PD-L1 negatively regulates CD4+CD25+Foxp3+ Tregs by limiting STAT-5 phosphorylation in patients chronically infected with HCV
Debora Franceschini, … , Mario U. Mondelli, Vincenzo Barnaba
Debora Franceschini, … , Mario U. Mondelli, Vincenzo Barnaba
Published February 23, 2009
Citation Information: J Clin Invest. 2009;119(3):551-564. https://doi.org/10.1172/JCI36604.
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Research Article

PD-L1 negatively regulates CD4+CD25+Foxp3+ Tregs by limiting STAT-5 phosphorylation in patients chronically infected with HCV

Abstract

CD4+CD25+Foxp3+ Tregs suppress autoimmune responses. In addition, they limit T cell responses during chronic infection, thereby minimizing T cell–dependent immunopathology. We sought to investigate how Tregs are regulated in the livers of patients chronically infected with HCV, where they control the balance between an adequate protective immune response and suppression of immunopathology. We found that, despite accumulating and proliferating at sites of infection in the livers of patients chronically infected with HCV, Tregs were relatively less expanded than CD4+CD25+Foxp3– effector T cells. The relative lower expansion of intrahepatic Tregs coincided with their upregulation of programmed death–1 (PD-1). PD-1 expression inversely correlated with both Treg proliferation and clinical markers of immune suppression in vivo. Consistent with the possibility that PD-1 controls Tregs, blockade of the interaction between PD-1 and programmed death–1 ligand 1 (PD-L1) enhanced the in vitro expansion and function of Tregs isolated from the livers of patients chronically infected with HCV. Blockade of the interaction between PD-L1 and B7.1 also improved the proliferation of these cells. Interestingly, both PD-1 and phosphorylated STAT-5 were overexpressed in intrahepatic Tregs in a parallel fashion in steady disease conditions, and in an alternate-fluctuating fashion during the course of severe hepatitis reactivation. Notably, PD-L1 blockade upregulated STAT-5 phosphorylation in Tregs ex vivo. These data suggest that PD-L1 negatively regulates Tregs at sites of chronic inflammation by controlling STAT-5 phosphorylation.

Authors

Debora Franceschini, Marino Paroli, Vittorio Francavilla, Melissa Videtta, Stefania Morrone, Giancarlo Labbadia, Antonella Cerino, Mario U. Mondelli, Vincenzo Barnaba

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Figure 6

PD-L1 blockade enhances IL-2–dependent proliferation of HCV-specific Tregs.

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PD-L1 blockade enhances IL-2–dependent proliferation of HCV-specific Tre...
(A) Single representative of 5 experiments, in which purified CFSE-labeled CD4+CD25+ Tregs from an HCV patient were stimulated with APCs alone or HCV-Ags/APCs and cocultured in a transwell plate system (denoted by lines) with Tresps that were stimulated or not with anti-CD3/CD28 in the presence or absence of anti–IL-2 mAb. In some cocultures, anti–HLA class I or –HLA class II mAb was added to the wells containing antigen-stimulated Tregs. After 6 d, cells were stained with mAbs to CD4, CD25, and Foxp3. Dot plot analyses are gated on CD4+CD25+ cells and show cells stained with both CFSE and anti-Foxp3. The percentage of cells is indicated in each quadrant. (B) Single representative of all the experiments shown in C, in which CFSE-labeled Tregs from an HCV patient were stimulated with APCs alone or HCV-Ags/APCs in the presence or absence (isotype control) of anti–PD-L1 mAb and/or Tresps. After 6 d, cells were stained with mAbs to CD4, CD25, and Foxp3. Dot plot analyses are gated on CD4+CD25+ cells and show cells stained with both CFSE and anti-Foxp3. The percentage of cells is indicated in each quadrant. (C) Percentage of CFSE-labeled Foxp3+ cells in Tregs upon antigen stimulation in the presence or absence of anti–PD-L1 and/or Tresps. Statistical analyses were performed with nonparametric Mann-Whitney U test for paired data. **P < 0.0040. Each symbol represents a single individual.