Structural Elements Recognized by Abacavir-Induced T Cells

Adverse drug reactions are one of the leading causes of morbidity and mortality in health care worldwide. Human leukocyte antigen (HLA) alleles have been strongly associated with drug hypersensitivities, and the causative drugs have been shown to stimulate specific T cells at the sites of autoimmune destruction. The structural elements recognized by drug-specific T cell receptors (TCRs) in vivo are poorly defined. Drug-stimulated T cells express TCRs specific for peptide/HLA complexes, but the characteristics of peptides (sequence, or endogenous or exogenous origin) presented in the context of small molecule drugs are not well studied. Using HLA-B*57:01 mediated hypersensitivity to abacavir as a model system, this study examines structural similarities of HLA presented peptides recognized by drug-specific TCRs. Using the crystal structure of HLA-B*57:01 complexed with abacavir and an immunogenic self peptide, VTTDIQVKV SPT5a 976–984, peptide side chains exhibiting flexibility and solvent exposure were identified as potential drug-specific T cell recognition motifs. Viral sequences with structural motifs similar to the immunogenic self peptide were identified. Abacavir-specific T cell clones were used to determine if virus peptides presented in the context of abacavir stimulate T cell responsiveness. An abacavir-specific T cell clone was stimulated by VTQQAQVRL, corresponding to HSV1/2 230–238, in the context of HLA-B*57:01. These data suggest the T cell polyclonal response to abacavir consists of multiple subsets, including T cells that recognize self peptide/HLA-B*57:01 complexes and crossreact with viral peptide/HLA-B*57:01 complexes due to similarity in TCR contact residues

Days to onset of hypersensitivity symptoms in patch test confirmed cases of abacavir HSR (range <2–19 days, median 8 days; n = 23).

<p>Days to onset of hypersensitivity symptoms in patch test confirmed cases of abacavir HSR (range <2–19 days, median 8 days; n = 23).</p

Assessment of abacavir responsive memory or naïve T cells.

<p><b>A</b>. Sorting gates used to collect total CD4+ and CD8+ memory phenotype (green polygon) and naïve phenotype T cells (blue rectangle) or <b>B</b>. CD8+ memory or naïve phenotype T cells only. <b>C</b>. Protocol used for the selection, priming and re-stimulation of sorted naïve and memory T cells from cryopreserved HLA-B*57:01 positive donor PBMC. <b>D</b>. Representative plots of in-vitro cultures of HLA-B*57:01 positive memory (left most pairs of plots) or naïve (right most pairs of plots) phenotype T cells derived from sorting strategy A. Cultures were re-stimulated with APCs treated with abacavir (C1R.B57.ABC) or untreated (C1R.B57), respectively. <b>E</b>. Representative plots of in-vitro cultures of HLA-B*57:01 positive memory (left most pairs of plots) or naïve (right most pairs of plots) phenotype CD8+ T cells derived from sort strategy B. Cultures were re-stimulated with APCs as described above.</p

Abacavir ELISpot responses.

<p><b>A</b>. Mean abacavir ELISpot responses for HLA B*57:01 positive or negative individuals, that were either abacavir naïve or treatment experienced. <b>B</b> Abacavir ELISpot responses in patch test confirmed abacavir HSR patients plotted relative to time from first exposure to abacavir (25-562 SFU/10⁶ PBMC, n = 12). Positivity of abacavir ELISpot responses was defined using a cut-off response of ≥ 10 SFU/10⁶ PBMC above background, and differences in proportions of positive responses were assessed using a Fishers exact test.</p

Abacavir responsive CD8+ T cells can be expanded from unsorted, memory and naïve phenotype T cells from abacavir-unexposed HLA-B*57:01 positive donors.

<p>CD8+/IFN-γ frequencies are compared across T-cell populations which had been cultured ± abacavir, and then re-stimulated with APCs treated with abacavir (yes) or untreated (no), respectively. <b>A</b>: CD8+/IFN-γ frequencies in unsorted PBMC from HLA-B*57:01 negative (n = 3) and positive donors (n = 8). CD8+/IFN-γ frequencies in HLA-B*57:01 positive donors according to memory and naïve phenotype in <b>B</b>: sorted CD4+ and CD8+ T cells (n = 8) and <b>C</b>: sorted CD8+ T cells only (4 samples, n = 3 donors). Each donor is represented by the same symbol in the different figures; group median CD8+ /IFN-γ+ T-cell frequencies are indicated by a horizontal line. Pairwise differences in observed frequencies according to abacavir stimulation are assessed by a donor-stratified Wilcoxon test.</p

Immunization of an HLA-B*57:01 donor with the yellow fever vaccine results in the expansion of populations of CD8+ T cells that detect both the wild type epitope and abacavir dependent variant epitopes.

<p>T cells expanded from a yellow fever vaccinated B*57:01 donor were labelled with anti-CD3 and CD8 and a mixture of K9<u>F</u>-B*57:01-PE, K9<u>A</u>-ABC-B*57:01-APC, and K9<u>V</u>-ABC-B*57:01-BV421 tetramers. CD8+ T cells were gated and the combination of K9<u>F</u>-B*57:01-PE and K9<u>A</u>-ABC-B*57:01-APC tetramer stained CD8+ T cells are shown in A. Subpopulations of tetramer stained cells are gated: P4 (pink); P5 (yellow); P6 (green); P7 (blue); P8 (orange); P9 (purple). The combinations of K9<u>F</u>-B*57:01-PE and K9<u>V</u>-ABC-B*57:01-BV421 tetramer stained CD8+ T cells are shown in B. The colors identify the position of the gated tetramer stained populations in panel A.</p