Search

Regulatory T (Treg) cells act as terminators of T cell immuniy during acute phase of viral infection; however, their role and suppressive mechanism in chronic viral infection are not completely understood. In this study, we compared the phenotype and function of Treg cells during acute or chronic infection with lymphocytic choriomeningitis virus. Chronic infection, unlike acute infection, led to a large expansion of Treg cells and their upregulation of programmed death-1 (PD-1). Treg cells from chronically infected mice (chronic Treg cells) displayed greater suppressive capacity for inhibiting both CD8+ and CD4+ T cell proliferation and subsequent cytokine production than those from naive or acutely infected mice. A contact between Treg and CD8+ T cells was necessary for the potent suppression of CD8+ T cell immune response. More importantly, the suppression required cell-specific expression and interaction of PD-1 on chronic Treg cells and PD-1 ligand on CD8+ T cells. Our study defines PD-1 upregulated on Treg cells and its interaction with PD-1 ligand on effector T cells as one cause for the potent T cell suppression and proposes the role of PD-1 on Treg cells, in addition to that on exhausted T cells, during chronic viral infection.

Antigenic and costimulatory signals trigger a developmental program by which naive CD8 T cells differentiate into effector and memory cells. However, initial cytokine signals that regulate the generation of effector and memory CD8 T cells are not well understood. In this study, we show that IL-12 priming during in vitro antigenic stimulation results in the significant increase of both primary and memory CD8 T cell population in mice after adoptive transfer of activated cells. The effect of IL-12 priming is closely associated with qualitative changes in CD8 T cells, such as reduced MHC I tetramer binding and CD69 expression, altered distribution of lipid rafts, decreased cytolytic activity, and less susceptibility to apoptosis. Furthermore, exogenous IL-12 priming improved the intrinsic survival properties of memory CD8 T cells, leading to better protective immunity and vaccine-induced memory CD8 T cell responses. However, the experiments with IL-12p40- and IL-12Rβ1-deficient mice showed similar levels of primary and memory CD8 T cell responses compared with wild-type mice, implying that endogenous IL-12 and/or IL-12R signaling in vivo is not critical for CD8 T cell immunity. Together, our results suggest that IL-12 can serve as an important, but dispensable regulatory factor for the development of CD8 T cells, and IL-12 priming could be useful in many medical applications.

IL-12p70 plays a pivotal role in regulating the Th1/Th2 balance in the initial stage of immune responses. In contrast, IL-12p40, which is produced excess over IL-12p70, has been known to down-regulate IL-12p70-mediated responses by acting as an antagonist. To investigate in vivo function of IL-12p40, RH7777 rat hepatoma cells were engineered to inducibly express mouse IL-12p40 under the tight control of doxycycline (dox). In the absence of dox, s.c. injection of these cells into syngeneic rat was shown to generate tumors. However, the induction of IL-12p40 by dox was sufficient for inhibiting tumor formation, as well as for tumor regression. Immunohistochemical analysis showed that macrophages, but not CD4+ T, CD8+ T, and NK cells, were predominantly recruited into tumor sites as early as 3 days after IL-12p40 induction. These results were further supported by the observation that IL-12p40, but not C-terminal deletion mutants by more than 5 amino acids, was able to chemoattract peritoneal macrophages in vitro, suggesting that IL-12p40, when produced in a large excess over IL-12p70 in vivo, can initially amplify the immune responses against tumors by directly recruiting macrophages. Our findings indicate that IL-12p40 may function as an effector molecule as well as an antagonist of IL-12p70.

Regulatory T (Treg) cells act as terminators of T cell immunity during acute phase of viral infection; however, their role and suppressive mechanism in chronic viral infection are not completely understood. In this study, we compared the phenotype and function of Treg cells during acute or chronic infection with lymphocytic choriomeningitis virus. Chronic infection, unlike acute infection, led to a large expansion of Treg cells and their upregulation of programmed death-1 (PD-1). Treg cells from chronically infected mice (chronic Treg cells) displayed greater suppressive capacity for inhibiting both CD8+ and CD4+ T cell proliferation and subsequent cytokine production than those from naive or acutely infected mice. A contact between Treg and CD8+ T cells was necessary for the potent suppression of CD8+ T cell immune response. More importantly, the suppression required cell-specific expression and interaction of PD-1 on chronic Treg cells and PD-1 ligand on CD8+ T cells. Our study defines PD-1 upregulated on Treg cells and its interaction with PD-1 ligand on effector T cells as one cause for the potent T cell suppression and proposes the role of PD-1 on Treg cells, in addition to that on exhausted T cells, during chronic viral infection.

Progressive loss of antiviral functions in antigen-specific CD8 T cell during chronic infection is caused by continuous upregulation and signaling by immunoinhibitory receptors including Programmed Death 1(PD-1). Using human and murine model systems of acute and chronic viral infections, we found that differentiation of naïve CD8 T cells into effector cells during acute infection was accompanied by a transient loss of DNA methylation of the PD-1 locus. Viral clearance and the subsequent effector to memory differentiation resulted in memory-specific DNA remethylation of the PD-1 locus. Conversely, chronic infection induced complete and sustained DNA demethylation of the PD-1 regulatory region in exhausted CD8 T cells. Surprisingly, reduction of virus levels and PD-1 expression in chronically infected animals did not result in remethylation and coincided with decreased expression of a specific de novo methyltransferase (Dnmt3a) isoform. The unmethylated PD-1 locus allowed for rapid PD-1 re-expression, potentially prematurely terminating antiviral functions. These studies provide a foundation for efforts towards reprogramming of PD-1 expression to rejuvenate exhausted T cells.

Blocking PD-1 signaling, by administering anti-PD-L1 or anti-PD-1 antibodies, has proven to be effective in partially restoring CD8+ T cell function during multiple chronic infection models. However since PD-L1 blockade does not completely rescue CD8+ T cell function, we have focused on finding treatments that synergize with PD-1 blockade. Our lab has previously shown that IL-2 administration can increase the frequency of LCMV-specific CD8+ T cells and decrease viral load. Using a chronic model of lymphocytic choriomeningitis virus (LCMV) we demonstrate that combined IL-2 administration and PD-L1 blockade induces massive increases in LCMV-specific CD8+ T cells and an increased ability to produce inflammatory cytokines to multiple epitopes, including subdominant epitopes, over PD-L1 blockade alone. Furthermore, combined treatment results in greater reduction of viral loads. Importantly, IL-7Rα (CD127), a marker of functional memory cells that is not seen on exhausted cells, even after anti-PD-L1 treatment, was up-regulated on a population of LCMV-specific CD8+ T cells after IL-2 or, even more drastically, after combined treatment. These results indicate that combined IL-2 and PD-1 blockade may prove a potential therapy for increasing CD8+ T cell responses and viral control in human chronic infections.

The PDL-1:PD-1 pathway is involved in T cell exhaustion after chronic viral infection. PD-1 can bind to either PDL-1 or PDL-2, whereas PDL-1 can bind to either PD-1 or B7.1. We tested the effect of combining standard aPDL-1 blockade with a4-1BB (3H3 agonistic antibody) treatment to determine T cell restoration effects in a model of chronic infection with lymphocytic choriomeningitis virus (LCMV). We chose 4-1BB because it has been shown to be an important costimulatory pathway. It appears that a4-1BB treatment enhances the effect of aPDL-1 blockade if given in a low dose (50 μg, once). Conversely, if a4-1BB is given in a high dose (200 μg, five times), it results in sudden increase of antigen-specific T cells by day 7, but massive T cell death by day 14 post treatment. These results show the importance for 4-1BB costimulation in aPDL-1 mediated T cell restoration.

Understanding the molecular basis of ineffective T cell response during chronic viral infections may provide new therapeutic targets. Our studies in lymphocytic choriomeningitis virus (LCMV) infection provided the first indication that programmed death-1 (PD-1) pathway regulate T cell dysfunction during chronic infection. These findings were extended to HIV, SIV, HBV and HCV infections and suggest that in vivo blockade of PD-1 pathway may provide a therapeutic approach for human chronic infections. However, further studies are needed to determine how best to modulate PD-1 signaling to activate anti-viral T cells while minimizing immunopathology. Understanding these signaling effects would be greatly advanced by direct imaging of PD-1 in relation to signaling structures in the immunological synapse. Using LCMV model, we investigated PD-1 localization at the T:DC synapse by direct imaging of PD-1 in relation to T cell receptor by confocal microscopy. We compared P14 (LCMV gp33-specific) effector and exhausted cells from mice infected with LCMV Armstrong and clone 13 strains, respectively. Here we show that PD-1:TCR co-localization at the T:DC synapse is different between exhausted and effector CD8 T cells. Our study provides important informations on molecular mechanisms behind T cell exhaustion and may help to identify novel targets to modify PD-1 signaling for the treatment of human chronic infections.