Statistical significance was determined by nonpaired, two-sided Student's t test or chi-squared test, where indicated. (A) Total mononuclear cells were harvested from mice infected with rJ or rJ.S Q600Y and stimulated ex vivo with S598 and S598 Q600Y peptides, respectively. Following stimulation, aliquots of cells were surface stained for CD8 and the indicated Vb chain followed by intracellular cytokine staining for IFN-c. (B) CNSderived cells from rJ.S Q600Y -infected mice were stimulated ex vivo with peptides corresponding to native S598 or S598 Q600Y epitopes. Following stimulation, cells were analyzed as described for (A). Data represent the fraction of IFN-c+CD8+ T cells expressing each Vb chain and are derived from cells pooled from 2-3 individual mice. (C) Alanine scanning of the native S598 determinant. CNS-derived mononuclear cells were recovered from rJ-infected mice 7 days p.i. and stimulated ex vivo in the presence of 500 mM TCEP and 1 mM of the indicated peptide then stained for CD8 and intracellular IFN-c. Data are normalized to the frequency of epitope-specific cells detected when stimulated with the native S598 determinant. (D) Alanine scanning of the S598 Q600Y determinant. Cells were harvested and tested as described for B except in this case the cells originated from the rJ.S Q600Y -infected CNS and were stimulated with 10 nM S598 Q600Y peptide. (E) Alanine scanning of the S598 determinant recognized by S598 Q600Y -primed, cross-reactive CTL. As in C, but cells were stimulated with 150 nM S598 peptide. For B-D, concentrations of peptide equivalent to 106 that required for half maximal stimulation were used; data are mean6SEM from four independent experiments. Note that the differential responses to RCAIFANI in panels C and D reflect the differing amounts of peptide used in the two assays. Found at: doi:10.1371/journal.ppat.1000186.s002 (0.76 MB TIF) Figure S3 Refined structures of WT and Q600Y S598-Aba bound to H-2K b . (A) View of the H-2K b antigen binding cleft from above. The HC is shown as a cartoon representation and coloured slate. The peptide is in stick format with carbon atoms coloured yellow. The unbiased F o -F c map density for the peptide contoured at 2.5 s is shown as a magenta mesh. (B) The same view as in A displaying key interactions (dashed lines) between H-2K b and S598-Aba. Selected residues of the HC are drawn in stick format (slate carbon atoms) and ordered water molecules are shown as red spheres. Peptide residues are labelled in italics. C and D, Equivalencies to A and B, respectively, for the H-2K b / S598 Q600Y -Aba structure. In these panels the HC is drawn in green and the peptide in cyan.  HIV-Specific T-Cells Accumulate in the Liver in HCV/HIV Co-Infection BACKGROUND AND AIMS: Hepatitis C Virus (HCV)-related liver disease progresses more rapidly in individuals co-infected with Human Immunodeficiency Virus-1 (HIV), although the underlying immunologic mechanisms are unknown. We examined whether HIV-specific T-cells are identified in the liver of HCV/HIV co-infected individuals and promote liver inflammation through bystander immune responses. METHODS: Ex-vivo intra-hepatic lymphocytes from HCV mono-infected and HCV/HIV co-infected individuals were assessed for immune responses to HIV and HCV antigens by polychromatic flow cytometry. RESULTS: HCV/HIV liver biopsies had similar frequencies of lymphocytes but lower percentages of CD4(+) T-cells compared to HCV biopsies. In co-infection, intra-hepatic HIV-specific CD8(+) and CD4(+) T-cells producing IFN-γ and TNF-α were detected and were comparable in frequency to those that were HCV-specific. In co-infected individuals, viral-specific CD8(+) T-cells produced more of the fibrogenic cytokine, TNF-α. In both mono- and co-infected individuals, intra-hepatic HCV-specific T-cells were poorly functional compared to HIV-specific T-cells. In co-infection, HAART was not associated with a reconstitution of intra-hepatic CD4(+) T-cells and was associated with reduction in both HIV and HCV-specific intra-hepatic cytokine responses. CONCLUSION: The accumulation of functional HIV-specific T-cells in the liver during HCV/HIV co-infection may represent a bystander role for HIV in inducing faster progression of liver disease. Approximately 25% of Human Immunodeficiency Virus-1 (HIV) infected individuals are also infected with Hepatitis C Virus (HCV) [1] . HIV adversely affects each stage of the natural history of HCV infection. Fewer individuals recover spontaneously from HCV infection when also infected with HIV [2] . Among those with persistent HCV infection, HIV co-infection is associated with higher HCV viremia and more rapid progression to cirrhosis and hepatocellular carcinoma [3] . A recent meta-analysis showed that HIV co-infection increased the risk of histological hepatic cirrhosis by two-fold and clinically decompensated liver disease by six-fold [4] . In addition, HCV co-infection is associated with increased incidence of HAART (highly active antiretroviral therapy) related liver injury [5] .