Figure 3.

ARG2-specific CD8⁺ T cells recognize A2S05 in the context of HLA-B8. (A) Generation of A2S05-specific T-cell cultures. ARG2-specific CD8⁺ T cells were expanded from three HDs and one patient with cancer (AA01, melanoma). The specificity of the T-cell cultures against A2S05 was assessed by intracellular cytokine staining for TNF-α and IFN-γ. CD107a was included as a marker of cytotoxicity. Bars show the percentage of CD8⁺ T cells expressing CD107a and secreting IFN-γ, TNF-α or both (DP) in response to Ctrl stimulation or A2S05 stimulation (pep). (B–D) ARG2-specific T cells from two donors (HD78 and HD93) were evaluated in IFN-γ ELISPOT with cancer cells lines prepulsed with A2S05 peptide. The same cell lines without peptide stimulation were included as Ctrls. 3×10⁴ ARG2-specific T cells were plated with 1×10⁴ cancer cells (E:T of 3:1). The cell lines were either HLA-A1⁺ (B), HLA-C7⁺, (C) or HLA-B8⁺ (D). T cells plated alone (−) or T cells plated with A2S05 (+pep) served as negative and positive Ctrls, respectively. Bars represent the mean IFN-γ spot count±SD of technical triplicates. (E) ⁵¹Cr-release assay of FM6 and FM6 stimulated with IFN-γ (100 U/mL) for 24 hours prior to the assay with A2S05-specific T cells from HD78. Error bars represent mean±SD of technical duplicates. Ctrl, control; DP, double positive; E:T, effector-totarget ratio; -HD, healthy donor; IFN-γ, interferon gamma; TNF-α, tumor necrosis factor alpha.