The main point of this study is that virus-specific CD8 T cells can persist indefinitely in chronically infected hosts. These T cells express activation/memory markers (CD44hi
), are receiving signals through the TCR (CD69hi
), and can proliferate in vivo, but are unable to elaborate antiviral effector functions and thus fail to control the infection. Although T cell unresponsiveness (anergy) has been extensively studied, cells of such a phenotype have not been described previously (44
). Moreover, the identification of these activated but effector-function–negative T cells reveals a new mechanism of silencing antiviral immune responses during chronic infections. These CD8 T cells seem to be engaged in a continual and ineffective effort much like the Greek king, Sisyphus, who was condemned forever to perform the fruitless task of pushing a large stone up a hill only to see it roll down again. Our studies show that these “Sisyphean” CD8 T cells are generated under conditions of chronic antigenic stimulus and are especially prevalent when there is inadequate CD4 help. Until now, deletion (exhaustion) of virus-specific CD8 T cells has been considered as a more general mechanism of tolerizing virus-specific immune responses during chronic infections (13
). Our studies documenting persistence of effector-function–negative antigen-specific CD8 T cells open new possibilities of therapeutic intervention for enhancing CD8 T cell immunity in chronically infected hosts. In addition, this study shows that within the same persistently infected host different mechanisms can operate to silence antiviral T cell responses; LCMV NP396-specific cells were deleted, whereas GP33-specific cells were maintained indefinitely but without any detectable CTL or cytokine secretion activity. Thus, strategies to enhance immune function during chronic infection need to be tailored towards individual epitopes.
In contrast to the strong correlation between CD4 help and CD8 T cell effector function, we found an inverse correlation between antigen persistence and responsiveness of virus-specific CD8 T cells. Of the three LCMV strains used in this study, clone 13 causes the most disseminated and persistent infection with the highest antigen load, followed by LCMV-t1b and then Armstrong, which only causes an acute infection (Figs. and and reference 19
). During the course of acute LCMV Armstrong infection (which only lasts about a week) and after viral clearance, all of the GP33-specific CD8 T cells remained functional (Fig. and reference 26
). In LCMV-t1b–infected +/+ mice (viral clearance in ~2 mo), ~60% of the GP33-specific CD8 T cells were unresponsive during the first month after infection, but by day 90 all of these cells had become functional (Fig. and data not shown). However, in clone 13– infected +/+ mice, ~80% of the GP33-specific T cells remained unresponsive for an extended period. Thus, the duration of exposure to viral antigen is a critical parameter that influences T cell responsiveness. Clone 13 causes the most prolonged infection and results in a pronounced “functional deficit” within the GP33-specific CD8 T cell population. Unlike LCMV-t1b infection, even after clone 13 infection has been controlled the GP33-specific T cells do not rapidly regain their capacity to elicit antiviral effector functions. None of the LCMV strains used in this study infect CD8 T cells; therefore, the unresponsiveness of GP33-specific cells is not due to a direct effect of LCMV replication within these cells. However, LCMV does replicate within professional APCs and the presentation of viral antigen by these cells can target them for destruction by virus-specific CTL (52
). Loss of professional APCs is least apparent during Armstrong infection and most widespread during clone 13 infection. Consequently, during systemic LCMV infection professional APCs are destroyed and large numbers of “nonprofessional” APCs may present viral antigen. Such conditions are likely to favor the induction of CD8 T cell unresponsiveness. Moreover, professional APCs may be aberrantly activated in the absence of CD4 T cell help and this may further promote the functional unresponsiveness of CD8 T cells (54
). Thus, a chronic antigenic stimulus in the absence of adequate CD4 help appears to be the worst possible scenario resulting in complete loss of CD8 effector functions and uncontrolled virus infection. Although such regulatory mechanisms are not well suited to deal with chronic infections and tumors, they may have evolved to prevent autoimmunity.
The degree of T cell activation is likely to be an important parameter that determines whether CD8 T cells are deleted or become functionally unresponsive (48
). The extent of T cell activation can be influenced by the abundance of antigen that is presented and also by the affinity of the TCR for the given MHC–peptide complex. Virus- infected cells will display both GP33 and NP396 epitopes to responding T cells. One possibility is that different amounts of GP33 and NP396 epitopes are presented in the periphery of chronically infected adult mice (58
). Consequently, GP33- and NP396-specific T cells may not be similarly activated and therefore the fate of these T cells could be differentially regulated. Alternatively, the on and off rates of GP33- and NP396-specific TCRs may differ. In this case even if both epitopes are present in similar concentrations on the infected cells, the responding T cells may undergo different activation programs and consequently succumb to different fates (i.e., deletion versus nonresponsiveness). Interestingly, GP33-specific T cells are not intrinsically resistant to deletion, and both GP33- and NP396-specific T cells are undetectable by tetramer staining in congenitally infected LCMV carrier mice (data not shown). These results suggest that GP33-specific CD8 T cells can undergo deletion during central (thymic) tolerance.
Our findings are clearly relevant for understanding how virus-specific CD8 T cell responses determine the outcome of persistent viral infections. We have shown that virus-specific T cells that express activation markers, such as CD44 and CD69, can be maintained in an unresponsive state during chronic infections. Therefore, simply detecting the physical presence of activated virus-specific CD8 T cells, which turn over in vivo, does not necessarily mean that these cells are operating to control the infection (60
). However, our data also demonstrate that not all virus-specific T cells need to be functionally competent in order to resolve a chronic infection. For example, even though NP396-specific T cells are deleted and only 10–20% of GP33-specific T cells are functional, infection with LCMV clone 13 is eventually resolved in +/+ mice. This suggests that for the control of persistent viral infections a threshold of functional antiviral T cells needs to be maintained. The number of functional antiviral CD8 T cells could fall below this threshold if too many virus-specific CD8 T cells are either deleted or become unresponsive, as occurs in the absence of CD4 T cell help. Such dampening of virus-specific CD8 T cell responses would result in a more protracted or even an uncontrollable infection.
The most striking finding of this study is that “activated” but effector-function–negative virus-specific CD8 T cells can persist indefinitely in chronically infected mice at remarkably high frequencies (1–2% of total CD8 T cells). It is likely that such effector-function–negative virus-specific CD8 T cells will also be found in chronic viral infections of humans. During HIV infection, CD8 CTLs play an important role in controlling the viral burden and both deletion and functional unresponsiveness may impair the efficacy of these responses (16
). Moreover, as progression to AIDS occurs, the numbers of CD4 T cells decline, and Rosenburg et al. (21
) reported that weak HIV-specific CD4 T cell responses correlate with higher viral loads. This is consistent with our data showing that CD4 T cells are required to maintain the effector functions of virus-specific CD8 T cells, and also suggests that if the number of functional virus-specific CD8 T cells falls below a threshold level, then an uncontrollable chronic infection can ensue. Since CD8 T cells are potent mediators of viral clearance, it is important to elucidate how to restore and sustain the effector functions of virus-specific T cells. Antiviral therapies that are designed to turn on the effector activity of preexisting virus-specific CD8 T cells may be successful at both clearing persistent infections and enhancing protective immunity to reinfection.