PBMC Cytotoxicity Assay

Human peripheral blood mononuclear cells (PBMC) represent a heterogeneous menagerie of immune cells isolated from peripheral blood. While they primarily consist of lymphocytes (B-cells, T-cells and NK cells), a smaller fraction of monocytic and dendritic cells are also present. Due to the overwhelming abundance of T-cells within the PBMC population (70-85%), these cells provide a useful and appropriate model with which to study the immunological mechanisms associated with drug-mediated hypersensitivity in vitro.

The PBMC cytotoxicity assay is an in vitro based method which utilizes PBMC isolated from consenting healthy donors using Ficoll and density gradient centrifugation. The cytotoxic potential of candidate drugs towards PBMC is assessed by measuring the levels of cellular ATP depletion and LDH release. Our recently established biobank of PBMC isolated from multiple healthy donors provides the opportunity to investigate donor-dependent PBMC cytotoxicity upon drug exposure. Furthermore, follow-up studies involving other endpoints, such as drug-induced cytokine release, can be performed using the same cryopreserved donor population.

Cyprotex deliver consistent, high quality data with the flexibility to adapt protocols based on specific customer requirements.

Background Information

Drugs designed for their therapeutic benefits can unfortunately also, on occasion, be associated with unwanted serious hypersensitivity reactions, which can sometimes be fatal in a small proportion of susceptible individuals¹.
Drug hypersensitivity is an unintended adverse drug reaction with an immunological aetiology to an otherwise safe and effective therapeutic agent.
In addition to inter-individual differences in detoxification pathways, the immunoregulatory system that preserves tolerance to neoantigens varies between individuals and can be influenced by genetic and environmental risk factors as well as disease.
Drug-specific T-cells are implicated in drug-induced end organ damage and have been isolated from cutaneous blister fluid and liver biopsies^{2, 3}.
The full immunological consequences of human drug exposure is extremely challenging to predict during early pre-clinical drug discovery and clinical trials.
This is partly because a complex interaction of specific individual characteristics (genetic and non-genetic), disease/tissue micro environment and environmental factors determine whether drug exposure will result in therapeutic benefit, adverse reactions or both⁴.
Furthermore, stress-signalling following drug exposure modulates the cellular and extracellular microenvironment crucial for adaptive immune activation.
The PBMC cytotoxicity assay, which utilises cells isolated from multiple individuals, provides a high throughput assessment of the cytotoxicity of candidate compounds in vitro. It can also provide an initial insight into how immune cells from different donors respond to candidate drugs in development.

In vitro high throughput assays utilising primary human immune cells will significantly enhance our capabilities to predict candidate drugs with potentials to cause rare but occasionally fatal hypersensitivity reactions during early stages of preclinical drug development.

Protocol

PBMC cytotoxicity protocol

Cell Line	PBMC
Donors	>6 donors available for multi-donor studies
Analysis Platform	Cellular ATP – Cytation 3 Cell Imaging Multi-Mode reader LDH release – SpectraMax ABS absorbance microplate reader
Test Compound Concentrations	8 point dose response curve with top concentration based on 100x C_max or solubility limit 3 replicates per concentration*
Compound Requirements	Maximum (dependent upon number of repeat doses) 150 µL of a DMSO* solution to achieve 200x top concentration maintained at 0.5% DMSO or equivalent amount in solid compound
Time Points	24-72 hour pre-incubation*
Quality Control	Negative control: 0.5% DMSO (vehicle)* Positive controls: 2 appropriate compounds
Data Delivery	Minimum effective concentration (MEC) and AC₅₀ value for cellular ATP content and LDH release

* other options available on request.

Data

Data from Cyprotex's PBMC Cytotoxicity Assay

Figure 1
Representative cellular ATP dose response graphs for (a) chlorpromazine, (b) minocycline, (c) abacavir and (d) isoniazid. Treatment of PBMC with chlorpromazine (AC₅₀ =17.1 µM; MEC = 8.6 µM) and minocycline (AC₅₀ = 336 µM; MEC = 196 µM) for 24 hours resulted in a decrease in cellular ATP. Abacavir and isoniazid showed no cytotoxic effect at 100 µM and 2000 µM top concentrations respectively. Error bars represent ± SD of the measurement while red dotted lines represent the range of control values.

Figure 2
Representative LDH release dose response graphs for (a) chlorpromazine, (b) minocycline, (c) abacavir and (d) isoniazid. Treatment of PBMC with chlorpromazine (AC₅₀ >100 µM; MEC = 10.7 µM) and minocycline (AC₅₀ = 934 µM; MEC = 103 µM) for 24 hours resulted in an increase in LDH release. Abacavir and isoniazid showed no cytotoxic effect at 100 µM and 2000 µM top concentrations respectively. Error bars represent ± SD of the measurement while red dotted lines represent the range of control values.

Figure 3
Dot plots demonstrating the AC₅₀ and MEC values collected for chlorpromazine and minocycline across six individual PBMC donors. Figures A & B and C & D show data obtained using cellular ATP and LDH assay respectively. Solid black lines represent the average of the measurement while error bars represent ± SD.

Table 1
MEC values derived from cellular ATP data for 27 reference compounds. Majority of these reference compounds are known to cause immune-mediated adverse drug reactions targeting the liver, skin or neutrophils (n = 6 donor PBMC). Chloramphenicol, dexamethasone, isoproterenol, pindolol, riboflavin and streptomycin have not been previously implicated in immune-mediated adverse drug reactions. NR represents no response at the top concentration tested. NA indicate no C_max data. Cells highlighted red indicate positive cellular ATP depletion at 25x C_max. Inter-individual variability was observed with regards to the cellular ATP responses for abacavir, acetaminophen, carbamazepine, chloramphenicol, clavulanic acid, erythromycin, naproxen, terbinafine and ticlopidine.

Table 2
MEC values derived from LDH release data for 27 reference compounds. Majority of these reference compounds are known to cause immune-mediated adverse drug reactions targeting the liver, skin or neutrophils (n = 6 donor PBMC). Chloramphenicol, dexamethasone, isoproterenol, pindolol, riboflavin and streptomycin have not been previously implicated in immune-mediated adverse drug reactions. NR represents no response at the top concentration tested. NA indicate no C_max data. Cells highlighted red indicate positive LDH release response at 25x C_max. Inter-individual variability was observed with regards to the LDH release for carbamazepine, chlorpromazine, clavulanic acid, erythromycin, isoproterenol and minocycline.

References

¹ Naisbitt DJ et al., (2020). Immune dysregulation increases the incidence of delayed-type drug hypersensitivity reactions. Allergy 75(4); 781-797
² Sullivan A et al., (2018). β-Lactam hypersensitivity involves expansion of circulating and skin-resident T_H22 cells. J Allergy Clin Immunol 141(1); 235-249
³ Mennicke M et al., (2009). Fulminant liver failure after vancomycin in a sulfasalazine-induced DRESS syndrome: fatal recurrence after liver transplantation. Am J Transplant 9(9); 2197-2202
⁴ Gibson A et al., (2018). Genetic and nongenetic factors that may predispose individuals to allergic drug reactions. Curr Opin Allergy Clin Immunol 18(4); 325-332