Activation of CD8+ T-Cell with abacavir and HLA-B*57:01 binding peptides and elimination of T-Cell responses through chemical modification

摘要

Adverse drug reactions are responsible for 6.5% of all hospital admissions. They also affect 10 to 20% of hospitalized patients. Drug-induced hypersensitivity reactions constitute an important major subtype of type B adverse drug reactions. Low molecular weight (i.e. penicillins, sulfamethoxazole and abacavir) and high molecular weight (biologics) drugs are both associated with the inadvertent activation of the adaptive immune system, which manifests as hypersensitivity in certain individuals. Pharmacogenomic research revealed that several forms of immunological drug reactions are strongly associated with specific human leukocyte antigens (HLA) alleles. The underlying mechanisms of HLA associated immunological drug reactions are still under exploration, however abacavir hypersensitivity represent a prototype model that exclusively occurs in individuals carrying the risk allele HLA- B*57:01. One hundred and ten CD8+ T-cell clones out of 1304 clones generated from 3 HLA-B*57:01 healthy volunteers displayed proliferative responses and/or secreted interferon gamma in the presence of abacavir which have been used for further mechanistic studies to evaluate mechanisms of drug antigen presentation. Approximately half of the clones were activated with abacavir in the absence of antigen presenting cells. Of the remaining clones, interestingly about half were activated with abacavir pulsed APC. The APC pulse negative clones are likely activated via a PI mechanism involving the direct binding of abacavir to surface HLA-B*57:01. In contrast, the pulse positive clones seem to be activated via the altered peptide repertoire mechanism involving abacavir binding to endogenous HLA-B*57:01. Some clones were also activated with APC from certain allogeneic donors expressing different HLA-B alleles. APC-dependent abacavir-specific clones were activated by the drug in the presence of APC from at least 3 allogenic different donors. Sixteen 6-amino substituted abacavir analogues were synthesized. Computational docking studies were completed to predict capacity for analogue binding within HLA-B*57:01. Abacavir-specific CD8+ clones were generated to study the association between HLA-B*57:01 analogue binding and T-cell activation. Antiviral activity and the direct inhibitory effect of analogues on proliferation were assessed. MHC class I-restricted CD8+ clones proliferated and secreted interferon gamma following abacavir binding to surface and endogenous HLA-B*57:01. Several analogues retained antiviral activity and showed no overt inhibitory effect on T-cell proliferation, but displayed highly divergent antigen-specific T-cell responses. Abacavir and N-propyl abacavir were equally potent at activating clones, while the closely-related analogues N-isopropyl and N-methyl isopropyl abacavir were devoid of T-cell activity. Docking abacavir analogues to HLA-B*57:01 revealed a quantitative relationship between drug-protein binding and the T-cell response. These studies demonstrate that the unwanted T-cell activity of abacavir can be eliminated whilst maintaining the favourable antiviral profile. A relatively large panel of abacavir-dependent HLA-B*57:01 binding self peptides were utilised to understand the mechanism of T-cell activation. Our data provides experimental evidence that a 9mer peptide (Peptide 15; NTVELRVKI) is responsible for the polyclonal CD8+ T-cell activation seen in abacavir hypersensitive patients. Furthermore, the discovery that most discovered unique altered self peptides are unable to induce CD8+ T-cell activation indicated that the immune response is highly peptide specific. Collectively the data generated enhances our understanding of how a single drug abacavir interacts with immunological receptors to initiate an unwanted and often pathogenic immune response. Even for this most highly HLA-restricted immunological drug reaction the nature of the drug receptor interaction is much more complex than initially anticipated. Thus, further studies are needed to determine how the results presented herein relate to other forms of immunological drug reaction.