Abacavir hypersensitivity syndrome (AHS) is a treatment-limiting and potentially life-threatening adverse event occurring in 5–9% of those initiating the antiretroviral agent abacavir . A cutaneous patch test (PT) has shown promise in characterizing patients with true AHS .
Genetic factors such as HLA-B*5701 and associated alleles carried on the 57.1 ancestral haplotypes have been identified as strong risk factors for AHS in Caucasians [3–5].
The high numbers of CD8 cells found in the skin of patients with skin rash and positive PT associated with AHS  as well as epidemiological studies associating an elevated CD8 count as a risk factor for AHS, support a role for CD8 cells in the pathogenesis of this disease . TNF-α levels were increased in response to abacavir in the whole blood of AHS cases in vitro, and were attenuated by CD8 T-cell depletion, suggesting an immunopathogenetic role of CD8 T cells in AHS .
Further testing was conducted to explore the durability of the PT, the association of the PT with genetic testing and abacavir-specific lymphocyte responses.
Repeat patch testing was performed according to previously published methods  on patients found to be PT positive a median of 23 months previous (range 7–42 months) with a history of AHS a median of 25 months previously (10–45 months). First-time immunological and genetic testing was performed on seven PT-positive patients and 11 abacavir-tolerant controls matched for age, sex, CD4 cell count and race. All patients gave informed consent for the study and DNA storage.
For the genetic and immune assays blood was collected into acid citrate dextran, peripheral blood mononuclear cells (PBMC) separated by density centrifugation, and intracellular cytokine staining (ICS) and proliferation (carboxy fluorescein diacetate succinimidyl ester; CFSE) assays set up simultaneously. For ICS, one million PBMC were incubated at 37°C, 5% carbon dioxide for one hour, either in media alone, with staphylococcal enterotoxin B (Sigma Aldrich Canada, Oakville, Ontario, Canada) at 2 mg/ml, or with abacavir at 0.1 and 1.0 mg/ml. Brefeldin A (GolgiPlug; Becton Dickinson Biosciences, San Jose, CA, USA) was added, cells were incubated for 5 h, washed and permeabilized using the BD Cytofix/Cytoperm kit (Becton Dickinson Biosciences), and stained with CD3 peridin chlorophyll protein, CD8 allophycocyanin, IFN-γ FITC, TNF-α phycoerythrin (Becton Dickinson Biosciences). CD3+/CD8− cells were assumed to be CD4 T cells. For CFSE proliferation , PBMC were incubated with CFSE (Molecular Probes, Eugene, OR, USA) at a final concentration of 1.5 mM at room temperature for 5 min. CFSE-labeled cells were then washed three times and re-suspended in a sterile 24-well tissue culture plate at 37°C, 5% carbon dioxide for 5 days with media alone, staphylococcal enterotoxin B or abacavir at the concentrations above. On day 5, cells were harvested, permeabilized, and stained with CD3 phycoerythrin, CD4 peridin chlorophyll protein, CD8 allophycocyanin. A positive response for both ICS and proliferation was defined as a response exceeding 0.05% of gated cells, and at least threefold greater than background. Flow cytometric acquisition and analysis was performed using FACSCaliber (BD Pharmingen, San Jose, CA, USA) with FlowJo version 4.5.8 software (Tree Star Inc., Ashland, OR, USA).
Genetic testing consisted of typing the major histocompatibility complex single nucleotide polymorphism markers using standard genetic assays and sequencing and the determination of the alleles specific to the 57.1 ancestral haplotype . Human leukocyte antigen class I and II high-resolution typing was performed using the previously published DNA sequencing methods .
Seven out of seven previously PT-positive cases showed positive PT at 24 h with all patients reporting local pruritus but no systemic symptoms. A positive PT on one patient 41 months from the original AHS is shown (Fig. 1a).
Abacavir-specific IFN-γ production was seen in two out of seven cases (29%) but no controls (P = 0.04). PT-positive and abacavir-tolerant controls did not differ for abacavir-specific CD4 cell proliferation, but CD8 T-cell proliferation was observed in five out of seven PT-positive cases (71%), and only in one out of 11 of the abacavir-tolerant controls (9%; P = 0.005) (Fig. 1b).
The distribution of MHC alleles was significantly different in the PT-positive group when compared with the abacavir-tolerant control group and significantly clustered to alleles carried on the 57.1 ancestral haplotype in the PT-positive group (Fig. 1c). HLA B*5701 was present in all seven PT patients but only in one of the abacavir-tolerant controls (P < 0.001). The Hsp70-Hom M493T allele (rs227956C) carried on the 57.1 ancestral haplotype was observed in six PT-positive and in none of the abacavir-tolerant controls (P < 0.001).
Vigorous PT responses at 24 h in all seven patients after an average interval of more than 2 years supports the durability of the abacavir PT in correctly classifying AHS. Abacavir-specific CD8 T-cell proliferation was seen in the majority of AHS cases, compared with one abacavir-tolerant control, supporting a direct role for CD8 T cells in the immunopathogenesis of AHS.
Alleles of the 57.1 ancestral haplotype clustered strongly in PT-positive versus abacavir-tolerant control cases (Fig. 1c). HLA-B*5701 was present in 100% of PT-positive patients. Furthermore, that 86% of abacavir PT-positive patients in our study carried the Hsp70-Hom variant compared with only 9% of abacavir-tolerant controls supports recent work suggesting that susceptibility to AHS is enhanced by the presence of the non-synonymous polymorphism Hsp70-Hom M493T (rs227956C) . One of the abacavir-tolerant control patients was positive for HLA-B*5701, Hsp70-Hom and associated alleles of the 57.1 ancestral haplotype, but lacked abacavir-specific CD4/CD8 cytokine and lymphocyte proliferation responses, supporting the theory that these genetic markers are not always associated with AHS, and implying the influence of other metabolic and immune factors. PT may thus also help clarify the predictiveness of genetic testing or identify novel genetic markers in non-Caucasian populations with a lower prevalence of HLA-B*5701 .
The abacavir PT shows durability over time, suggesting that it may be useful when the history of AHS is remote. CD8 proliferation in response to abacavir is a new finding, which combined with the genetic findings implicates the role of HLA-B*5701-restricted CD8 cells in the pathogenesis of AHS. A good correlation between patch, immunological and genetic testing suggests that these tests may complement each other in the clinical setting and enhance the ability to diagnose and prevent the occurrence of AHS.
The authors would like to thank Sunnybrook Pharmacy Manufacturing for the preparation of the petrolatum-based abacavir product for the patch testing, Sunnybrook dermatology nurses for their help in the patch testing process, and Dr Jack Uetrecht for the purification of abacavir for in-vitro immunological testing.
Sponsorship: This work was funded by the Canadian Dermatology Foundation.
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