Detection of Primary T Cell Responses to Drugs and Chemicals in HLA-Typed Volunteers: Implications for the Prediction of Drug Immunogenicity

Toxicological Sciences, Dec 2016

A number of serious adverse drug reactions are caused by T cells. An association with HLA alleles has been identified with certain reactions, which makes it difficult to develop standardized preclinical tests to predict chemical liability. We have recently developed a T cell priming assay using the drug metabolite nitroso sulfamethoxazole (SMX-NO). We now report on reproducibility of the assay, establishment of a biobank of PBMC from 1000 HLA-typed volunteers, and generation of antigen-specific responses to a panel of compounds. Forty T cell priming assays were performed with SMX-NO; 5 gave weak responses (1.5–1.9) and 34 showed good (SI 2.0–3.9) or strong responses (SI  > 4.0) using readouts for proliferation and cytokine release. Thus, SMX-NO can be used as a model reagent for in vitro T cell activation. Good to strong responses were also generated to haptenic compounds (amoxicillin, piperacillin and Bandrowski’s base) that are not associated with an HLA risk allele. Furthermore, responses were detected to carbamazepine (in HLA-B*15:02 donors), flucloxacillin (in 1 HLA-B*57:01 donor) and oxypurinol (in HLA-B*58:01 donors), which are associated with HLA-class I-restricted forms of hypersensitivity. In contrast, naïve T cell priming to ximelagatran, lumiracoxib, and lapatinib (HLA-class II-restricted forms of hypersensitivity) yielded negative results. Abacavir, which activates memory T cells in patients, did not activate naïve T cells from HLA-B*57:01 donors. This work shows that the priming assay can be used to assess primary T cell responses to drugs and to study mechanisms T cell priming for drugs that display HLA class I restriction. Additional studies are required to investigate HLA-class II-restricted reactions.

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Detection of Primary T Cell Responses to Drugs and Chemicals in HLA-Typed Volunteers: Implications for the Prediction of Drug Immunogenicity

A number of serious adverse drug reactions are caused by T cells. An association with HLA alleles has been identified with certain reactions, which makes it difficult to develop standardized preclinical tests to predict chemical liability. We have recently developed a T cell priming assay using the drug metabolite nitroso sulfamethoxazole (SMX-NO). We now report on reproducibility of the assay, establishment of a biobank of PBMC from 1000 HLA-typed volunteers, and generation of antigen-specific responses to a panel of compounds. Forty T cell priming assays were performed with SMX-NO; 5 gave weak responses (1.5–1.9) and 34 showed good (SI 2.0–3.9) or strong responses (SI  > 4.0) using readouts for proliferation and cytokine release. Thus, SMX-NO can be used as a model reagent for in vitro T cell activation. Good to strong responses were also generated to haptenic compounds (amoxicillin, piperacillin and Bandrowski’s base) that are not associated with an HLA risk allele. Furthermore, responses were detected to carbamazepine (in HLA-B*15:02 donors), flucloxacillin (in 1 HLA-B*57:01 donor) and oxypurinol (in HLA-B*58:01 donors), which are associated with HLA-class I-restricted forms of hypersensitivity. In contrast, naïve T cell priming to ximelagatran, lumiracoxib, and lapatinib (HLA-class II-restricted forms of hypersensitivity) yielded negative results. Abacavir, which activates memory T cells in patients, did not activate naïve T cells from HLA-B*57:01 donors. This work shows that the priming assay can be used to assess primary T cell responses to drugs and to study mechanisms T cell priming for drugs that display HLA class I restriction. Additional studies are required to investigate HLA-class II-restricted reactions. HLA, T cells, drug hypersensitivity. The mechanisms involved in the etiology of adverse drug reactions (ADRs) are complex with both drug and patient-specific factors contributing towards susceptibility. Up to 20% of ADRs are classified as hypersensitivity reactions where the immune response causes an unexpected clinical reaction (White et al., 2015). Hypersensitivity reactions frequently involve the skin but may also involve other organs such as the liver. Most chemicals, drugs, and their metabolites are too small to act as conventional T cell antigens. However, they can become bound to carrier proteins which are large enough to be antigenic or in some instances they may bind directly to immune receptors resulting in cell activation (Adam et al., 2011). The role of T cells in the development of drug hypersensitivity has been studied for several drugs including abacavir, carbamazepine, sulfamethoxazole, piperacillin, and allopurinol (Chessman et al., 2008; El-Ghaiesh et al., 2012; Ko et al., 2011; Naisbitt et al., 2001), but the critical factors determining which individuals among the treated population will suffer from drug hypersensitivity remains poorly understood. Genome wide screens have identified an association with several different HLA alleles and drugs which cause both skin and liver reactions (Chung et al., 2004; Daly et al., 2009; Mallal et al., 2002). HLA molecules are key proteins that regulate T cell-mediated immunity. HLA class I molecules present antigens to CD8 T cells and HLA class II molecules present antigens to CD4 T cells. The relationship between HLA-restricted responses and the aetiology of drug hypersensitivity has been clearly demonstrated for HLA-B*57:01 which is associated with abacavir and flucloxacillin hypersensitivity. Abacavir-specific CD8+ T cells respond only to antigen presented in the context of HLA-B*57:01 (Chessman et al., 2008). In patients with drug-induced liver injury, flucloxacillin-protein adducts are presented to T cells by HLA-B*57:01 via a hapten mechanism, whereas in normal volunteers when naïve T cells are primed in vitro, the primed T cells can respond to flucloxacillin presented on number of different alleles via a direct interaction (Monshi et al., 2013; Yaseen et al., 2015). In order to investigate the role of specific HLA alleles in drug hypersensitivity we have established a biobank of peripheral blood mononuclear cells (PBMCs) isolated from HLA-typed volunteers (Alfirevic et al., 2012). We initially recruited 400 volunteers during 2009–2010. These volunteers are representative of the North-West population and consist of 64% female and 36% male volunteers with a mean age of 29 years (9 ± 10 years, range 18–60 years). The ethnicity of the volunteers also reflects regional diversity which is primarily Caucasian (84%) with Asian Indians (6%), Chinese (4%), and Blacks (1%) as minor populations. Typing of HLA–A, B, C, DRB1, and DQB1 alleles showed that all major Caucasian haplotypes were represented (Alfirevic et al., 2012). We have now extended the biobank by an additional 600 volunteers, who were recruited during 2014 and here we report on the characteristics of these new volunteers. Once the biobank was established, we develope (...truncated)


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Faulkner, Lee, Gibson, Andrew, Sullivan, Andrew, Tailor, Arun, Usui, Toru, Alfirevic, Ana, Pirmohamed, Munir, Naisbitt, Dean J., Kevin Park, B.. Detection of Primary T Cell Responses to Drugs and Chemicals in HLA-Typed Volunteers: Implications for the Prediction of Drug Immunogenicity, Toxicological Sciences, 2016, pp. 416-429, Volume 154, Issue 2, DOI: 10.1093/toxsci/kfw177