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. Author manuscript; available in PMC: 2015 Dec 18.
Published in final edited form as: Immunity. 2014 Dec 8;41(6):1001–1012. doi: 10.1016/j.immuni.2014.12.011

Figure 2. Caspase-8 activity is upregulated in HIV-specific CD8+ T cells and is associated with disease progression.

Figure 2

(A) Real-time qPCR measurement of caspase-8 expression relative to GAPDH expression from sorted peptide-stimulated HIV-specific Tetramer+ CD8+ T cells from ECs (n = 7) and CPs (n = 7). Filled symbols represent HLA-B*2705 KK10 tetramer+ responses and open symbols represent HLA-B*5701 KF11 tetramer+ responses. Relative expression was calculated using the 2−ΔΔ Ct method (Livak et al., 2001). Statistical analysis was made using the Mann-Whitney test. (B) Representative caspase-8 mean fluorescence intensity (MFI) values of KK10-specific CD8+ T cells in three HLA-B*2705 HIV+ patients with diverse viral loads. (C) Relative mean fluorescence intensity (MFI) of caspase-8 activity on HIV-specific CD8+ T cells from ECs (n = 9), VC (n = 7) and CPs (n = 15). Filled symbols represent HLA-B*2705 KK10-specific response, open symbols represent HLA-B*5701 KF11-specific responses and gray symbols represent non-HLA B*2705/non-HLA B*5701 responses. Horizontal bars indicate mean MFI of caspase-8 activity. Kruskal-Wallis test was used for comparison among all groups of subjects; the Dunns post-test was used for comparisons between groups. (D) Positive correlation between MFI of caspase-8 activity of HIV-specific CD8+ T cells and viral load (n = 31). (E) Negative correlation between MFI of caspase-8 activity of HIV-specific CD8+ T cells and CD4+ T cell count (n = 31). (F) Caspase-8 activity decreases in HIV-specific CD8+ T cells following initiation of HAART (n = 9). Statistical comparisons were made using the Wilcoxon matched pairs test. (G) Positive correlation between MFI of caspase-8 activity and MFI of PD-1 expression on HIV-specific CD8+ T cells (n = 21). (H) Mean fluorescence intensity of caspase-8 activity of A*0201 SL9-specific CD8+ T cells compared with A*0201 CMV pp65 NV9-specific CD8+ T cells and total CD8+ T cells in patients naïve from anti-retroviral therapy (n = 18), and total CD8+ T cells from A*0201 HIV-seronegative controls (n = 9). Repeated measures analysis of variance was used for comparison CMV Tet+, HIV Tet+ and HIV Total CD8+ given that these were paired observations. Mann-Whitney test was used for comparison of HIV+ and HIV− Total CD8+. (I) Negative correlation between MFI of caspase-8 activity of HIV-specific CD8+ T cells and percentage of CFSE lo Tetramer+ cells following peptide stimulation (n = 13). Correlation statistics for D, E, G and I were calculated using the Spearman correlation. (J) Representative CCR7 and CD45RA staining of total CD8+ T cells (gray) and HIV tetramer+ CD8+ T cells (black) from an HIV+ CP to identify naïve, central memory (Tcm), effector memory (Tem) and terminal effector (Temra) cells. (K) Representative FACS plot of KK10 tetramer+ CD8+ T cells identifying caspase-8 negative (Casp8-; red) and positive (Casp8+; blue) cells. (L, M, N) Representative histograms of CD45RA, CCR7 and CD27 staining of KK10 tetramer+ Casp8− CD8+ T cells (red), KK10 tetramer+ Casp8+ CD8+ T cells (blue) and total CD8+ T cells (gray). (O) Summary of phenotypic data for HIV tetramer+ Casp8− and Casp8+ CD8+ T cells analyzed for CD45RA, CCR7 and CD27 (n = 10). Horizontal bars indicate mean percentage of HIV tetramer+ Casp8− and Casp8+ CD8+ T cells that were positive for the indicated marker. Statistical comparisons were made by the Wilcoxon matched pairs test.