RESULTS/DISCUSSION
In this study, we included PBMC and BFC samples that had been
cryogenically stored from four patients with confirmed SCAR (Cases 1-4)
including SJS, TEN, DRESS, and generalised bullous fixed drug eruption
(GBFDE) identified from previous prospective studies
(Supplementary Methods ). All patients had a Naranjo score of 4
or higher[11], a minimum Scorten score of 2 and a minimum Alden
score of 4 for SJS/TEN[12, 13]. Cases 2 and 4 had one implicated
drug while Cases 1 and 3 had three implicated drugs and all cases were
receiving the implicated drug at time of rash onset (Table 2). The
latency period for cases (defined as time between drug commencement and
rash onset) ranged from 0-38 days with a median value of 18.5 days. PBMC
and BFC collection delay for testing had means of 24 and 22 days,
respectively. Case 4 had a delayed collection latency for PBMC and BFC
of 48 and 49 days, respectively. Baseline demographics, clinical
features and biological sampling are shown in Tables 1 and 2 .
IFN-γ ELISpot was performed in matched PBMC and BFC samples from Cases
1-4, as per previously published methods[7] andSupplementary Methods (Figures 1 and 2 ,Supplementary Figures 1 and 2) . Two of these patients displayed
positive ELISpot results (defined as SFU ≥ 50U/million cells
[7, 8])
upon ex vivo challenge with suspected drugs for both PBMC and BFC
(Cases 1-ceftriaxone and 4-oxypurinol), while Cases 2 and 3 only
displayed a positive result with BFC (Figure 1).
Case 1 BFC tested positive for both doses of ceftriaxone (200 and
2000µg/mL), whilst matched PBMC only tested positive to the highest
dose, with half of the response elicited in BFC. Case 4 BFC tested
positive at both concentrations (5 and 50µg/mL) of allopurinol in
addition to its metabolite, oxypurinol, while PBMC only showed a
positive response to oxypurinol. This suggests that BFC analysis can
provide higher sensitivity to drug-allergy testing than PBMC. This is
further supported by analysis of Cases 2 and 3 whereby positive IFN-γ
release ELISpot responses were detected using BFC but not PBMC samples.
Case 2 BFC displayed positivity to all doses of meloxicam (2, 20 and 200
µg/mL) and Case 3 only to the highest dose of sulfamethoxazole
(SMX-500µg/mL), its metabolite 4-Nitro-SMX (100µg/mL), as well as to the
commercial product (Bactrim©; trimethoprim-sulfamethoxazole) at 50 and
250µg/mL of the sulfamethoxazole component, respectively.
Flow cytometry was used to investigate whether different cellular
compositions of matched BFC and PBMC for Cases 1, 3 and 4 could account
for differences in ELISpot sensitivities (Figure 2,
Supplementary Figure 1-2) . We found that BFC samples were enriched for
total T (CD3+) cells and for IFN-γ-secreting cells, relative to matched
PBMC (Figure 2A, Supplementary Figure 1 and 2 ). The total
proportions of CD4+, CD8+, or double negative (DN) T cells varied across
individuals (Figure 2A, Supplementary Figure 1 ), likely
reflecting differences in the pathology and/or treatments, with Case 1
displaying a strong bias for CD4+ T cells, which is typical of
DRESS[14]. In Contrast, Case 4 BFC were enriched for CD8+ T cells
relative to matched PBMC, which may be associated with a delayed BFC
sampling, compared to other cases (Table 2 ), possibly
reflecting CD8+ T cells egress from peripheral blood[8]. Cases 3 and
4 BFC showed an enrichment for T cell populations with a tissue
residency/recruitment (CD69+CD103+) phenotype, which have been
implicated in SCAR[8] (Figure 2A, Supplementary Figure 1 ).
Case 3 BFC samples further displayed higher proportions of memory
(CD45RO) and activated (CD69) T cells, relative to PBMC, whilst
remaining similar for Cases 1 and 4, which may partly account for the
differences in ELISpot sensitivities between the two samples
(Figure 1 ). As unconventional T cells (not HLA-restricted) are
also known to produce IFN-γ, and their role in SCAR remains
unexplored[15], we assessed the proportions of γδ T cells,
mucosal-associated invariant T (MAIT) cells and CD56-expressing T cells
(T cells expressing natural killer (NK) markers, likely including
natural killer T (NKT) cells)[16]. While MAIT cells and γδ T cells
did not show preferential recruitment into BFC (Figure 2A and
Supplementary Figure 1 ), they were found among IFN-γ+ populations
(Figure 2B, Supplementary Figure 2B ), representing a
large proportion of Case 3 PBMC (38.4% and 11.4%, respectively). IFN-γ
secreting cells also comprised NK-like T cells (CD56+CD3+), and NK cells
(CD56+CD3-) - Case 3 PBMC. Overall, IFN-γ-secreting cells comprised
CD4+, CD8+ and DN (CD4-CD8-) T cells, with preferential enrichment for
CD8+ T cells in BFC from Cases 3 and 4 and displayed memory and
activated phenotypes (CD45RO+/CD69+) (Figure 2,Supplementary Figure 2B ). Overall BFC samples display T
lymphocytes that have been recruited from the blood or adjacent tissue
with an activated phenotype and cytokine secreting capacity. This leads
to higher proportions of cells with an IFN-γ secreting capacity (when
compared to blood), which may reflect higher representations of the
drug-antigen-specific clones. Collectively, these results suggest a
higher sensitivity for BFC samples in ELISpot-testing relative to PBMC,
likely reflecting differences in their cellular composition.
Ex vivo drug-allergy diagnostics have an increasing evidence base
and clinical demand[3]. By analysing samples from four SCAR patients
with distinct drug-allergies and clinical manifestations that are
presumed to be T-cell-mediated, this study provides impetus for further
work to explore alternative sampling sources for drug-allergy
diagnostics. At present there is no gold-standard diagnostic for
causality assessment in SCAR, and previous studies, whilst showing
promising sensitivity[3], remain limited. Our results suggest higher
sensitivity for BFC analysis relative to matched PBMC using ex
vivo IFN-γ release ELISpot [3]. Whilst limited by low numbers and
cell viability, the rare nature of both blister fluid capture and SCAR
cases that have been accurately phenotyped provides a unique insight
into the diagnostic potential for this IFN-γ release ELISpot assay.
Our results are consistent with recruitment of known populations
involved in the pathology (T cells with a memory/activated phenotype and
cytokine-secreting capacity) into blisters. We further reveal that,
relative to BFC, PBMC may have lower representation of cells with an
IFN-γ-secretion capacity[7, 8]. How much IFN-γ detection by ELISpot
is due to direct activation of drug-specific cells or bystander
secretion of non-specific cells remains to be understood, and it may
vary with the drug causing SCAR. It is possible that some drug-specific
cells may produce cytokines other than IFN-γ (such as TNF, IL-4, IL-17)
upon activation that have not been tested. Whilst we also assessed
IL-17-secretion using flow cytometry, our results do not seem to suggest
that this could be a key contributor for the responses studied
(Supplementary Figure 3 ). This may require ELISpot assays for
other cytokines or markers yet to be identified, or even more generic
activation assays using cellular activation markers like CD69, CD107a.
Thus, we recommend that clinicians sample BFC, whenever available, for
testing with ELISpot assays in drug-allergy diagnostics, whilst
retaining correlation with PBMC results. This may prove to be an
invaluable resource for future studies aiming at characterising the
immunopathogenesis and HLA (or HLA-like) restriction of these
drug-induced allergies, including drug-presentation pathways, cell
populations involved, and cytokine-outputs. Such knowledge may
ultimately lead to improved diagnostics for SCAR patients, improving
efforts to lower the significant morbidity and mortality associated with
SCAR.