 |
|
Molecular & Genomic Testing
HIV Reservoir & Virology
Immunology
Sample Processing & Biobanking
Therapeutic Modalities
A killing assay reports that capacity as specific lysis at a defined effector-to-target ratio, which is the net output of the entire effector population. We measure cell-mediated killing by T cells and NK cells, native and engineered, using flow cytometry to score target cell death by viability dye uptake within a labeled target population, with effector phenotype, intracellular function, and secreted cytokines available from the same experiment. Programs supported include CAR-T and TCR-T, bispecific antibodies and T cell engagers, therapeutic vaccines, oncolytic viruses, NK cell therapeutics, and virology research, including HIV cure and remission studies.
A cytotoxicity assay returns one number per condition. That number is the net output of a population, and it is silent about which cells produced it. Two effector products with the same specific lysis can differ in the proportion of effectors that engaged at all, in whether killing was concentrated in a small highly functional subset or distributed broadly, and in the differentiation state of the cells doing the work. None of that changes the lysis value. All of it changes what the result predicts.
Phenotyping the Cells That Were There
Function at Single-Cell Resolution
What Was Released into the Well
We score target cell death by flow cytometry using viability dyes, so the effectors are present in the same acquired sample as the targets. Adding markers to that panel extends the readout from how much killing occurred to which cells were present when it occurred, with the effectors interrogated in the state in which they killed rather than the state in which they were dispensed. This can resolve which subsets persisted through co-culture, their memory and differentiation distribution, activation markers acquired during the assay, and inhibitory receptor expression accompanying target engagement. Effector products are often heterogeneous at dispensing, and a shift in subset composition over co-culture indicates which cells survived the encounter rather than only which were present at the start.
Intracellular cytokine staining on the same cells adds the functional layer. Degranulation markers and cytotoxic effector proteins such as granzyme B and perforin report the mechanism engaged. IFN-γ, TNF-α, and IL-2 report whether killing was accompanied by the broader effector program. The distinction that usually matters most is between monofunctional and polyfunctional effectors: a product in which multiple cytokines are produced by the same cell that kills is not the same product as one in which cytotoxic and cytokine-producing cells are largely separate populations, even where lysis is equivalent.
The supernatant is already present at harvest and carries what neither flow readout captures. Intracellular staining requires blocking secretion, so it reports what a cell retained at one timepoint under artificial conditions. Multiplex bead-based profiling reports what was actually released over the full co-culture, integrated across every cell in the well, including bystander populations outside the flow panel. The two readouts can disagree in interpretable ways. Strong intracellular signal with little in the supernatant may reflect accumulation without release. Abundant supernatant cytokine with few cells scoring by ICS suggests production concentrated in a small subset, or release earlier than the ICS window sampled. A multiplex panel also surveys chemokines and regulatory cytokines from a small volume without consuming cells or detectors, which often identifies which pathways are worth resolving at single-cell level in a later run.
Accelevir measures cell-mediated killing in a CAP-accredited, CLIA-certified laboratory that supports programs from preclinical studies through clinical trials. Killing assays are analytically qualified rather than run as CAP or CLIA tests, and they are executed under the same quality framework that governs the rest of the laboratory, including SOP-governed procedures, documented sample chain of custody, and controlled data review. We generate a qualification report for each method and provide it with the study report, so that sponsors can evaluate it against the requirements of their own filings.
For mouse and non-human primate studies, we work with the vivarium of your choice to align sample collection plans with the scoped immune assay testing plan, coordinate sample transfer logistics, and report results from the same methods we use for human samples, which keeps preclinical and clinical data comparable without redeveloping the assay between stages.
Accelevir is a scientist-run laboratory, with one team from protocol design through final report, so an assay design can be adapted as a program’s biology and critical questions evolve. To scope a cell killing assay for your program, contact our scientific team.
Because target cell death is scored by flow cytometry, effector markers can be added to the same panel. One run reports how much killing occurred, which effector subsets were present when it occurred, what activation and inhibitory receptors they expressed, and which cytotoxic effector proteins and cytokines they contained.
The co-culture supernatant is profiled by multiplex bead-based assay, so the inflammatory signature accompanying killing is measured on the same material rather than inferred from a parallel experiment. For CAR-T and T cell engager programs, this is the readout most often used to characterize cytokine release.
Digital PCR can be run on the same samples, which supports construct persistence measurement for engineered products and reservoir quantification for HIV cure and remission studies.
Cytolytic function is concentrated in two lineages that reach the same endpoint by different routes. CD8+ T cells recognize peptide presented on MHC class I, form a synapse with the target cell, and release perforin and granzymes that trigger apoptosis, with Fas ligand contributing in some settings. Recognition is antigen-specific and MHC-restricted, so the target must be matched to the effector for killing to occur at all. Unlike CD8+ T cells, NK cells do not rely on antigen-specific recognition to identify targets. Instead, their activity is guided by the balance of activating and inhibitory signals, enabling them to recognize and eliminate cells with reduced MHC class I expression, a process known as missing-self recognition. NK cells also contribute to antibody-dependent cellular cytotoxicity (ADCC), using CD16 to recognize and kill antibody-coated target cells. CD4+ T cells can acquire cytolytic function under some conditions and can be included in the panel, although they are not the primary effector population in most programs.
Engineering changes recognition without changing the killing machinery. A chimeric antigen receptor replaces MHC-restricted TCR recognition with direct binding to a surface antigen, and a bispecific engager bridges an endogenous T cell to a target cell. In both cases killing becomes independent of HLA matching while the perforin and granzyme mechanism remains the same. The consequence is felt in assay design rather than in biology, since it determines which target cells are appropriate and what the effector-to-target ratio refers to.
Because target cell death is scored by flow cytometry rather than by bulk release, effectors and targets are acquired in the same sample, and a single run can report which effector subsets were present, what activation and inhibitory receptors they expressed, and which cytotoxic effector proteins they contained. For engineered products, construct-positive cells can be identified in the same panel where a suitable detection reagent is available, so killing can be attributed to the transduced fraction rather than to total cells.
Target cells are labeled with a cell tracker dye so they can be distinguished from effectors in the acquired sample. Effectors are added across several ratios spanning a range, since killing is dose-dependent and a single ratio can fall at either end of the response. Co-cultures are incubated for a defined period, and target cell death is scored by viability dye uptake within the tracker-positive population. Specific lysis is calculated by subtracting death observed in no-effector wells from death in the co-culture, so the reported value reflects effector-mediated killing rather than background. Controls include no-effector wells for spontaneous target death, effector-only wells to account for effector autofluorescence and dye spillover into the target gate, and a positive killing control. Acceptance criteria are set for target viability at the start of co-culture and for effector recovery at the end. Results are reported as specific lysis as a function of effector-to-target ratio.
The protocol skeleton is shared across effector types, but target selection is not. T cell killing is MHC-restricted and antigen-specific, so targets must present the relevant antigen on a matched HLA background, typically autologous or HLA-matched cells presenting peptide or expressing the antigen of interest. NK cell killing does not require antigen presentation and is governed by the balance of activating and inhibitory ligands on the target, so NK assays are commonly run against a susceptible reference line, with HLA-null targets used to assess missing-self recognition. Redirected formats, including CAR-T and bispecific engagers, decouple killing from MHC restriction, and target selection follows antigen expression rather than HLA matching. Within those constraints, target cells can be tumor cell lines, patient-derived tumor cells, or antigen-presenting cells pulsed with peptide, including autologous or HLA-matched B cell lines, which are a standard target for antigen-specific CTL assays in virology. Where mechanism needs to be confirmed rather than assumed, co-cultures can be run with and without blocking antibodies or small-molecule inhibitors, so that killing attributable to a particular receptor or pathway can be separated from the total.
Whether the effectors are native or engineered changes how the assay is set up and reported. Native antigen-specific T cells are typically rare in an unmanipulated sample, so an expansion or enrichment step usually precedes the assay, and killing is attributed to the population as a whole. Engineered products carry a defined construct, so the construct-positive fraction is known and the effector-to-target ratio can be normalized to construct-positive cells rather than total cells. The two conventions are not interchangeable, and the basis for the ratio should be reported with the result. Engineered formats also allow a control that native formats do not: an untransduced or mock-transduced effector population from the same donor, which separates construct-dependent killing from background cytotoxicity. Native effector assays rely instead on an irrelevant-antigen or unpulsed target condition for the same purpose. These distinctions are settled at study design.
For immunostimulatory cytokines, activation, proliferation, and cytokine response describe how the therapy changes T cell and NK cell function. For oncolytic virus programs, pairing killing with viral load and immune profiling may help distinguish target cell death that is virus-driven from death that is immune-mediated.
In virology, the question is often whether effector cells can eliminate infected cells rather than tumor targets, which changes the target rather than the method. Antigen-specific CTL assays use autologous or HLA-matched targets presenting viral peptide or expressing viral antigen, and B cell lines are a standard choice. For HIV cure and remission research, killing readouts can be run alongside reservoir quantification by digital PCR on matched participant samples, so that effector function and the size of the persistent reservoir are measured on the same material.
For combination trials that pair a checkpoint inhibitor with a cell therapy or a bispecific, running the killing assay with paired phenotyping and cytokine profiling characterizes whether the combination changed effector function, shifted the composition of the responding population, or produced a different inflammatory signature.
