In the clinic, skin tests are the only diagnostic tool routinely used for hypersensitivity reactions with numerous publications providing guidelines for carrying out these skin tests (Barbaud et al., 2001; Barbaud et al., 1998; Brockow et al., 2002). There are three classic methods of skin tests; skin prick, intradermal and patch tests. Skin prick tests, whereby a needle with the suspected allergen pricks the skin, is followed up with intradermal testing (injection of up to 50µl of drug solution) if results are negative (Torres et al., 2003). Skin prick and intradermal test are normally carried out where the reaction is thought to be IgE mediated/immediate hypersensitivity, whereas patch tests are normally performed in cases of delayed hypersensitivity (Demoly et al., 2005). Suspect chemical entities are applied to the upper back with the use of adhesive tape with reactions noted (Barbaud et al., 1998). However these traditional skin test often give false-negative results, have low sensitivity and in the case of intradermal tests, are invasive (Barbaud et al., 2001; Strauss et al., 2001). Re-challenging patients in provocation tests are useful for the confirmation of culprit drug following inconclusive results after skin testing. This must be carried out under careful medical observation due to the risk of re-activation of symptoms and so in severe reaction i.e. in SJS, these test are discouraged (Romano et al., 2004).
The diagnosis of delayed hypersensitivity, particularly T-cell mediated reactions, can be complemented by the use of in vitro techniques which help
55
characterise clinical symptoms and highlight the drug responsible for such a reaction. These tests are not widely used in the clinic due to various reasons including the availability of freshly isolated peripheral blood mononuclear cells, the expertise required to conduct such assays/training researchers to carry out the tests and the lack of high throughput methods. The use of patient cells ex vivo is however at the centre of drug hypersensitivity research. The in vitro techniques described below can offer a safe alternative to often invasive skin tests and importantly in research, allow a better understanding of the molecular mechanisms of the reactions seen in the clinic.
1.9.1 Lymphocyte transformation test (LTT)
The lymphocyte transformation test (LTT) is used to detect drug-specific T-cell responses and the most commonly utilised in vitro assay for patients with suspected drug allergies (Nyfeler et al., 1997). Peripheral blood mononuclear cells (PBMCs) are isolated from hypersensitive patients, exposed to the culprit drug and the proliferation of memory T-cells previously sensitised are measured (Luque et al., 2001; Nyfeler et al., 1997; Pichler et al., 2004). Sensitized lymphocytes generate cytokines including IL-2, undertake blastogenesis and the subsequent proliferative response is measured and quantified through the incorporation of 3H-thydmidine during the synthesis of
DNA (Merk, 2005). The stimulation index (SI), the ratio of antigenic proliferation in comparison to proliferation in the absence of antigen i.e. cell culture medium, is used to express lymphocyte proliferation. An SI of 2 or more is considered a positive response.
56
The LTT can be used in delayed and also immediate hypersensitivity reactions though particular drugs have higher specificity including the β-lactam antibiotics, sulphonamides and anticonvulsants (Luque et al., 2001; Merk, 2005). Tolerant or naive individuals would not display positive responses. Studies have shown the usefulness of LTTs in diagnosing penicillin allergy in delayed hypersensitivity, with sensitivities between 60% and 75% (Rozieres et al., 2009). Although non-invasive, the length of the assay (six days) and the requirement of radioactive isotopes do not translate well clinically.
1.9.2 Enzyme-linked immunospot (ELISpot)
The enzyme-linked immunospot (ELISpot) assay has a shorter duration than the LTT (four days), is sensitive and allows the detection and characterization of drug-specific cytokine secreting T-cells (Czerkinsky et al., 1988). T-cells secrete an array of cytokines following activation. Pro-inflammatory (generally Th1) cytokines such as IFN-γ, TNF-α, IL-2, IL-6 and IL-12, Th2 anti- inflammatory cytokines such as IL-4, IL-5, IL-10, IL-13and TGF-β and other secreted molecules such as granzyme B, perforin and Fas ligand can be investigated (Lehmann et al., 2012; Lochmatter et al., 2009; Romagnani, 1992; Romagnani, 2006). This assay provides more detailed information on the biological function of the T-cells that mediate the hypersensitivity reaction. IFN-γ is one of the major cytokines implicated in numerous delayed hypersensitivity reactions and drug-induced exanthemas. Drug-specific IFN-γ secreting T-cells have been detected and quantified (Naisbitt et al., 2005; Teraki et al., 2003; Yoshimura et al., 2004). While playing an important role in Th1 responses and serving as a cytotoxicity marker, IFN-γ can also contribute to the
57
Th2 response through its ability to upregulate MHC II molecules on the surface of CD4+ T-cells (Beeler et al., 2006). Sensitivity from PBMC cultures in the IFN-γ ELISpot can reach 90% and up to 100% specificity can be seen in penicillin- induced MPE (Porebski et al., 2011; Rozieres et al., 2009).
Multiplex, or Luminex, assays also quantify drug-specific cytokine secretion by T-cells (Beeler et al., 2006; Chen et al., 2009). Capture antibodies directed at each target molecule/cytokine are bound to colour-coded beads and are detected and quantified by a fluorescently labelled reporter. As each antibody is coupled with a specific bead, multiple beads can be added and therefore multiple cytokines can be examined in a single sample.
1.9.3 Flow cytometry
Flow cytometry allows for the identification of multiple cell surface markers and subsequently the phenotypic analysis and characterisation of cells involved in drug hypersensitivity. Cell surface markers on activated T-cells can be used to detect drug-specific T-cell populations and thus can be utilised in the diagnosis of hypersensitivity (Hari et al., 2001). The surface molecule CD69 is one such marker that has been used as a marker of delayed drug hypersensitivity and is comparable to LTT in terms of sensitivity and specificity (Beeler et al., 2008). Other cell surface markers of drug-specific T-cell activation include CD25, CD40L CD71 and HLA-DR which are upregulated in sensitized individuals (Beeler et al., 2006; Porebski et al., 2011).
As with the LTT, flow cytometry can also be used to measure the proliferative response of previously sensitised memory T-cells. Drug-specific T-cells are stained with the fluorescent dye carboxyfluoescein succinimidyl ester (CFSE),
58
which binds to amino groups of intracellular proteins and becomes integrated within the lipid membrane. During a drug-specific proliferative response, the intensity of CFSE fluorescence will decrease by half with each cell division and so the the number of divisions can be determined and evaluated (Beeler et al., 2006).
Following stimulation by a drug or antigen Th1, IFN-γ secreting cells and Th2, IL-4 secreting cells play an important role in the development and nature of the immune response (Del Prete et al., 1993; Mosmann et al., 1996). Intracellular cytokine staining, ICS, uses flow cytometry to evaluate such cytokine secretion as these two subsets of cells do not possess cell surface markers. Fluorescent antibodies for cytokines, including IFN-γ and IL-4, form the basis of ICS and with flow cytometry we can determine their involvement in drug induced immune response.
1.9.4 T-cell cloning
In some individuals it can be difficult to detect drug-specific T-cells as part of a PBMC culture using the above described methods. The use of T-cell clones in assays adapted from those previously described provides an alternative allowing research to focus specifically on antigen-specific cells. Using the well- established method of serial or limiting dilution, long lived drug-specific T-cells can be isolated from allergic individuals and expanded to generate drug-specific T-cell clones (Mauri-Hellweg et al., 1995; Staszewski, 1984). Figure 1.8 provides further details of the processes involved in the generation of drug-specific T-cell clones. T-cell clones can then be fully characterised in terms of phenotype, for the expression of specific markers, proliferative capacity and their cytokine
59
secretion prolife determined. This allows the pathomechanism of drug hypersensitivity to be investigated.
Figure 1.8T-cell cloning
Timeframe and process involved in the generation of antigen-specific T-cell clones