstrating that CD32 together with CD16 are involved in ADCC provide support for the potential application of IgG2 antibody in immunotherapy.

Impaired NK cell function has been reported in various types of malignancy. Alterations in the expression of activating and inhibiting receptors, increased MHC class I expression, down-regulated expression in the signal transducing ζ chain, CD16 and cytotoxic machinery were reported to contribute to NK cell dysfunction^15,16,17. Although reduced expression of CD16 on NK cells was commonly observed in many malignancies, there was no significant down-regulation of CD16 expression on NK cells from B-CLL patients in our study (data not shown). Nevertheless, these cells still exhibited a reduced ADCC ability compared to NK cells from healthy individuals possibly due to other factors mentioned above. On the contrary, CD16+ monocytes from these patients were as capable as CD16+ monocytes from healthy individuals in terms of ADCC.

Unlike NK cells where IL-12 and IL-15 activates and enhances their cytolytic ability, the ADCC capacity of CD16+ monocytes was unaffected by these cytokines. A previous study showed that IFNγ could enhance monocyte/macrophage ADCC activity but only via FcγRI⁴³. While TLR agonists such as CpG can enhance the cytolytic ability of NK cells⁴⁴, LPS and R848, which ligate TLR4 and TLR7/8 respectively, specifically enhanced the ADCC activity of CD16+ monocytes. R848 has been shown to activate NK cell cytotoxicity after 18 hrs⁴⁵ but no enhancing effect was detected at the 5 hr time point used in our study. TLR8 agonist, in particular, promoted ADCC by monocytes through a IL-12-induced granzyme B expression and secretion after 12 hrs⁴⁶. However, no granzyme B protein was detectable when CD16+ monocytes were treated with R848 for 5 hrs in our study (data not shown) and IL-12 treatment also did not enhance ADCC at this time-point. Both HMGB1 and S100A9 are self-derived molecules well-known as damage-associated molecular patterns (DAMPs), which are released at sites of tissue damage or regions of necrotic cells⁴⁷. Treatment of cancer with therapeutic antibodies is routinely performed in conjunction with chemotherapy or surgery, which leads to tissue damage and death in the tumour environment. As such, monocytes recruited to the tumour site and activated locally by DAMPs might then be able to promote killing of the remaining cancer cells coated with the therapeutic antibody. Both HMGB1 and S100A9 as well as TLR agonists and IFNγ could also activate monocytes to release pro-inflammatory cytokines including TNFα^48,49,50. Specifically, IL-10 and TGF-β over-production were shown to decrease NK cell mediated functions including ADCC, down regulation of CD16 expression and IFNγ production^16,51,52, our data however showed that the enhancement of CD16 expression on CD16− monocytes by these mediators conferred these monocytes with ADCC activity, which might be favorable for cancer immunotherapy.

Previous studies showed that FcγR engagement can induce β2-integrin activation on murine macrophages for optimal phagocytic activity but played no role in ADCC by in vitro differentiated human macrophages^53,54. However, β2-integrin appears to be involved in ADCC by CD16+ monocytes in our study. Besides promoting the release of proinflammatory cytokines, stimuli like LPS and S100A9 may potentially be enhancing ADCC activity of CD16+ monocytes through regulating the activity of CD11b, the binding partner of β2-integrin^55,56.

The production of TNFα by activated macrophages and monocytes has been well described. The involvement of TNFα in ADCC by macrophages through antibody neutralization assay had also been reported in numerous studies²⁹. Nevertheless, the exact mechanism is still unclear. The TNFα secreted by CD16+ monocytes upon engagement of the FcγR could be involved in the activation of b2-integrins in an autocrine fashion similar to that reported for neutrophils⁵⁷. In addition, as shown for breast cancer cells, the secreted TNFα also induced ICAM1 expression on the tumor cells in our study (data not shown)⁵⁸. Together, these would result in further cell-cell interaction to promote target cell lysis. Most importantly, only target cells in direct contact with the CD16+ monocytes will undergo ADCC because the clustering of antigens on the target cell surface through engaging the FcγR on the CD16+ monocytes promoted TNFR surface expression, predisposing these target cells to TNFα-mediated cell death. A finding that has not yet been reported.

Moreover, CD16+ monocytes have been reported to expand during infection, autoimmune disease and certain cancers such as colorectal, gastric and breast^59,60. It will therefore be interesting to understand how this biological observation might link with clinical outcomes, and in particular whether higher numbers of CD16+ monocytes might favor better responses to therapeutic antibody treatment. Interestingly, a study by Romano et al. showed that melanoma patients who responded to treatment with ipilimumab had a significantly higher proportion of CD16+ monocytes as compared with non-responding patients³⁶. Another study showed that CD16+ myeloid cells infiltration into the tumour mass in colorectal cancer patients represents a strong, novel and independent prognostic prosurvival factor⁸. Further studies are required to determine how the preferential expansion of this subset influences the progress of the different diseases. Moreover, we have shown the potential for human CD16+ monocytes to be effective mediators of ADCC against a range of cell types in vitro and therefore exploration of ways to exploit this potential in vivo could prove valuable in the clinical setting.