PBMC Proliferation Assay

T-lymphocyte mediated drug hypersensitivity reactions mainly target the skin and liver^1,2. These reactions are dependent on antigen presentation to drug-specific T-lymphocytes which result in their activation, expansion, differentiation and the targeting of tissues and organs by activated T-lymphocytes.

The peripheral blood mononuclear cells (PBMC) proliferation assay is an in vitro method which utilizes PBMC isolated from consenting healthy donors using Ficoll and density gradient centrifugation. This assay is also suitable for evaluating the priming of naïve T-lymphocytes within the PBMC population and for the assessment of immunogenicity and cross reactivity of small chemical molecules, biologics and peptides. In addition, inter-individual variability in immune responses can be explored by using multiple HLA-typed donor PBMC. Furthermore, follow-up studies involving other endpoints, such as drug-induced cytokine release profiles can be performed using the same cryopreserved donor PBMC.

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

Background Information

Drug hypersensitivity reactions are rare off-target, immune-mediated and mostly delayed-type reactions involving drug-specific T-lymphocytes.
Common classes of drugs implicated in hypersensitivity include antibiotics, anticonvulsants, antivirals, non-steroidal anti-inflammatory drugs and biologics.
These reactions are difficult to predict during preclinical drug discovery due to the involvement of specific individual characteristics (genetic and non-genetic) that are present in only susceptible individuals³.
Current animal models of immunogenicity fail to recapitulate the complexity of the human immune system.
In vitro drug-induced T-lymphocyte proliferation using PBMC isolated from drug hypersensitive individuals have provided supporting evidence for the diagnosis of clinical cases of drug hypersensitivity reactions^4,5.
Retrospective genome-wide association studies (GWAS) have identified multiple human leukocyte alleles (HLA) linked to various forms of drug hypersensitivity reactions.
Unfortunately, these HLA-associated hypersensitivity reactions are currently difficult to prospectively identify using existing immune based in vitro assays during early drug development.
Hence, this highlights the importance of using relevant human immune cells to screen potentially immunogenic drugs during preclinical drug discovery.
Our high throughput PBMC proliferation assay evaluates the immunogenic potential of candidate drugs using multiple HLA-typed PBMC donors cryopreserved in our immune cell biobank.
This assay is non-radioactive and assesses the immunogenic potential of candidate drugs (biologics and small chemicals molecules) based on antigen-induced PBMC proliferation and corresponding increase in cellular ATP.

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 proliferation protocol

Cell Type	PBMC
Donors	>6 donors available for multi-donor studies
Analysis Platform	PBMC proliferation – fluorescent detection using Alamar blue cell viability reagent Cellular ATP – CellTiter-Glo^® (Promega) with a Cytation 3 Cell Imaging Multi-Mode Reader (BioTek)
Test Article Concentrations	8 point dose response curve with top concentration based on 100x C_max or solubility limit 3 replicates per concentration*
Test Article 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 PBMC proliferation and cellular ATP content

* other options available on request.

Data

Data from Cyprotex's PBMC Proliferation Assay

Figure 1
Representative PBMC proliferation (blue line) and cellular ATP (green line) dose response graphs for (a) phytohemagglutinin (PHA), (b) purified anti-CD3 antibody, (c) abacavir and (d) terbinafine. Treatments with PHA (AC₅₀= 2.86 µg/mL; MEC = 1.97 µg/mL) and purified anti-CD3 (AC₅₀ = 0.012 µg/mL; MEC = <0.006 µg/mL) for 72 hours resulted in an increase in PBMC proliferation with a corresponding increase in cellular ATP. Abacavir had no effect on either PBMC proliferation or cellular ATP levels at 100 µM while terbinafine caused a decrease in PBMC viability at >100 µM.

Figure 2
Dot plots demonstrating the MEC values collected for PHA and purified anti-CD3 antibody treatments across 5 individual PBMC donors. Figures 2a and 2b show data obtained for PBMC proliferation and cellular ATP assays respectively. Solid blue lines represent the average of the measurement while error bars represent ± standard deviation.

Table 1
MEC values derived from PBMC proliferation and cellular ATP data for 14 reference compounds (n = 5 donor PBMC). Abacavir, 4-nitrososulfamethoxazole, sulfamethoxazole, carbamazepine, ticlopidine, lapatinib, lumiracoxib and terbinafine are known to cause immune-mediated adverse drug reactions targeting the liver and skin. Pindolol, riboflavin and streptomycin have not been previously implicated in immune-mediated adverse drug reactions. NR in green cells represents no response at the top concentration tested. Values in red cells indicate where a proliferative or cellular ATP response was observed and the arrows show the direction of the observed response.

References

¹ 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
² 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.
³ 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.
⁴ Pichler WJ and Tilch J, (2004). The lymphocyte transformation test in the diagnosis of drug hypersensitivity. Allergy 59(8); 809-820
⁵ Nyfeler B and Pichler WJ, (1997). The lymphocyte transformation test for the diagnosis of drug allergy: sensitivity and specificity. Clin Exp Allergy 27(2); 175-181