The Role of In Vitro Detection of Drug-Specific Mediator-Releasing Cells to Diagnose Different Phenotypes of Severe Cutaneous Adverse Reactions

Abstract

Propose: The purpose of this study was to investigate panels of enzyme-linked immunospot assays (ELISpot) to detect drug-specific mediator releasing cells for confirming culprit drugs in severe cutaneous adverse reactions (SCARs).

Methods: Frequencies of drug-induced interleukin-22 (IL-22)-, interferon-gamma (IFN-γ)-, and granzyme-B (GrB)-releasing cells were measured by incubating peripheral blood mononuclear cells (PBMCs) from SCAR patients with the culprit drugs. Potential immunoadjuvants were supplemented to enhance drug-induced mediator responses.

Results: Twenty-seven patients, including 9 acute generalized exanthematous pustulosis (AGEP), 10 drug reactions with eosinophilia and systemic symptoms, and 8 Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) were recruited. The average frequencies of drug-induced IL-22-, IFN-γ-, and GrB-releasing cells were 35.5±16.3, 33.0±7.1, and 164.8±43.1 cells/million PBMCs, respectively. The sensitivity of combined IFN-γ/IL-22/GrB ELISpot was higher than that of IFN-γ ELISpot alone for culprit drug detection in all SCAR subjects (77.8% vs 51.9%, P < 0.01). The measurement of drug-induced IL-22- and IFN-γ releasing cells confirmed the culprit drugs in 77.8% of AGEP. The measurement of drug-induced IFN-γ- and GrB-releasing cells confirmed the culprit drugs in 62.5% of SJS/TEN. Alpha-galactosylceramide supplementation significantly increased the frequencies of drug-induced IFN-γ releasing cells.

Conclusion: The measurement of drug-induced IFN-γ-releasing cells is the key for identifying culprit drugs. The additional measurement of drug-induced IL-22-releasing cells enhances ELISpot sensitivity to identify drug-induced AGEP, while the measurement of drug-induced GrB-releasing cells could have a role in SJS/TEN. ELISpot sensitivity might be improved by supplementary alpha-galactosylceramide.

Trial registration: ClinicalTrials.gov Identifier: NCT02574988.

Keywords: Drug allergy, diagnosis, in vitro; IFN-γ; T cell; cytokine.

Conflict of interest statement

There are no financial or other issues that might lead to conflict of interest.

Copyright © 2021 The Korean Academy of Asthma, Allergy and Clinical Immunology • The Korean Academy of Pediatric Allergy and Respiratory Disease.

Figures

Fig. 1. Representative figures of drug-induced IL-22, IFN-γ, and GrB releasing cells as demonstrated by ELISpot assay after stimulating PBMCs with the suspected drugs in patients with a history of amoxicillin/clavulanate-induced AGEP (patient No. 4), phenytoin-induced SJS (patient No. 25), and leflunomide-induced DRESS (patient No. 14).

IL, interleukin; AGEP, acute generalized exanthematous pustulosis; IFN-γ, interferon-gamma; SJS, Stevens-Johnson syndrome; GrB, granzyme B; DRESS, drug reaction with eosinophilia and systemic symptoms.

Fig. 2. Frequencies of drug-induced mediator releasing cells in different phenotypes of severe cutaneous adverse reactions. The average frequencies of drug-induced IL-22 releasing cells in AGEP subjects were significantly higher than those in non-AGEP SCAR subjects, while those of drug-induced IFN-γ releasing cells were significantly lower.

SFU, spot-forming units; PBMCs, peripheral blood mononuclear cells; IL, interleukin; IFN-γ, interferon-gamma; GrB, granzyme B; AGEP, acute generalized exanthematous pustulosis; DRESS, drug reaction with eosinophilia and systemic symptoms; SJS, Stevens-Johnson syndrome; TEN, toxic epidermal necrolysis. *P values < 0.05.

Fig. 3. The sensitivity of different ELISpot assays to identify the culprit drugs in severe cutaneous adverse reactions (n = 27). The sensitivity of the three-mediator combination to confirm the culprit drugs in overall SCAR subjects was significantly higher than that of IFN-γ measurement alone (77.8% versus 51.9%, respectively).

ELISpot, enzyme-linked immunospot assays; SCARs, severe cutaneous adverse reactions; AGEP, acute generalized exanthematous pustulosis; DRESS, drug reaction with eosinophilia and systemic symptoms; SJS, Stevens-Johnson syndrome; TEN, toxic epidermal necrolysis; IFN-γ, interferon-gamma; IL, interleukin; GrB, granzyme B. *P value < 0.01.

Fig. 4. The effects of potential immunoadjuvants on the frequencies of drug-induced mediator releasing cells in severe cutaneous adverse reactions (n =27). The average frequencies of drug-induced IFN-γ releasing cells upon incubation with the culprit drugs alone were 33.0 ± 7.1 SFU/106 cells, while supplementation with α-GalCer significantly increased the frequencies to 72.3 ± 18.5 SFU/106 cells.

IL, interleukin; IFN-γ, interferon-gamma; GrB, granzyme B; PD-1, programmed cell death protein 1; TIM3, T-cell immunoglobulin and mucin domain 3; CTLA4, cytotoxic T-lymphocyte-associated protein 4; α-GalCer, alpha-galactosylceramide; SFU, spot-forming units; PBMCs, peripheral blood mononuclear cells. *P value < 0.05 compared to IFN-γ alone, †P value < 0.05 compared to GrB alone.