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Supporting Information Table I Characterization of abc-specific TCCs

Supporting Information Figure 1. The CD8+ T cell reactivity to abc depends on the presence of HLA-B*5701. (A) Gating strategy for the IFN-γ secretion analysis of CD3+ T cells upon abc-specific restimulation. Abc-specific T cell lines were induced and restimulated as described in the Material and Methods section. In order to exclude CFSE labelled APCs, CFSE-negative cells were selected from the lymphocyte pool followed by the selection of CD3+ cells. (B – E) CD3+ cells were gated according to their expression of CD8 and IFN-γ. (B – D) In all HLA-B*5701+ donors, IFN-γ secretion in response to abc was observed exclusively in the CD8+ population. The strength of the responses amongst the different individuals was comparable, irrespective of a prior exposure to the drug or the donor's HIV state. (E) HLA-B*5701-negative individuals do not exhibit abc reactive T cells. Numbers in squares indicate percent IFN-γ+ CD8+ T cells. Representative data of three independent experiments are shown.

Supporting Information Figure 2. ADH isoenzymes are not expressed in keratinocytes. Although not present in our in vitro system, we also analyzed the ADH expression in keratinocytes by reverse transcriptase PCR. In contrast to the control liver cell line (Huh-7), relevant ADH expression was not detected in keratinocytes from healthy donor 74 (HD74). Only ADH5, a formaldehyde-dehydrogenase not described to be involved in abc metabolism, was ubiquitously expressed. Amplification of the housekeeping-gene actin served as an internal positive control. The data are representative of two independent experiments.

Supporting Information Figure 3. Proteasome inhibition abrogates protein processing and reactivity of peptide specific CD8+ T cells. In order to verify the efficacy of the used proteasome inhibitors, Lactacystin and Bortezomib, inhibition assays were performed with APCs expressing the Hepatitic C Virus (HCV) derived NS3/4A protein [36]. Briefly, a HLAA* 0201 restricted T cell line was generated against the peptide epitope CV9 (CINGVWCTV) of the NS3/4A protein. Specificity of the T cell line was assessed after a 6 h peptide specific restimulation by flow cytometry, using CD107a expression as a marker for degranulation. T1 (mock transfected) and T1/NS3–4A cells (expressing the NS3–4A protein) were used as APCs [36]. After peptide stripping by a short acid treatment, these APCs were incubated with control medium, Lactacystin (50 μM) and Bortezomib (10 nM), respectively, for 16 hours. Then, APCs were used to stimulate the CV9 specific T cell line in the presence of the corresponding inhibitors. In the absence of Lactacystin and Bortezomib, T1/NS3–4A cells prompted the degranulation of specific T cells, demonstrating peptide presentation after proteasomal processing of NS3/4A. In the presence of the proteasome inhibitors, the T cell reactivity was inhibited, reflecting a block of the NS3/4A protein processing. CV9 peptidepulsed T1/NS3–4A cells served as positive control. Data are shown as mean ± SD across two independent experiments performed in triplicates.

Supporting Information Figure 4. The reactivity to abc is specific. In order to verify the specificity of the reactivity to abc, a sulfamethoxazole-specific TCC was stimulated with sulfamethoxazole and abc, respectively, and the response was assessed in a Ca2+ influx assay. Two concentrations of sulfamethoxazole were added to the TCC in the presence of autologous APCs (black arrow). These stimuli resulted in immediate and strong TCC responses. Moreover, the clone was stimulated with autologous abc-pulsed APCs (grey arrow), which did not result in TCC activation. TCC incubated with APCs in medium served as a negative control. Representative data of two independent experiments are shown.

Supporting Information Figure 5. Activated CD8+ TCCs pass through a transient annV+ state. In order to verify that the annV+ state of abc-TCCs is a marker of activation [33] and does not signify the onset of early apoptosis, TCC-2C/HD589 was co-stained with propidium iodide and annV-AlexaFluor647. The clone was stimulated with 10 ∝g/ml and 50 ∝g/ml abc for 4 h and 50 h, respectively. (A) Abc-specific stimulation resulted in a prominent shift in the annV fluorescence intensity, demonstrating the TCC activation after 4 h. However, annV fluorescence intensity decreased to background levels 50 h post activation. This finding demonstrates the reversibility of the annV+ state. (B) The clone remained propidium iodide negative under the applied stimulatory conditions, revealing that annV-positivity is due to TCC activation and not due to the induction of apoptosis. Shown are representative data of three independent experiments.

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