Ifn γ capture reagent

Interferon-γ (IFN-γ) positive abacavir reactive cells were detected in PBMC from abacavir unexposed donors following stimulation with C1R.B57 APC treated ± abacavir, as described above, for four hours. Cells were then labelled with an IFN-γ capture reagent and IFN-γ was captured for forty-five minutes, according to manufacturer’s directions (Miltenyi Biotec). Cells were then washed and labelled with anti-IFN-γ-PE, anti-CD3-Pacific Blue and anti-CD8-APC-H7 (Becton Dickinson). Cells were either analyzed immediately by flow cytometry or following enrichment of positive cells using anti-PE microbeads and magnetic column separation (Miltenyi Biotec) and then analyzed by flow cytometry [19 (link)]. A lymphocyte gate was set using FSC and SSC parameters. CD3+ lymphocytes were then analysed for CD8 and IFN-γ expression. Positive gates were set above the level of the background staining of anti- IFN-γ-PE on unstimulated CD3+ PBMC.

In order to characterize in details neoantigen recognition by neoepitope-specific T-cells, we established neoepitope-specific T-cell clones. For CD8⁺ T-cells, presensitized T-cells that showed neoantigen-specific reactivity in ELISPOT assays were restimulated by mutated peptide-pulsed T-APC and IFN-γ-producing T-cells were labeled using IFN-γ-capture reagent (Miltenyi Biotec) and sorted by flow-cytometry as described [86 (link)]. For CD4⁺ T-cells, presensitized T-cells were similarly restimulated in the presence of monensin (Sigma) and phycoerythrin (PE)-labeled anti-CD154 monoclonal antibody (mAb) as described [87 (link)] and CD154-expressing cells were sorted. Isolated T-cells were polyclonally expanded by PHA stimulation in the presence of allogeneic irradiated PBMC, IL-2 and IL-7. Purity and clonality of T-cells were tested by low-resolution TCR spectratyping using Vβ subtype-specific antibodies (Beckman Coulter). For some oligoclonal T-cell cultures containing different Vβ-expressing T-cells, cells were sorted again based on Vβ expression. Reactivity of neoepitope-specific T-cell clones were tested by ELISA and/or intracellular cytokine staining [88 (link)].