Differences in cellular maturation between MTB- and CMV-specific CD4 T cells are diminished during active TB disease and are associated with diametrically opposed production of IL-2 and MIP-1β. (A) Surface expression of the maturation markers CD27, CD45RO, and CD57 on total CD4 T cells (black) is shown for representative subjects as a density plot overlay. MTB-specific CD4 T cells in the absence (blue, HIV⁻) or presence (green, HIV⁺) of active TB disease and for CMV-specific CD4 T cells (red). The percentage of CD27⁺CD45RO⁺, CD27⁻CD45RO⁺, and CD57⁺ subsets within MTB- and CMV-specific CD4 T cells from HIV⁻ subjects (blue, n = 17) and HIV⁺ subjects with active TB (green, n = 11; bottom). (B) Flow cytometric analysis of IFN-γ, IL-2, MIP-1β, and TNF production within pathogen-specific CD4 T cells. PBMCs from subjects with latent MTB infection (defined by positive response to region of difference 1 [RD1] antigens) or from HIV⁺ subjects with active TB were stimulated with MTB-antigens (PPD or a mix of PPD and RD1 peptide pools) or CMV whole antigen. The pie charts show the fraction of cells with 1, 2, 3, or 4 functions. The color-coded circles indicate the proportion of the 4, 3, 2, and 1 functional responses response that are contributed by the single cytokines IFN-γ (red), IL-2 (blue), Mip-1β (green), and TNF (pink). The fraction of cells that produce MIP-1β (right top) or IL-2 (bottom right) is shown as percentage of total cytokine-positive CD4 T cells. The median is indicated. Further delineation of the 16 different possible cytokine combinations is shown (bottom). (C) Intracellular staining of IL-2 (y axis) and MIP-1β (x axis) within CD27⁺CD45RO⁺, CD27⁻CD45RO⁺CD57⁻, and CD27⁻CD45RO⁺CD57⁺ CD4 T cells after stimulation with PPD or CMV is shown for one representative subject (left) and for all studied responses further delineated by IL-2 and MIP-1β production and presented as percentage of total cytokine-positive response (right). Statistical analysis was performed using the Mann-Whitney test (***, P < 0.0005; **, P < 0.005; *, P < 0.05).