xCELLigence Immunotherapy Kit – B Cell Killing Assay
The Promise and Challenge of Cancer Immunotherapy
The high specificity and potent cytotoxicity of immune system effector cells make them promising agents for extirpating B cell cancers. Though the list of efficacious cell-mediated immunotherapies is growing, realizing the full therapeutic potential of this field will require: (1) continued elucidation of the mechanisms underlying cancer cell recognition and immune cell-mediated killing, and (2) an ability to screen immunotherapy constructs/conditions using patient-derived effector and/or target cells to identify optimal treatment regimens. Fundamental to both of the above is the ability to quantitatively monitor the potency of immune cell-mediated killing of target B cells under controlled conditions in vitro. Traditional cell killing assays suffer from drawbacks that prevent them from meeting this need efficiently.
Download our B Cell Killing Assay Manual! Download PDF
Shortcomings of Traditional Assays
Immune cell-mediated killing can be studied by measuring the activation of effector cells or their secretion of cytotoxic molecules (perforin, granzymes, etc.). While these readouts are indeed useful, they don’t necessarily correlate with target cell killing efficiency – which is the ultimate measure of therapeutic efficacy. Assays focused on the response of target cells primarily monitor the release of either previously added labels (such as 51Cr or fluorescent dyes) or endogenous biomolecules (GAPDH, LDH, etc.) upon target cell lysis. Besides the potential artifacts associated with using exogenous labels, the time frame over which such labels are useful is extremely narrow (due to the spontaneous leakage of label out of target cells). Moreover, release assays as a whole suffer from low sensitivity, low efficiency/throughput, and the fact that only endpoint data (mere snapshots in a cell response continuum) is produced.
Using xCELLigence to Monitor the Efficacy of B Cell Immunotherapies
ACEA’s xCELLigence® Real-Time Cell Analysis (RTCA) instruments utilize gold microelectrodes embedded in the bottom of microtiter wells to non-invasively monitor the status of adherent cells. Parameters analyzed include cell number, cell size/shape, and cell-substrate attachment strength. Though they are not naturally adherent, B cells can be immobilized on the plate bottom by pre-coating the wells with anti-CD40 antibody. Upon exposure to diverse immunotherapies (NK cell, T cells, CART, oncolytic virus, checkpoint inhibitors, etc.) the B cell response is automatically recoded by the xCELLigence® instrument as shown in detail below. The major distinguishing features of this technology include enhanced sensitivity, the preclusion of labels, simple workflow and, importantly, continuous kinetic measurement of target B cell health/behavior.
Step 1: The target B cells are first seeded in the wells of an electronic microtiter plate (E-Plate®) that have been pre-coated with anti-CD40 antibody. Interaction of the B cells with the gold microelectrodes impedes the flow of electric current between electrodes. This impedance value, plotted as a unitless parameter called “Cell Index”, increases as cells proliferate and then plateaus as cells approach 100% confluence.
Step 2: When added subsequently, non-adherent effector cells (i.e. immune cells) in suspension do not cause impedance changes in and of themselves (due to lack of adherence to the gold microelectrodes).
Step 3: If effector cells and/or antibodies induce the destruction of the target B cells, this cytolytic activity can be sensitively and precisely detected. The continuous acquisition of impedance data for each well of an E-Plate enables the generation of real-time killing curves for multiple conditions simultaneously.
- Label-Free: Allowing for more physiological assay conditions; labeling or secondary assays aren’t required.
- Real-Time: Quantitative monitoring of both fast (hours) and slow (days) killing kinetics.
- Sensitive: Capable of evaluating low effector cell to target cell ratios that are physiologically relevant.
- Simple Workflow: Requires only the addition of effector cells to target cells (in the presence or absence of antibodies); homogeneous assay without additional sample handling.
- Automatic Data Plotting: RTCA software enables facile data display and objective analysis, precluding the subjective data vetting that is common to imaging-based assays.
Figures 1A & B demonstrate that B cells are effectively immobilized when E-Plate wells are pre-coated with anti-CD40 antibody. As expected, this immobilization is specific for CD40+ B cells but not for CD40– NK cells (Figure 1B) or T cells (not shown here). As seen in Figure 1C, when left untreated the immobilized Daudi B cells proliferate on the well bottom – giving rise to a steady increase in the cellular impedance signal. However, upon addition of increasing quantities of T cells (TALL-104) the impedance signal decreases in a dose dependent manner that is consistent with T cell-mediated lysis of the B cells.
Figure 1. Using xCELLigence Real-Time Cell Analysis to monitor killing of B cells. (A) By coating E-Plate wells with anti-CD40 antibody, B cells can be immobilized. (B) Daudi B cells, non-adherent under normal conditions, proliferate on E-Plate well bottoms that have been pre-coated with anti-CD40. The presence or absence of anti-CD40 coating has no impact on NK cells which are CD40-. The same is true for T cells (not shown here). (C) In a time- and dose-dependent manner NK92 cells kill immobilized Raji B cells.