1Massachusetts General Hospital, Ragon Institute of MGH, MIT, and Harvard, 2Thayer School of Engineering, Dartmouth College
McAndrew, E. G., Dugast, A., Licht, A. F., Eusebio, J. R., Alter, G., Ackerman, M. E. Determining the Phagocytic Activity of Clinical Antibody Samples. J. Vis. Exp. (57), e3588, doi:10.3791/3588 (2011).
Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 μm fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple FcγRs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of FcγRs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.
1. Culture Phagocytic Cells
2. Prepare Biotinylated Antigen
3. Prepare Antigen Saturated Beads
NOTE: Saturation of beads must be determined experimentally. This can be accomplished by identifying bead-coating conditions that yield maximal phagocytosis when beads are subsequently opsonized with a control monoclonal antibody.
4. Prepare Antibody Samples
NOTE: Proper precautions regarding handling human samples must always been taken.
5. Plating the Experiment
NOTE: Signal to noise may be improved by determining the optimal bead:antibody:THP-1 cell ratios for a given antigen and antibody source.
6. Flow Cytometric Analysis
7. Representative Results
There should be clear differentiation of antibody samples from affected and unaffected subjects. Figure 1A presents the FACS histograms of an antibody sample from an HIV negative (black trace) and an HIV positive (gray trace) subject, and demonstrates the increased phagocytosis driven by the presence of antigen-specific antibodies.
Optimal sensitivity of the assay is dependent on saturation of the beads with biotinylated antigen. Figure 1B presents the phagocytosis observed when beads coated with differing amounts of antigen were opsonized with 3 control monoclonal antibodies (including a non-binding antibody, triangles), and establishes 2μg antigen/μl beads as a saturating concentration for this antigen.
A dose-response curve is presented in Figure 2, and demonstrates the differential capacity of subject antibody samples to induce phagocytosis over an antibody concentration range of 0.05-5 μg/ml. This differential phagocytosis may be driven by either differences in titer or Fc domain properties such as IgG subclass and glycosylation state.
When THP-1 cells are imaged by fluorescent microscopy, there is clear evidence of bead phagocytosis. Figure 3 presents two 63x still images of THP-1 cells after incubation with antibody opsonized (green) and non-opsonized (red) beads, demonstrating the lack of phagocytic uptake in the absence of antibody. When time lapse microscopy is performed, the antibody-specific phagocytic uptake of fluorescent beads is even more striking (Movie 1, 20x magnification).
Previous work has confirmed internalization of the beads associated with the cells, and experiments with primary monocytes have agreed well with phagocytic scores (iMFI values) in this high throughput assay (data not shown).
Figure 1. Assay Quality Control. 1A, Flow cytometry histograms of phagocytosis for an antibody sample from an HIV negative subject (black trace) and an HIV positive subject (gray trace). 1B: Experimental determination of the optimal bead coating conditions for a sample antigen. Antigen-specific monoclonal antibodies (circle, square) demonstrate maximal phagocytosis of beads coated with >2 μg antigen/μl of beads, while a control antibody (triangle) demonstrates no phagocytic activity.
Figure 2. Phagocytosis Dose-Response Curve. Clinical antibody samples from HIV positive (treated, untreated, and exhibiting control of viral replication in the absence of anti-retroviral therapy) and HIV negative subjects drive phagocytosis of gp120 (HIV envelope) coated beads differentially.
Figure 3. Efficient Internalization. Microscopy confirms the internalization of antibody-opsonized beads (green), while non-opsonized beads remain in solution (63x magnification).
Movie 1. Time-lapse microscopy of antibody-driven phagocytosis was performed over the course of 14 hours and allows visualization of the phagocytic activity of the THP-1 cells utilized in the high-throughput assay. Green fluorescent beads are antibody opsonized, red fluorescent beads provide a negative control. Click here to watch the movie.
The assay described here allows high-throughput analysis of the phagocytic activity of clinical antibody samples by utilizing a monocytic cell line long utilized to study phagocytic processes10 and plate based automated flow cytometry. This assay is advantageous over others in its ability to precisely characterize antigen-specific antibody subsets, allowing not only for study of differences in phagocytosis driven by disease-specific antibodies from different subjects, but also study of multiple antigen specificities within the same subject. Because antibody recruitment of innate effector cells, including monocytes and other antigen presenting cells strongly impacts disease outcome11-13, and is biologically variable depending antibody geometry, IgG subclass, and glycosylation at Asparagine297 of the Fc domain14-16, this method provides for evaluation of a critical antibody mechanism of action. Furthermore, because antibody-mediated phagocytosis is exploited by some pathogens during the course of infection7,17,18, this assay holds promise in evaluation of the process of antibody-dependent enhancement, and highlights the dual nature of phagocytosis as a potentially protective, as well as potentially detrimental antibody activity.
The assay described can be utilized to analyze antibodies from clinical samples of patient populations, but also of vaccinees, and may be adapted to utilize serum rather than purified IgG. Additionally, cytokine secretion in response to phagocytosis can be analysed from the culture supernatant, and the influence of specific FcR can be determined by using FcR-blocking antibodies, allowing not only differences in phagocytic potency to be determined, but downstream signaling events, with insight into mechanism, and may help to resolve and refine understanding of this immune mechanism.
No conflicts of interest declared.
The authors wish to acknowledge funding from NIH 3R01AI080289-02S1, and a Harvard University Center for AIDS Research Scholar Fellowship under NIH/NIAID 2P30AI060354-07.
|EZ-Link Micro Sulfo-NHS-LC-Biotinylation Kit||Thermo Fisher Scientific, Inc.||
|Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-50 membrane||EMD Millipore||UFC905024||Filter unit size may vary according to antigen size|
|FluoSpheres NeutrAvidin labeled microspheres, 1.0 Ám, yellow-green fluorescent (505/515)||Invitrogen||F-8776||Manufacturers produce this product in various colors|
|Melon Gel IgG Purification Kit||Thermo Fisher Scientific, Inc.||45212||Care should be taken to verify the purity of Ab samples by SDS-page.|