Bystander activation by contaminants or non-TCR stimulation has limited impact on upregulation of AIM markers.
(A,B) OX40/CD25 expression of PBMCs cultured in the presence of increasing amounts of LPS (25–2500 EU/ml), with an unstimulated (UN) and SEB condition as negative and positive control. (A) Example plots from a representative donor cultured with or without LPS. (B) Data shown as fold-increase (LPS/UN); n = 3 donors. (C) Experimental setup for transwell plate experiment, where antigen-exposed PBMCs were placed within the top well of a transwell plate, and antigen-naïve PBMCs were placed within the bottom well, with the two wells separated by a 0.4 μm pore-size permeable membrane. The cultures were then left unstimulated (UN) or stimulated with antigen (Ag) for 24 hours at 37°C. (D) Representative flow cytometry plot of the antigen-experienced PBMCs in the top well of the transwell plate (left column), the antigen-naïve PBMC in the bottom well of the transwell plate (middle column), and the antigen-naïve PBMC control in a separate plate (right column) after 24 hours in culture; with the unstimulated condition (UN) on the top row and antigen stimulated (Ag) on the bottom row. Gated on total CD4 T cells. (E) Quantification of the OX40+CD25+ signal for the three culture conditions, unstimulated (UN) or stimulated with antigen (Ag). n = 3 antigen-naïve donors. (F) Diagram of the CD4 T cell line bystander activation experiment from panels G-K. Briefly, cell tracer-labeled (CellVue+) PBMCs were cocultured with an autologous CD4 T cell line at different ratios and stimulated with peptide antigens for 18 hr. (G,H) Example flow plots show the frequency of antigen-specific CD4 T cells CellVue+ PBMCs in the top row compared to the total frequency of antigen-specific cells within the coculture (CD4 T cell line + CellVue+ PBMCs) in the bottom row, at indicated ratios of CD4 T cell line and CellVue+ PBMCs. Antigen-specific cells are identified as CD69+CD40L+ (G) or OX40+CD25+ (H) respectively. (I,J) Quantification of data shown in (G,H) for CD69/CD40L (I) and OX40/CD25 expression (J) respectively. The total frequency of antigen-specific cells in the culture (CD4 T cell line + CellVue+ PBMCs) is indicated by grey bars; the frequency of CellVue+ autologous CD4 T cells from PBMCs is indicated by the colored dots/lines. (K) Data shown as a relative change in the percentage of antigen-specific CD4 T cells from the CellVue+ PBMCs at each ratio, compared to the PBMC alone condition. n = 3 independent experiments and donors, with HIV-Gag or hCMV pp65 peptide stimulation. Bars represent mean +/- SEM, dotted line at 100% (i.e. no change).
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