*with HCV. an Paroxetine mesylate expansion of germinal Paroxetine mesylate center Tfr. Notably, expansion was Paroxetine mesylate mediated by TGF–containing exosomes released from HCV-infected hepatocytes as blockade of exosome-associated TGF- or inhibition of exosome release abrogated Tfr expansion. Conclusion These results show that liver-derived exosomes play a pivotal role in the accumulation of Tfr cells, likely leading to suppression of Tfh responses in HCV-infected patients. Our study identifies a novel pathway in which HCV infection in hepatocytes exacerbates Tfr cell responses to subvert antiviral immunity. co-culture system, we demonstrate that Tfr from healthy subjects undergo expansion following exposure to infected hepatocytes. The expansion of Tfr cells was accompanied by the acquisition of an enhanced regulatory phenotype and leads to the functional suppression of Tfh cells. Increases in Tfr responses were driven by a novel pathway involving release of TGF–containing exosomes from HCV-infected hepatocytes. These findings highlight the accumulation of Tfr in the livers of HCV patients, potentially inhibiting protective Tfh and B cell responses at the site of infection, and contributing to viral persistence. Materials and Methods Human Subjects Intrahepatic leukocytes from explanted liver tissue of HCV-infected patients (n=8), non-viral hepatitis patients (n=6; non-alcoholic steatohepatitis, alcoholic liver disease, autoimmune hepatitis), and healthy control subjects (n=7) and sera were provided by Dr. Hugo Rosen (University of Colorado). Briefly, intrahepatic leukocytes were isolated from liver tissues by first dissecting tissues into small fragments and then incubating with collagenase type IV as previously described.(37) Intrahepatic leukocytes were then cryopreserved and shipped from the University of Colorado. All participants of this study provided written informed consent and IRB protocol 06-0566 was approved by the Colorado Multiple Institutional Review Board. Hepatocytes, HCV, and PBMC co-cultures The human hepatoma cell line Huh7.5.1 was maintained in complete DMEM. One day following seeding of hepatocytes, cells were infected with HCV (JFH-1 strain, genotype 2a) at a multiplicity of infection (MOI) of 0.1. JFH-1 was kindly provided by Dr. Wakita (Tokyo Metropolitan Institute). For co-culture, cryopreserved PBMCs previously isolated from the buffy coats of healthy subjects (Virginia Blood Services, Richmond, VA) were re-suspended in complete RPMI and added to uninfected or HCV-infected hepatoma cells on day 4 post-infection or cultured alone for 4 days. In some experiments, tonsillar MNCs were co-cultured with uninfected or infected hepatocytes. Cryopreserved primary human hepatocytes (PHHs; Thermo Fisher Scientific) were cultured with Williams Medium E and Hepatocyte Maintenance Supplement Pack on Collagen I-coated plates according to manufacturer protocols (Thermo Fisher Scientific) and were inoculated with Furin HCV-infected patient sera. PHHs and PBMCs exhibited greater than 80% viability following cryopreservation and during experimentation. Flow cytometry and T cell isolations Cells were stained with Zombie Aqua Fixable Viability dye (Biolegend). For identification of liver Tfr cells, surface staining was performed with the following antibodies: CD45-PerCP (Tonbo;2D1), CD4-APC/Cy7 (Tonbo;RPA-T4), CD14-APC (eBioscience;61D3), CD56-APC (eBioscience; CMSSB), CD11b-APC (Biolegend;ICRF44), CXCR5-BV421 (Biolegend;J252D4), PD-1-PE/Cy7 (Biolegend;EH12.2H7), and CD25-FITC (BD Biosciences;BC96). Following fixation with the Foxp3/Transcription Factor Fixation/Permeabilization Kit (eBioscience), cells were stained with Foxp3-PE (eBioscience;236A/E7). For intracellular cytokine analysis, co-cultures were stimulated with 0.1g/mL PMA and 0.5 g/mL ionomycin (Sigma) in the presence of GolgiPlug (BD Biosciences) for 4-6 hours. Cells were then surface stained with the following antibodies: CD4-APC/Cy7, CXCR5-BV421, and PD-1-PE/Cy7. Following fixation with CytoFix/CytoPerm (BD Bioscience), cells were stained with IFN–FITC Paroxetine mesylate (Biolegend;4S.B3), Paroxetine mesylate IL-21-PE (eBioscience;eBio3A3-N2), or IL-17-PerCPeFluor710 (eBioscience;BL168). Intracellular staining of Tfr cells was performed by staining with Foxp3-APC (eBioscience;236A/E7), IL-10-PE/Cy7 (Biolegend;JES3-9D7), and CTLA-4-PE (eBioscience;14D3). CD4 T cell isolations were performed by depleting CD14+ monocytes using CD14 microbeads (Miltenyi) followed by positive selection with CD4 microbeads (Miltenyi). For depletion or sorting of Tfr, enriched CD4 T cells were stained with CD4-APC/Cy7, CXCR5-BV421, CD25-FITC or CD25-PE (Biolegend;BC96),.