Stimulation with abacavir and selected peptides can enhance the allogenicity of the induced TCL.

<p>In order to generate TCL, PBMC from donors ID-207 (A) and ID-145 (B) were stimulated in the presence of abacavir (10 µg/ml) and the mentioned peptide (10 µg/ml). TCLs were re-challenged with autologous PHA-blasts (Autol.), autologous PHA-blasts pulsed with abacavir (Autol^abc), or PHA-blasts pulsed with abacavir and the mentioned peptide (Autol^abc/xx9), or 721.221 cells expressing HLA-B*58∶01 (B*58∶01), 721.221 cells expressing HLA-B*58∶01 and presenting the mentioned peptide (B*58∶01^XX9), or 721.221 cells expressing HLA-B*57∶01 (B*57∶01). Reactivity was monitored by means of CD107a up-regulation on flow cytometry after 6 hours of stimulation. C. TCL SI9+ abc from ID-145 was cloned by limiting dilution. The table shows the number of generated TCC with reactivity to abacavir and cross-reactivity to HLA-B*58∶01. D. Calcium influx was measured by fluorescence measurement of the Fluo4-AM calcium sensitive dye. TCC B10 from ID-145 was stimulated after 300 seconds of baseline measurement (black arrow) with HLA-B*57∶01 expressing 721.211 cells (grey line), or HLA-B*57∶01 expressing 721.221 cells pulsed with abacavir (red line), or with 721.221 cells expressing HLA-B*58∶01 (orange line). Alternatively, after 600 seconds of baseline measurement (blue arrow) abacavir in solution (50 µg/ml) was added to the TCC in the presence of HLA-B*57∶01 expressing 721.221 cells (blue line). Representative data of two independent experiments are shown.</p

Abacavir-reacting T cells are found in the naïve and memory CD8⁺ T cell pools.

<p>CD8⁺ T cells from two HLA-B*57∶01⁺ donors were isolated by magnetic sorting according to their expression of memory (CD45RO) and naïve (CD45RA, CCR7) markers. (A) Negatively selected memory and (B) naïve CD8⁺ T cells were stimulated with abacavir (10 µg/ml). After 14 days, cells were re-challenged with abacavir (10 µg/ml) and reactivity was monitored by flow cytometry. Plots show CD3⁺ T cells and percentages relate to the CD8⁺ CD107a⁺ T cell population. Representative data from two independent experiments of ID-576 are shown.</p

Peptide candidates possibly involved in HLA-B*58∶01 allogenicity.

<p>Peptide sequences were selected from the publication of Norcross et al, 2012 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095339#pone.0095339-Norcross1" target="_blank">[8]</a>. Peptides were considered only if they were eluted from cells treated with abacavir and were absent from abacavir untreated cells. IC50 values for HLA-B*57∶01 and HLA-B*58∶01 were calculated using the netMHC pan server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095339#pone.0095339-Hoof1" target="_blank">[12]</a>.</p>§<p>Peptide with Trp, Phe or Tyr at their C-term were excluded from the candidate list.</p>†<p>Name was given arbitrary with the letter of the first and the last residue of the peptide, followed by the number of amino acids.</p>‡<p>The ratio value corresponds to the IC50 for HLA-B*58∶01 divided by IC50 for HLA-B*57∶01. It may point to a difference of peptide affinity to both allotypes.</p

Molecular modelling of HLA-peptide complexes.

<p>A. HLA B*57∶01 peptide binding cleft with bound abacavir (orange) is shown empty (upper panel) or filled with the SI9 peptide (lower panel). B. The position of Arg97 is highlighted in yellow on the HLA-B*58∶01 molecule without (upper panel) or with the anchored SI9 peptide (lower panel). C An overlay of the {HLA-B*57∶01+abc} complex (turquoise) and HLA-B*58∶01 (purple) with modelled SI9 peptide is shown. SI9 highlighted in orange is bound to {HLA-B*57∶01+abc} and SI9 highlighted in purple is bound to HLA-B*58∶01. Abacavir and Arg97 found in the F9 pocket are highlighted in orange and yellow, respectively. The 3D conformation of SI9 is very similar between {HLA-B*57∶01+abc} and HLA-B*58∶01.</p

Abacavir-reacting CD8⁺ T cells are detectable after 13 days of <i>in vitro</i> culture and are observed in 100% of the tested HLA-B*57∶01⁺ individuals.

<p>A. PBMC from healthy donors (HD) were cultured <i>in vitro</i> with abacavir (10 µg/ml) for 14 days as explained in materials and methods. Reactivity was monitored after a drug-specific <i>in vitro</i> restimulation assay by flow cytometry. CD107a served as marker for T cell reactivity. Representative data from ID-207 are shown as mean ± SD. Experiment was performed in duplicates. B. PBMC from HLA-B*57∶01⁺ HD (n = 13), HLA-B*57∶01⁻ HD (n = 8) and HLA-B*57∶01⁺ HIV⁺ patients (n = 7) were induced with abacavir (10 µg/ml) for 14 days <i>in vitro</i>. T cell reactivity was monitored by means of IFNγ secretion after a drug-specific restimulation. p = 1.00, two tailed Mann-Whitney test.</p

Allo-reactivity of abacavir-reacting T cell clones.

<p>Abacavir-reacting TCC from three individuals were tested for allo-reactivity against a panel of EBV-BLCL from 22 HD (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095339#pone-0095339-t001" target="_blank">table 1</a>) in a 16 hour IFNγ ELISpot assay. A. An example of a TCC from ID-635 without allo-reactivity is shown. The experiment was performed in triplicates and data are shown as mean ± SD. B. TCC 3L from ID-635 shows allo-reactivity against EBV-BLCL from donor ID-601. The experiment was performed in triplicates and data are shown as mean ± SD C. Allo-reactivity of TCC ID-635 3L was confirmed by IFNγ ELISpot. TCC was stimulated with autologous EBV-BLCL (first line), abacavir-pulsed autologous EBV-BLCL (second line), EBV-BLCL from donor ID-601 (third line) and 721.221 cells expressing HLA-B*58∶01 (forth line). (D) TCC allo-reactive against HLA-B*58∶01 (upper panel) or without allo-reactivity to HLA-B*58∶01 (lower panel) were stimulated with 721.221 cells expressing HLA-B*57∶01 in the absence (left) or in the presence of abacavir (middle), or with 721.221 cells expressing HLA-B*58∶01 (right). After four hours of stimulation TCC were analyzed for CD107a up-regulation and IFNγ secretion by flow cytometry. Plots show gated CD3⁺ CD8⁺ T cells. Representative data of three independent experiments are shown.</p

Summary of HLA-B*58∶01 allo-reacting TCC from abacavir-reacting CD8⁺ TCC.

<p>Summary of HLA-B*58∶01 allo-reacting TCC from abacavir-reacting CD8⁺ TCC.</p

Abacavir-reacting T cells can be induced <i>in vitro</i> in the absence of APC.

<p>PBMC, CD3⁺ sorted T cells, and 14 days old PHA-blast from ID-453 were cultured in the presence of abacavir (10 µg/ml). Drug reactivity was monitored on day 14 in a degranulation (CD107a) assay after a six hours drug-specific restimulation assay. Plots are gated on CD3⁺ T cells and percentages relate to the CD8⁺ CD107a⁺ T cell population. Representative data from three independent experiments are shown.</p

Abacavir does not induce DC maturation.

<p><i>In vitro</i> generated DC were incubated with increasing concentrations of abacavir or NiSO₄ (250 mM) for 24 hours. A. DC were harvested and the expression of co-stimulatory molecules was analyzed by flow cytometry. Data are shown as mean fluorescence intensity and represent mean ± S.E.M. from DC of 4 individuals. Experiments were performed in triplicates. B. Cell culture supernatants were analyzed for cytokines by multiplex analysis. Data represent mean ± S.E.M. from DC of 4 individuals. Experiments were performed in triplicates.</p