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13.2C: Antibody-dependent Cellular Cytotoxicity (ADCC) by Natural Killer Cells - Biology

13.2C: Antibody-dependent Cellular Cytotoxicity (ADCC) by Natural Killer Cells - Biology


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Learning Objectives

  1. Discuss how antibodies defend the body by way of ADCC by Natural Killer cells. (Include what classes or isotypes of immunoglobulins are involved, the role of the Fab portion of the antibody, the role, if any, of the Fc portion of the antibody, and the role of any complement proteins, if any, involved.)

Natural killer (NK) cells are capable of antibody-dependent cellular cytotoxicity or ADCC. NK cells have receptors on their surface for the Fc portion of certain subclasses of IgG. When the antibody IgG is made against epitopes on "foreign" membrane-bound cells, such as virus-infected cells and cancer cells, the Fab portions of the antibodies react with the "foreign" cell. The NK cells then bind to the Fc portion of the antibody (Figure (PageIndex{13}).2.1).

Figure (PageIndex{13}).2.1: Destruction of Virus-Infected Cells by NK Cells through Antibody-Dependent Cellular Cytotoxicity (ADCC), (left) Step 1: The Fab portion of the antibody binds to epitopes on the "foreign" cell. The NK cell then binds to the Fc portion of the antibody. (right) Step 2: The NK cell is then able to contact the cell and release pore-forming proteins called perforins and proteolytic enzymes called granzymes. Granzymes pass through the pores and activate the enzymes that lead to apoptosis of the infected cell by means of destruction of its structural cytoskeleton proteins and by chromosomal degradation. As a result, the cell breaks into fragments that are subsequently removed by phagocytes. Perforins can also sometimes result in cell lysis.

The NK cell then releases pore-forming proteins called perforins, proteolytic enzymes called granzymes, and chemokines. Granzymes pass through the pores and activate the enzymes that lead to apoptosis of the infected cell by means of destruction of its structural cytoskeleton proteins and by chromosomal degradation (Figure (PageIndex{13}).5.1; right panel and Figure (PageIndex{12}).5.2). Perforins can also sometimes result in cell lysis. (When NK cells are carrying out ADCC, they are sometimes also referred to as killer cells.)

Figure (PageIndex{13}).2.2: NK cells release pore-forming proteins called perforins and proteolytic enzymes called granzymes. Granzymes pass through the pores and activate the enzymes that lead to apoptosis, a programmed suicide of the infected cell. Apoptosis occurs when certain granzymes activate a group of protease enzymes called caspases that destroy the protein structural scaffolding of the cell, degrade the cell's nucleoprotein, and activate enzymes that degrade the cell's DNA. As a result, the infected cell breaks into membrane-bound fragments that are subsequently removed by phagocytes. If very large numbers of perforins are inserted into the plasma membrane of the infected cell, this can result in a weakening of the membrane and lead to cell lysis rather than apoptosis. An advantage to killing infected cells by apoptosis is that the cell's contents, including viable virus particles and mediators of inflammation, are not released as they are during cell lysis.

Video: YouTube animation illustrating the detailed cellular mechanism behind apoptosis. https://www.youtube.com/watch?v=9KTDz-ZisZ0

Explain how IgG can work with NK cells to kill virus-infected cells.

Outside Links

  1. Flash animation of ADCC contact by NK cells.
  2. html5 version of animation for iPad showing ADCC contact by NK cells
  3. Flash animation of apoptosis by NK cells.
  4. html5 version of animation for iPad showing apoptosis by NK cells.

Summary

NK cells are capable of antibody-dependent cellular cytotoxicity or ADCC. When IgG is made against epitopes on "foreign" membrane-bound cells, such as virus-infected cells and cancer cells, the Fab portions of the antibodies react with epitopes on the "foreign" cell and then NK cells bind to the Fc portion of the antibody. The NK cell then releases pore-forming proteins called perforins and proteolytic enzymes called granzymes. Granzymes pass through the pores and activate the enzymes that lead to apoptosis of the infected cell and the cell breaks into fragments that are subsequently removed by phagocytes.

Questions

Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.

  1. Discuss how antibodies defend the body by way of ADCC by NK cells. (Include what classes or isotypes of immunoglobulins are involved, the role of the Fab portion of the antibody, the role, if any, of the Fc portion of the antibody, and the role of any complement proteins, if any, involved.)
  2. Antibody-dependent cellular cytotoxicity (ADCC) is a result of:
    1. Antibodies sticking infected cells and cancer cells to phagocytes.
    2. Antibodies sticking infected cells and cancer cells to cytotoxic T-lymphocytes (CTLs).
    3. Antibodies sticking infected cells and cancer cells to NK cells.
    4. MAC lysing the membranes of infected cells and cancer cells.
  3. During ADCC, the Fab portion of the antibody _____________while the Fc portion _______________.
    1. binds to epitopes of an antigen; activates the complement pathway.
    2. activates the complement pathway; binds to epitopes of an antigen.
    3. binds to epitopes of an antigen; binds to cytotoxic T-lymphocytes.
    4. binds to epitopes of an antigen; binds to NK cells.
  4. NK cells kill the cells they bind to by:
    1. Triggering apoptosis.
    2. Dumping the contents of their lysosomes on the cell.
    3. Producing cytolytic exotoxins that lyse the cell.
    4. Inducing extracellular killing by eosinophils.

A novel method for evaluating antibody-dependent cell-mediated cytotoxicity by flowcytometry using cryopreserved human peripheral blood mononuclear cells

Analyzing the cytotoxic functions of effector cells, such as NK cells against target cancer cells, is thought to be necessary for predicting the clinical efficacy of antibody-dependent cellular cytotoxicity (ADCC) -dependent antibody therapy. The 51 Cr release assay has long been the most widely used method for quantification of ADCC activity. However, the reproducibilities of these release assays are not adequate and they do not allow evaluation of the lysis susceptibilities of distinct cell types within the target cell population. In this study, we established a novel method for evaluating cytotoxicity, which involves the detection and quantification of dead target cells using flowcytometry. CFSE (carboxyfluorescein succinimidyl ester) was used as a dye to specifically stain and thereby label the target cell population, allowing living and dead cells, as well as both target and effector cells, to be quantitatively distinguished. Furthermore, with our new approach, ADCC activity was more reproducibly, sensitively and specifically detectable, not only in freshly isolated but also in frozen human peripheral blood mononuclear cells (PBMCs), than with the calcein-AM release assay. This assay, validated herein, is expected to become a standard assay for evaluating ADCC activity which will ultimately contribute the clinical development of ADCC dependent-antibody therapies.


Role of the NK cell-mediated ADCC in the antitumor immune response

Robust and efficient antitumor immune responses can lead to complete tumor regression or long-term control of tumor growth. 1 These successful immune responses require the coordinated activity of a variety of innate and adaptive immune cells. Type 1 cytotoxic responses are considered the most effective to fight tumors. This sort of immune response usually starts with the release of interleukin (IL)-12 by dendritic cells and/or other myeloid cells. CD4 + T lymphocytes differentiated under the effect of the transcription factor T-bet while secreted IFNγ has a key role as well. Prompted by these influences, CD8 + T lymphocytes recognizing cognate antigen differentiate into cytotoxic T cells with the ability to kill tumor cells displaying the specific antigens. Tumor progression is also controlled by macrophages with an M1 phenotype and by natural killer (NK) cells. NK cell and M1 macrophage antitumor cytotoxicity can be redirected by IgG1 antibodies. Antibodies may recognize with high-affinity cell surface tumor antigens and recruit effector immune functions to the tumor. The fragment crystallizable region (Fc) offers binding sites for C1q that can trigger complement-dependent cytotoxicity (CDC) as well as binding sites for a surface receptor expressed on NK cells and macrophages named CD16A (FcγRIIIA), that will elicit the antibody-dependent cellular-mediated cytotoxicity (ADCC). The density of IgG antibody coating the target cell is a critical factor in this process.

The activation of NK cells is highly regulated by a set of stimulating and inhibitory receptors expressed on the target cell membrane. In humans, the main inhibitory receptors are the killer immunoglobulin receptor family (KIRs) and CD94/NKG2A heterodimer. The differential expression of KIRs members clonally defines NK cells subpopulations that recognize diallelic polymorphisms in subgroups HLA-B and HLA-C allotypes. The lack of recognition of class I HLA by KIRs on tumor cells allows for cytotoxic activity of NK cells in fulfillment of the missing self hypothesis. 2 The CD94/NKG2A system recognizes HLA-E molecules which become expressed on the target cell surface only by the occupation of its binding cleft by peptides corresponding to residues −22 to −14 of the leader sequences of certain HLA-A, HLA-B and HLA-C moieties. Thus, this inhibitory system becomes operational to inhibit NK cells only when they express normal levels of classical HLA class I molecules. The activatory receptors of NK cells to mediate spontaneous or natural cytotoxicity include NKp46, NKp44, NKp30, NKG2D, DNAM-1 and FcγRIIIA/CD16A. The latter receptor binds to the Fc region of antibodies coating target cells and activates the spatially oriented exocytosis of cytotoxic granules releasing perforins and granzymes, and the paracrine production of IFNγ to attain ADCC.

It should not be forgotten that mouse models have revealed an important component of macrophage involvement via FcγRI and FcγRIV in ADCC in the tumor microenvironment. 3 It is clear that at this location, macrophages and other myeloid cells are much more abundant than NK cells. Moreover, macrophage-mediated ADCC has been involved in the immunomodulatory effects of anti-CTLA-4 mAb by depleting regulatory T lymphocytes. 4


Introduction

Antibodies have a bifunctional role within the immune system. This role is physically built into their structure through two parts: the fragment antigen binding (Fab), for recognizing antigen, and the fragment crystallizable (Fc), for recruiting effector immune cells. The process by which antibody-coated cells direct effector cells to attack and kill an opsonized target is known as antibody dependent cellular cytotoxicity (ADCC). This is accomplished through ligation with Fc gamma receptors (FcγRs), which forms a conduit of communication between the target cell (TC) and immune effector cell (1). The FcγRs are an assortment of transmembrane receptors expressed to varying levels on primarily innate, but also some adaptive, immune cells (2). The ability of antibodies to recruit ADCC is a highly desirable trait for therapeutic and vaccine development, and NK cells are of central focus due to their proclivity for ADCC and as a front-line defense immune cell (3𠄶). While our understanding of antigen-antibody recognition and Fc-FcγR interaction are each quite extensive in isolation, there is still a gap in knowledge about how these two important aspects of antibodies interplay, especially in vivo. Combined with frequent incongruency between available in vitro and in vivo data regarding antibody effector function as well as the generally complicated nature of the human immune system, we are left with a looming question: what makes an effective antibody for recruiting NK cell ADCC?

Answering the question above requires a much better understanding of the underlying molecular basis of antibody and cellular effector functions. A good place to start is at the point of initial contact between an NK cell and TC, known as the immune synapse (IS). This is the point where activating receptors on the NK cell surface bind to the Fc domain of antigen-engaged antibodies and initialize a cascade of events that lead to NK cell activation and ultimately target-cell death. Extensive studies of the T cell receptor have provided valuable insight into the organization of the T cell IS (7�), but much less is known about the NK cell immune synapse (NKIS).

Antibodies are necessary for clustering activating receptors in the early stages of ADCC. Structural biology has been instrumental in providing a much more detailed view of this initial interaction of antibody and antigen, especially in the context of viral antigens from HIV, influenza and ebolavirus. Depending on the location of antibody epitopes, the Fc domain of the antibody can differ vastly in how it is presented to a surveying NK cell. Many other variables, including antigen shape, size, and density as well as lipid environment and mobility, can also affect Fc presentation. Further, all these variables can change with antibody isotype, subclass and glycosylation as well as FcγR isotype, cellular subclass, FcγR expression and diversity as well as FcγR glycosylation and alleles (2).

With an increasing number of antibody therapeutics, vaccines and immunotherapies entering the clinical market (11), a greater understanding of NK cell mediated ADCC will guide precision medicine and create more effective drugs. In this review, I will focus on current efforts to understand NK cell ADCC, with a particular focus in the context of virally infected cells. I will explore how advances in microscopy techniques as well as the increasing accessibility of big data technologies such as transcriptomics, proteomics, and metabolomics are challenging our understanding of classical immunology and paving a way to fill the gap between in vitro and in vivo observations. Such advances will reveal new avenues for vetting therapeutics with the greatest chance of success in patients.


RESULTS

Circulating CD16+ Mononuclear Cells in FCGR3A-158V/F Genotyped Donors.

The number of peripheral blood CD16+ mononuclear cells, CD16+ monocytes, CD3+CD16+ T cells, and CD3-CD16+ NK cells were analyzed by flow cytometry in 54 blood donors simultaneously tested for the FCGR3A-158V/F polymorphism. Of the 54 donors, 7 and 21 were homozygous for FCGR3A-158V and FCGR3A-158F, respectively, and 26 were heterozygous (Table 1) ⇓ . The three groups were not different in terms of sex or age. When all samples were analyzed together, we found that 13.8% of PBMCs, 9.4% of monocytes, and 14.7% of lymphocytes were CD16+. The vast majority of CD16+ PBMCs were NK cells (77.1%), whereas CD16+ monocytes and CD3+CD16+ lymphocytes represented 11.9% and 9.7% of these cells, respectively. No difference was found among the three genotype groups in terms of number of circulating CD16+ mononuclear cells, CD16+ monocytes, CD3+CD16+ T cells, and CD3-CD16+ NK cells (Table 1) ⇓ . The level of CD16 expression on monocytes was slightly higher than that observed on NK cells whatever the genotype, whereas it was very low on CD3+CD16+ T cells. Finally, higher binding of anti-CD16 3G8 mAb to NK cells and monocytes was observed in FCGR3A-158V homozygous and heterozygous donors compared with homozygous FCGR3A-158F donors (Table 1) ⇓ . Given that the binding of several other anti-CD16 mAbs is similar on NK cells from VV and FF donors (2) , it is likely that the difference in binding of 3G8 reflects a greater affinity of this mAb for the V allotype.

Enumeration of circulating CD16+ mononuclear cells according to FCGR3A genotype a

Influence of FCGR3A-158V/F Polymorphism on Rituximab-Binding Properties of NK Cell FcγRIIIa.

It has been assumed that the FCGR3A-158V/F polymorphism affects FcγRIIIa affinity for IgG1 (2) . To address this question, we compared the ability of rituximab (IgG1) to inhibit the binding of FITC-conjugated 3G8 mAb (29) with NK cells from VV and FF donors. A 50% inhibition of 3G8 mAb binding to VV and FF NK cells was achieved with less than 0.15 mg/ml and more than 0.8 mg/ml rituximab, respectively (Fig. 1) ⇓ . The difference in the ability of rituximab to block 3G8 mAb binding to FcγRIIIa on VV and FF NK cells cannot result from a difference in the affinity of 3G8 mAb binding to FcγRIIIa allotypes. Indeed, the higher binding of 3G8 mAb to VV NK cells (Table 1) ⇓ should lead to less rather than more blockade of 3G8 mAb binding in the presence of rituximab. These findings thus indicate that FcγRIIIa expressed on NK cells from VV donors binds rituximab with higher affinity than FcγRIIIa expressed on NK cells from FF donors.

Binding of rituximab to NK cells from FCGR3A-158 VV and FF donors. Purified NK cells from homozygous VV and FF blood donors were incubated with varying concentrations of rituximab for 30 min at 4°C followed by FITC-conjugated anti-CD16 3G8 mAb and then analyzed by flow cytometry. Percentages of inhibition of 3G8 binding were calculated as described in “Materials and Methods,” and the results are expressed as the mean ± SD (n = 3 VV and 3 FF).

Binding of Rituximab to Daudi Cells.

To study the in vitro susceptibility of B cells to ADCC in the presence of rituximab, Daudi cells (HLA class I negative and CD20 positive) were used as target cells. To study the binding of rituximab, Daudi cells were incubated with rituximab concentrations ranging from 0.0002 to 2 μg/ml followed by FITC-conjugated antihuman IgG F(ab′)2 that did not recognize the membrane IgM on Daudi cells (Fig. 2) ⇓ . Weak binding of rituximab was detected with 0.0006 μg/ml rituximab. Binding then increased dramatically with increasing concentrations reaching a maximum at 0.2 μg/ml.

Binding of rituximab to Daudi cells. Daudi cells were incubated with varying concentrations of rituximab followed by FITC-conjugated goat antihuman IgG F(ab′)2 and then analyzed by flow cytometry. Fluorescence intensity is displayed on the X axis (in log scale) and cell number on the Y axis (results are from one representative experiment among three).

Cytolytic Potential of NK Cells from VV and FF Donors in Response to Optimal FcγRIIIa Stimulation.

As expected, Daudi cells were resistant to lysis in the absence of rituximab but were killed efficiently by PBMCs and peripheral blood lymphocytes in the presence of 0.2 μg/ml rituximab (not shown). In addition, lysis did not increase in the presence of 20 μg/ml rituximab, and Daudi cells were not killed by purified monocytes or by purified T cells, but they were killed very efficiently by purified NK cells in the presence of rituximab (not shown). We next addressed the question of the influence of FCGR3A polymorphism on the cytotoxic response of NK cells to optimal FcγRIIIa engagement. Daudi cells express CD32/FcγRII, which binds mouse IgG1 (30) . They may therefore be used as targets in a redirected killing assay in the presence of anti-CD16 3G8 mAb. Daudi cells were resistant to lysis in the presence of anti-CD56 T199 mAb (data not shown), whereas they were killed very efficiently in the presence of 3G8 mAb (Fig. 3) ⇓ . Although 3G8 mAb bound slightly more efficiently to NK cells from VV donors (Table 1) ⇓ , the lysis observed with VV and FF NK cells in the presence of 3G8 mAb was similar and was also equivalent to that observed in the presence of saturating amounts of rituximab (Fig. 3) ⇓ .

Lysis of Daudi cells by NK cells from VV and FF donors after optimal FcγRIIIa stimulation. 51 Cr-labeled Daudi cells were incubated for 4 h at 37°C with NK cells from homozygous VV (solid lines) and FF (dotted lines) donors in the presence of 0.2 μg/ml rituximab (⋄) or 0.2 μg/ml 3G8 mAb (▪). Cytotoxicity against Daudi cells is expressed as the mean ± SD percentage of specific lysis (n = 6 VV and 6 FF)

Influence of FCGR3A-158V/F Polymorphism on the Concentration-Effect Relationship of Rituximab-Dependent NK CellMediated Cytotoxicity.

The influence of the FCGR3A-158V/F genotype on the lysis of Daudi cells was then analyzed in the presence of decreasing concentrations of rituximab (0.2 to 0.0002 μg/ml), with NK cells from six VV and six FF donors (E:T ratio = 2.5:1). As expected, for each donor, the observed lysis of Daudi cells increased with increasing concentrations of rituximab and reached a plateau at high concentrations (Fig. 4) ⇓ . The relationship between Daudi cell lysis and rituximab concentrations was analyzed for each individual using an Emax model. Basal lysis and maximal lysis induced by rituximab with NK cells from VV and FF donors were not different: E0 values obtained with VV and FF NK cells were 11.3 ± 9.9% and 8.3 ± 3.5% specific lysis, respectively, P = 1 whereas Emax values were 47.4 ± 6.2% and 41.9 ± 9.5% specific lysis, respectively, P = 0.3939. By contrast, the rituximab concentration resulting in 50% lysis of target cells obtained with NK cells from VV donors was on average 4.2 times lower than that obtained with NK cells from FF donors: EC50s obtained with VV and FF NK cells were 0.00096 ± 0.00058 and 0.00402 ± 0.00236 μg/ml, respectively, P = 0.0043.

Relationship between rituximab concentration and Daudi cell lysis by NK cells. 51 Cr-labeled Daudi cells were incubated for 4 h at 37°C with NK cells from homozygous VV and FF donors (E:T ratio = 2.5:1) in the presence of varying concentrations of rituximab. Cytotoxicity against Daudi cells is expressed as the mean percentage of (lysis in presence of rituximab − basal lysis in absence of rituximab) of each triplicate (□, VV ▪, FF) and as predicted by the Emax model (see “Materials and Methods” solid lines, VV, n = 6 dotted lines, FF, n = 6). Observed and model-predicted basal lysis (E0), which was not different between VV and FF, was subtracted to display rituximab-dependent lysis.


Identification of Dominant Antibody-Dependent Cell-Mediated Cytotoxicity Epitopes on the Hemagglutinin Antigen of Pandemic H1N1 Influenza Virus

Fig 1 HAI and NI titers of convalescent-phase plasma samples from seven H1N1-infected human subjects. Each plasma sample was tested for anti-HA antibodies by HAI assay. The NI titer against pandemic H1N1 A/HK/01/2009 virus was determined. Fig 2 Binding of purified IgG antibodies to H1N1-infected Raji cells by flow cytometry. All IgG samples were tested at 10 μg/ml. A nonspecific IgG sample was a secondary-antibody-only control (without a primary antibody added). Fig 3 Percent ADCC of seven convalescent-phase plasma samples and their purified IgG antibodies. (A) Plasma samples were diluted 1:10,000 and 1:2,000 (B) purified IgG antibodies were tested at 0.5 μg/ml and 2.5 μg/ml. Each sample was tested in duplicate, and the average standard variation was 5%, as displayed by the error bars.

Construction of recombinant yeast library and sorting of induced-yeast library against six IgG samples.

Epitope mapping of purified IgG antibodies and identification of dominant ADCC epitopes on H1N1 HA.

Fig 4 Epitope mapping of six purified IgG samples. (A) The amino acid frequencies for all six IgG samples were mapped onto the HA ectodomain (B) six IgG samples were categorized into the ADCC ++ (samples M1036 and M1037), ADCC + (samples M1024, M1027, and M1039) and ADCC − (sample M1089) groups, and the amino acid frequencies in each group were averaged and mapped to the H1N1 HA ectodomain. The y axes represent the frequency of each amino acid in positive clones. The x axes represent the amino acid position on H1N1 HA.
HA epitopeAmino acid positionsAmino acid sequence Avg frequency (%) for the following groups (samples):
ADCC ++ (M1036 and M1037)ADCC + (M1024, M1017, and M1039)ADCC − (M1089)
E192–117 SWSYIVETSSSDNGTCYPGDFIDYEE 51.86 ± 3.1638.21 ± 0.7224.11 ± 2.55
E2124–159 SVSSFERFEIFPKISSWPNHESNKGVTAACPHAGAK 20.66 ± 2.2332.14 ± 2.9310.25 ± 1.19
E3470–521 LKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVK 15.57 ± 1.456.54 ± 1.1740.17 ± 4.09

Confirmation of dominant ADCC epitopes on HA.

Fig 5 Percent ADCC of IgG from samples M1036 and M1037 after depletion with monoclonal yeast expressing E1 or E2, or both. Each depleted IgG sample was tested at a final concentration of 2.5 μg/ml. Undepleted IgG from samples M1036 and M1037 and IgG from samples M1036 and M1037 depleted with recombinant yeast expressing E3 were included as controls.

Measure Antibody-Dependent Cell-mediated Cytotoxicity (ADCC)

ADCC is a simple but important mechanism for the immune system to target diseased or infected cells. Antibodies bind to specific antigens on the surface of the target cell (see Fig 1). PBMCs or natural killer (NK) cells, express Fc receptors on their cell surface and act as the effector cells. Interaction between the Fc region of the antibody and the Fc receptor induces the effector cell to degranulate, releasing IFN-γ, granzymes, and other cytotoxic compounds that lyse the target cell.

ADCC is not only a natural part of the adaptive immune response, but animal experiments have shown that it can also be seen as an important mechanism of action of therapeutic monoclonal antibodies (1), including the breast cancer drug trastuzumab, and rituximab, a drug used to treat diseases which show overactive, dysfunctional, or excessive numbers of B cells (e.g. lymphomas).

Cell lines to build up cellular ADCC screening assays

Fig 2: Principle of BPS’s ADCC cell lines

To enable researchers to build up a cellular ADCC screening system, BPS Biosciences have developed 2 reporter cell lines, which can replace NK cells or PBMCs in such a cellular assay (see Fig 2). The system is based on Jurkat cells that stably express human FcγRIIIa (CD16a), the receptor for the Fc region of human IgG. The FcγRIIIa on the Jurkat cells binds to the IgG on the surface of the target cell. This crosslinking causes the Jurkat cells to activate NFAT transcription, which induces the expression of luciferase and can be easily detected using the ONE-Step™ Luciferase Detection Reagents.

The effectiveness of ADCC depends on how well the effector cells are activated after the engagement of FcγRIIIa. Human FcγRIIIa displays dimorphism at amino acid 158 – one allele (V158) encodes a high Fc affinity receptor variant, while the other (F158) encodes a lower Fc affinity receptor variant. BPS offers 2 different ADCC cell lines expressing either of these Fc receptors to allow selective antibody binding analyses using each type of receptor.

Get more information about our ADCC cell lines – just leave your questions or comments in the form below!

(1) Clynes, RA, Towers, TL, Presta, LG, Ravetch, JV Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets Nat Med. 6 (4): 443-446 (2000)


13.2C: Antibody-dependent Cellular Cytotoxicity (ADCC) by Natural Killer Cells - Biology

a Department of Pharmaceutical Sciences, Northeastern University, 360 Huntington Avenue, Boston, MA, USA
E-mail: [email protected]

b Department of Electrical and Computer Engineering, College of Engineering, and College of Computer and Information Science, Northeastern University, 360 Huntington Avenue, Boston, MA, USA

c Barnett Institute of Chemical and Biological Analysis, Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Avenue, Boston, MA, USA

d Department of Electrical and Computer Engineering, College of Engineering, and Khoury College of Computer Science, Northeastern University, 360 Huntington Avenue, Boston, MA, USA

Abstract

Natural killer (NK) cells have emerged as an effective alternative option to T cell-based immunotherapies, particularly against liquid (hematologic) tumors. However, the effectiveness of NK cell therapy has been less than optimal for solid tumors, partly due to the heterogeneity in target interaction leading to variable anti-tumor cytotoxicity. This paper describes a microfluidic droplet-based cytotoxicity assay for quantitative comparison of immunotherapeutic NK-92 cell interaction with various types of target cells. Machine learning algorithms were developed to assess the dynamics of individual effector-target cell pair conjugation and target death in droplets in a semi-automated manner. Our results showed that while short contacts were sufficient to induce potent killing of hematological cancer cells, long-lasting stable conjugation with NK-92 cells was unable to kill HER2 + solid tumor cells (SKOV3, SKBR3) significantly. NK-92 cells that were engineered to express FcγRIII (CD16) mediated antibody-dependent cellular cytotoxicity (ADCC) selectively against HER2 + cells upon addition of Herceptin (trastuzumab). The requirement of CD16, Herceptin and specific pre-incubation temperature served as three inputs to generate a molecular logic function with HER2 + cell death as the output. Mass proteomic analysis of the two effector cell lines suggested differential changes in adhesion, exocytosis, metabolism, transport and activation of upstream regulators and cytotoxicity mediators, which can be utilized to regulate specific functionalities of NK-92 cells in future. These results suggest that this semi-automated single cell assay can reveal the variability and functional potency of NK cells and may be used to optimize immunotherapeutic efficacy for preclinical analyses.


Establishment and functional characterization of novel natural killer cell lines derived from a temperature-sensitive SV40 large T antigen transgenic mouse

Natural killer (NK) cells belong to an important lymphocyte population that eliminates transformed cells and invading pathogens without any prior sensitization. NK cells possess not only natural killing activity against non-self and altered-self cells but also exhibit cytokine production and antibody-dependent cell-mediated cytotoxicity (ADCC). Despite their important roles in the innate immune system, little is known about the details of NK cell biology. In spite of that several murine NK cell clones have been established, studies have mainly focused on their natural killing activity but not their cytokine production or ADCC. In this study, we established and characterized eight novel, immortalized murine NK cell clones derived from a temperature-sensitive SV40 large-T antigen transgenic mouse. These NK cell lines continuously proliferated for more than 30 months in a culture medium supplemented with interleukin 2. All cell lines contained azurophilic granules in the cytoplasm, and a few clones retained the NK cell functions, such as natural killing activity, cytokine production, and ADCC. In addition, one clone could serve as a host for transient as well as stable gene transfection. Taken together, these findings indicate that the cell lines could constitute useful tools for detailed analysis of murine NK cell biology.


Natural killer cells inhibit Plasmodium falciparum growth in red blood cells via antibody-dependent cellular cytotoxicity

Antibodies acquired naturally through repeated exposure to Plasmodium falciparum are essential in the control of blood-stage malaria. Antibody-dependent functions may include neutralization of parasite–host interactions, complement activation, and activation of Fc receptor functions. A role of antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells in protection from malaria has not been established. Here we show that IgG isolated from adults living in a malaria-endemic region activated ADCC by primary human NK cells, which lysed infected red blood cells (RBCs) and inhibited parasite growth in an in vitro assay for ADCC-dependent growth inhibition. RBC lysis by NK cells was highly selective for infected RBCs in a mixed culture with uninfected RBCs. Human antibodies to P. falciparum antigens PfEMP1 and RIFIN were sufficient to promote NK-dependent growth inhibition. As these results implicate acquired immunity through NK-mediated ADCC, antibody-based vaccines that target bloodstream parasites should consider this new mechanism of action.



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