Inhibitory marker coexpression correlates inversely with polyfunctionality. (A) 10 HIV⁺ subjects from Duke University were studied. Representative plots of intracellular cytokine staining combined with SL9/HLA A*0201, NV9/HLA A*0201, TM9/HLA B*0702, TM10/HLA B*0702, or TW10/HLA B*5701 tetramer staining of CD8⁺ T cells are shown. Cells were independently stimulated with individual epitopic peptides for 6 hours. Pooled data lower panels represent the percentage of nonfunctional CD8⁺ T cells with PD-1⁺CD160⁺2B4⁺LAG-3⁻, PD-1⁺CD160⁻2B4⁺LAG-3⁻, and PD-1⁻CD160⁻2B4⁺LAG-3⁻ expression profiles within tetramer⁺ or any cytokine⁺ populations (HIV-specific responses, n = 7; CMV-specific responses, n = 10). (B) The fraction of the total response composing cells expressing each of the 7 possible combinations of IFN-γ, IL-2, and TNF-α production within the PD-1⁺CD160⁺2B4⁺LAG-3⁻, PD-1⁺CD160⁻2B4⁺LAG-3⁻, and PD-1⁻CD160⁻2B4⁺LAG-3⁻ populations. Cells were stimulated with the HIV-1 Gag peptide pool (n = 47) or the CMV pp65 peptide pool (n = 60), respectively. (C) Comparisons between polyfunctionality and the percentage of PD-1⁺CD160⁺2B4⁺LAG-3⁻, PD-1⁺CD160⁻2B4⁺LAG-3⁻, and PD-1⁻CD160⁻2B4⁺LAG-3⁻ expression profiles on HIV-specific CD8⁺ T cells as a function of pVL (n = 47). Lines indicate correlations detected by Prism software Version 5.0c.