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. 2014 Jun 1;30(6):535–541. doi: 10.1089/aid.2012.0195

FIG. 1.

FIG. 1.

Lymphocyte activation gene-3 (LAG-3+) expression on HIV-specific CD8+ T cells does not express CD107a or produce interferon (IFN)-γ. (A) Peripheral blood mononuclear cells (PBMCs) were stimulated for 6 h with HIV antigens Gag B, Nef B, or CMV pp65 in the presence of anti-CD107a PE and Golgi-Stop or (B) restimulated following an initial 24-h stimulation, as described in Materials and Methods, then stained with Vivid viability dye for dead/live cell exclusion, anti-CD3 AmCyan, anti-CD4 PerCP Cy5.5, anti-CD8 APC-Cy7, anti-LAG-3 FITC, and IFN-γ APC, and analyzed by flow cytometry. Samples were gated on the CD3+/CD8+ lymphocyte population and then the percent of CD107a and IFN-γ-positive CD8+ T cells was determined. (C) Representative plots of the frequency LAG-3 on Gag- or Nef-specific CD8+ T cells and CMV pp65-specific CD8+ T cells expressing CD107a and IFN-γ are shown. Staphylococcus enterotoxin B (SEB) is used as a positive control. No significant overlap between Lag-3 expression and CD107a or IFN-γ was observed. (D) LAG-3+CD8+ T cell expression after antigen peptide stimulation compared to no antigen-stimulated control. Gag B-specific LAG-3+CD8+ T cells were statistically significant compared to Nef B or CMV pp65 (p<0.0001 and p=0.0002, respectively). Results were expressed as the percentage of CD107a or IFN-γ-positive CD8+ T cells (percent positive=percent Ag-specific – percent negative control). Responses ≥0.1% and two times the background were considered positive. p values <0.05 were considered significant (n=35).