B7/CD28 family molecules play a central role in the generation and modulation of the adaptive T cell immune response. A balance of costimulatory and coinhibitory signaling governs the activation and function of the responding T cells (reviewed in (21
)). Classically, it is the B7 ligand expressed on the antigen presenting cell signaling to the CD28 family receptor on the T cell that directs the T cell response. However, a number of studies have also identified B7 ligand expression on T cells (16
), and linked its expression to an enhancement of the T cell response (16
). Expression of CD80 on a human CD4+ T cell clone enhanced a mixed lymphocyte reaction (MLR) to resting peripheral blood responder T cells (16
) and expression of CD86 on anti-CD3 stimulated and paraformaldehyde fixed human T cells enhanced interferon-gamma production and proliferation of naïve CD4+ T cells responding to suboptimal concentrations of anti-CD3 (18
). Furthermore, fixed CD86+ but not CD86- T cells induced an MLR response that was partly decreased by neutralizing anti-CD86 monoclonal antibodies (18
). Despite these studies, the importance of B7 ligand expression on virus specific CD8+ T cells is not known.
In the acute phase of infection, the inhibitory receptor, PD-1, is often highly expressed on HCV specific CD8+ T cells (7
), so we hypothesized that other costimulatory signals are important at this early phase of infection to enable an effective immune response. In the current study, we demonstrate that the B7 ligand, CD86, is highly expressed on HCV specific CD8+ T cells during the early acute phase of infection and not during the later phase of acute infection or during chronic infection even at the site of infection in the liver. Significant CD86 expression on HCV specific CD8+ T cells was not detected at any later time points for all of the acutely infected patients developing chronic infection nor for any other chronically infected patients that we evaluated. For some patients in the acute phase of HCV infection, HCV specific CD8+ T cells also expressed CD80, though expression was not seen in other patients with acute infection. Currently, why CD80 is expressed on HCV specific CD8+ T cells from some patients with acute infection but not others is not well understood. We hypothesize that this may be related to differing kinetics of CD86 and CD80 expression or to different levels of signaling required for expression of CD86 and CD80.
In this study, we investigated the significance of B7 ligand expression on T cells and found that high-level expression was delayed after brief in vitro culture (5-7 days) and was linked with recent common gamma-chain cytokine signaling. This was in contrast with other “activation markers” such as CD69, CD38, HLA-DR or CD25 whose expression could rapidly (1-3 days) be induced by TCR stimulation alone after brief in vitro culture of PBMC. Hence, our findings support the hypothesis that B7 ligand expression on T cells is a unique marker that identifies recent stimulation via TCR in the presence of sufficient supportive cytokine. The lack of B7 ligand expression on liver infiltrating HCV specific CD8+ T cells in chronic infection, despite high-level expression of activation markers, highlights a critical deficit in supportive cytokine signaling that contributes to the waning immune response to HCV. Recent studies in mice demonstrate IL-7 can be produced by hepatocytes themselves and that IL-7 is important in regulating the expansion of T cells in response to LPS (39
). Though hepatocyte IL-7 was not found to be important in pathogen-specific CD8+ T cell proliferation in this study (39
), improved understanding of the cytokine milieu in the liver of patients with hepatotropic viral infection is clearly important.
IL-2, in particular, has been shown to be important in the generation of effective immune responses to HCV infection, and secretion of IL-2 by CD4+ T cells during the acute phase infection is critical for sustained and effective adaptive CD8+ T cell responses (38
). CD4+ T cells from patients with self-limited evolution of infection produced considerably more IL-2 in response to HCV recombinant proteins compared with patients with chronically evolving disease (38
). In the chimpanzee model of HCV infection, depletion of CD4+ T cells prior to infection led to an inability to clear viremia (42
). On progression to chronic HCV infection, a preferential loss of IL-2 secreting CD4+ T cells has been noted (43
), and HCV specific CD8+ T cells from the peripheral blood of patients with chronic infection have an impaired ability to proliferate that can be rescued in vitro by exposure to IL-2 (44
). Our study further supports the critical loss of IL-2 during progression to chronic infection and identifies ex vivo pSTAT5 signaling and expression of B7 ligands (CD86 and CD80) as important markers of recent effective signaling. Though our study highlights the role of common gamma-chain cytokines such as IL-2 in the expression of CD86 on T cells, future studies will need to investigate the role of other inflammatory cytokines in the expression of CD86 or CD80 on T cells during HCV infection. Furthermore, determining whether the level of CD86 expression or the timing of CD86 expression is a determinant of viral clearance versus persistence will require longitudinal studies with larger numbers of acute patients.
Studies of liver infiltrating HCV specific CD8+ T cells are critical to understand the failure of the immune response seen in most patients with HCV infection. Previous studies have demonstrated high activation state of these cells in the liver but poor functionality in the chronic phase of infection (7
). A number of factors likely contribute to the waning immune response and include high-level expression of PD-1 (11
), infiltration by Tregs ((45
) and reviewed by (46
)), and a loss of CD4+ T cell help (43
). We hypothesize that a central feature of each of these mechanisms is the loss of IL-2 signaling on HCV specific CD8+ T cells. Recent studies on the mechanism of action of PD-1 signaling indicate the possibility that PD-1 signaling might directly prevent STAT5 phosphorylation via activation of the SHP-2 phosphatase (45
). In addition, one of the proposed mechanisms of action of Tregs is to act as an “IL-2-sink” and depleting the immunological milieu of supportive cytokine (48
). Our study is the first characterize pSTAT5 on virus specific CD8+ T cells using tetramers. Our findings indicate an early loss of pSTAT5 that occurs in the acute phase of infection and a lack of high-level pSTAT5 in liver infiltrating HCV specific CD8+ T cells despite persistent infection and persistent activation. Future studies will need to determine the relative contribution of PD-1 signaling, Treg infiltration and CD4+ T helper cell loss in the reduction of pSTAT5 in HCV specific CD8+ T cells.
Clearly, differences in the acute versus chronic immune response are evident in HCV infection and this can be seen in the differing clinical responses and degree of liver injury as measured by ALT in the patients infected with HCV. Based on our study, we hypothesize that other B7 molecules, and in particular, CD86, that are expressed at this early phase of infection provide costimulatory signals via T:T interactions, that enhance the immune response at this early stage of infection. In this study, we assessed for the ability of HCV specific CD8+ T cells expressing CD86 to function as antigen presenting cells in a T:T dependent manner by sorting on CD3+CD8+ T cells from fresh PBMC from a808 (day 0) and culturing in the presence of HCV peptide, HCV peptide plus IL-2, and HCV peptide plus anti-CD86 with/without IL-2 (data not shown). We were unable to demonstrate an ability of HCV specific CD8+ T cells to present antigen in this manner, indicating that these T cells functioned poorly in presenting antigen despite expression of B7 ligands (data not shown). Though we did not see evidence of direct antigen presentation by HCV specific CD8+ T cells to other T cells, we hypothesize that expression of CD86 on HCV specific CD8+ T cells is important for other T:T interactions by providing costimulation to neighboring T cells interacting with an antigen presenting cell or via an cell autonomous costimulatory signal. Unfortunately, given the difficulty in separating the effect of CD86 expression on antigen presenting cells from CD86 expression on T cells during in vitro assays, we were unable to directly demonstrate this effect.
In this study, we found that CD86 expression was lost early during HCV infection () despite persistent high-level PD-1 expression on HCV specific CD8+ T cells in acute infection (7
). This loss of CD86 expression also coincided with decreased liver inflammation as measured by ALT levels. Thus, we hypothesize that, as HCV progresses to chronic infection, the persistent negative signals via receptors such as PD-1 and the loss of positive signals via CD86 on T cells, tip the balance in favor of a waning response. Net negative costimulatory/coinhibitory signals to T cells at this phase of infection may be adaptive for a host that is unable to clear a virus, and waning CD86 expression may be a mechanism to prevent further high-level liver damage. If, indeed, CD86 expression on HCV specific CD8+ T cells is shown to provide direct costimulation to other HCV specific CD8+ T cells in acute infection, prolonging or modulating CD86 expression on these T cells may also be a mechanism that can be utilized to enhance future therapies for patients with chronic HCV infection.