Dissection of antiviral and immune regulatory functions of tumor necrosis factor receptors in a chronic lymphocytic choriomeningitis virus infection

The effector function of CD8 T cells is mediated via cell-mediated cytotoxicity and production of cytokines like gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). While the roles of perforin-dependent cytotoxicity, IFN-gamma, and TNF-alpha in controlling acute viral infections are well studied, their relative importance in defense against chronic viral infections is not well understood. Using mice deficient for TNF receptor (TNFR) I and/or II, we show that TNF-TNFR interactions have a dual role in mediating viral clearance and downregulating CD8 and CD4 T-cell responses during a chronic lymphocytic choriomeningitis virus (LCMV) infection. While wild-type (+/+) and TNFR II-deficient (p75(-/-)) mice cleared LCMV from the liver and lung, mice deficient in TNFR I (p55(-/-)) or both TNFR I and TNFR II (double knockout [DKO]) exhibited impaired viral clearance. The inability of p55(-/-) and DKO mice to clear LCMV was not a sequel to either suboptimal activation of virus-specific CD8 or CD4 T cells or impairment in trafficking of LCMV-specific CD8 T cells to the liver and lung. In fact, the expansion of LCMV-specific CD8 and CD4 T cells was significantly higher in DKO mice compared to that in +/+, p55(-/-), and p75(-/-) mice. TNFR deficiency did not preclude the physical deletion of CD8 T cells specific for nucleoprotein 396 to 404 but delayed the contraction of CD8 T-cell responses to the epitopes GP33-41 and GP276-285 in the viral glycoprotein. The antibody response to LCMV was not significantly altered by TNFR deficiency. Taken together, these findings have implications in development of immunotherapy in chronic viral infections of humans.     

Maintenance, loss, and resurgence of T cell responses during acute, protracted, and chronic viral infections

The acute phase of many viral infections is associated with the induction of a pronounced CD8 T cell response which plays a principle role in clearing the infection. By contrast, certain infections are not as readily controlled. In this study, we have used the well-defined system of lymphocytic choriomeningitis virus (LCMV) infection of mice to determine quantitative and qualitative changes in virus-specific CD8 T cell responses that rapidly resolve acute infections, more slowly control protracted infections, or fail to clear chronic infections. Acute LCMV infection elicits potent, functional, multi-epitope-specific CD8 T cell responses. Virus-specific CD8 T cells also expand, albeit to a lesser extent, during protracted LCMV infection. Under these conditions, there is a progressive diminution in the capacity to produce IL-2, TNF-alpha, and IFN-gamma. Changes in cytotoxic activities are also detectable but differ depending upon the specificity of the responding cells. As the infection is slowly resolved, a resurgence of cytokine production by virus-specific CD8 T cells is observed. CD4-deficient mice cannot control infection with certain strains of LCMV, but do mount multi-epitope-specific CD8 T cell responses that also lose effector capabilities; however, they are not maintained indefinitely in an unresponsive state as these cells become deleted over time. Overall, our findings suggest that constant high viral loads result in the progressive diminution of T cell effector functions and subsequent physical loss of the responding cells, whereas if the viral load is brought under control a partial restoration of CD8 T cell functions can occur.     

Long-Term CD4 Th1 and Th2 Memory following Acute Lymphocytic Choriomeningitis Virus Infection

CD4 T cells play a central role in viral immunity. They provide help for B cells and CD8 T cells and can act as effectors themselves. Despite their importance, relatively little is known about the magnitude and duration of virus-specific CD4 T-cell responses. In particular, it is not known whether both CD4 Th1 memory and CD4 Th2 memory can be induced by viral infections. To address these issues, we quantitated virus-specific CD4 Th1 (interleukin 2 [IL-2] and gamma-interferon) and Th2 (IL-4) responses in mice acutely infected with lymphocytic choriomeningitis virus (LCMV). Using two sensitive assays (enzyme-linked immunospot assay and intracellular stain) to measure cytokine production at the single-cell level, we found that both CD4 Th1 and Th2 responses were induced during primary LCMV infection. At the peak (day 8) of the response, the frequency of LCMV-specific CD4 Th1 cells was 1/35 to 1/160 CD4 T cells, and the frequency of Th2 cells was 1/400. After viral clearance, the numbers of virus-specific CD4 T cells dropped to 1/260 to 1/3,700 and then were maintained at this level indefinitely. Upon rechallenge with LCMV, both CD4 Th1 and Th2 memory cells made an anamnestic response in vivo. These results show that unlike some microbial infections in which only Th1 or Th2 responses are seen, an acute viral infection can induce a mixed CD4 T-cell response with long-term memory.     

Antiviral CD4 and CD8 T-cell memory: differences in the size of the response and activation requirements

Following acute lymphocytic choriomeningitis virus (LCMV) infection, there is a potent antiviral CD8 T-cell response that eliminates the infection. This initial CD8 T-cell response is followed by a period of memory during which elevated numbers of virus-specific CD8 T cells remain in the mouse. CD4 T cells are also activated after LCMV infection, but relatively less is known about the magnitude and duration of the CD4 response. In this study, we used intracellular staining for interferon-gamma to measure both CD4 and CD8 responses in the same mice at the single cell level. After LCMV infection, there was an increase in the number of activated CD4 T cells and an associated increase in the number of virus-specific CD4 T cells. At the peak of this expansion phase, the frequency of virus-specific CD4 T cells was 1 in 20 (0.5-1.0 x 10(6) per spleen). Like the CD8 response, long-term CD4 memory could be found up to a year after the infection with frequencies of approximately 1 in 260 (0.5-1.5 x 10(5) per spleen). However, the magnitude of virus-specific CD8 T cells was greater than virus-specific CD4 T cells during all phases of the immune response (expansion, death, and memory). At day 8, there were 20- to 35-fold more virus-specific CD8 T cells than CD4 T cells. This initial difference in cell number lasted into the memory phase as there remained a ten- to 20-fold difference in the CD8 and CD4 responses. These results highlight the importance of the expansion phase in determining the size of the memory T-cell pool. In addition to the difference in the magnitude, the activation requirements of CD8 and CD4 T-cell responses were different: CD8 T responses were not affected by blockade of CD40-CD40 ligand interaction whereas CD4 responses were reduced 90%. So while there is long-term memory in both the CD8 and CD4 compartments, the rules regulating the activation of CD8 and CD4 T cells and the overall magnitude of the responses are different.     

CD4+ T cells are required to sustain CD8+ cytotoxic T-cell responses during chronic viral infection.

In this study, we have examined the relative contributions of CD4+ and CD8+ T cells in controlling an acute or chronic lymphocytic choriomeningitis virus (LCMV) infection. To study acute infection, we used the LCMV Armstrong strain, which is cleared by adult mice in 8 to 10 days, and to analyze chronic infection, we used a panel of lymphocyte-tropic and macrophage-tropic variants of LCMV that persist in adult mice for several months. We show that CD4+ T cells are not necessary for resolving an acute LCMV infection. CD4+ T-cell-depleted mice were capable of generating an LCMV-specific CD8+ cytotoxic T-lymphocyte (CTL) response and eliminated virus with kinetics similar to those for control mice. The CD8+ CTL response was critical for resolving this infection, since beta 2-microglobulin knockout (CD8-deficient) mice were unable to control the LCMV Armstrong infection and became persistently infected. In striking contrast to the acute infection, even a transient depletion of CD4+ T cells profoundly affected the outcome of infection with the macrophage- and lymphocyte-tropic LCMV variants. Adult mice given a single injection of anti-CD4 monoclonal antibody (GK1.5) at the time of virus challenge became lifelong carriers with high levels of virus in most tissues. Unmanipulated adult mice infected with the different LCMV variants contained virus for prolonged periods (> 3 months) but eventually eliminated infection from most tissues, and all of these mice had LCMV-specific CD8+ CTL responses. Although the level of CTL activity was quite low, it was consistently present in all of the chronically infected mice that eventually resolved the infection. These results clearly show that even in the presence of an overwhelming viral infection of the immune system, CD8+ CTL can remain active for long periods and eventually resolve and/or keep the virus infection in check. In contrast, LCMV-specific CTL responses were completely lost in chronically infected CD4-depleted mice. Taken together, these results show that CD4+ T cells are dispensable for short-term acute infection in which CD8+ CTL activity does not need to be sustained for more than 2 weeks. However, under conditions of chronic infection, in which CD8+ CTLs take several months or longer to clear the infection, CD4+ T-cell function is critical. Thus, CD4+ T cells play an important role in sustaining virus-specific CD8+ CTL during chronic LCMV infection. These findings have implications for chronic viral infections in general and may provide a possible explanation for the loss of human immunodeficiency virus-specific CD8+ CTL activity that is seen during the late stages of AIDS, when CD4+ T cells become limiting.     

Viral persistence alters CD8 T-cell immunodominance and tissue distribution and results in distinct stages of functional impairment

Chronic viral infections often result in ineffective CD8 T-cell responses due to functional exhaustion or physical deletion of virus-specific T cells. However, how persisting virus impacts various CD8 T-cell effector functions and influences other aspects of CD8 T-cell dynamics, such as immunodominance and tissue distribution, remains largely unknown. Using different strains of lymphocytic choriomeningitis virus (LCMV), we compared responses to the same CD8 T-cell epitopes during acute or chronic infection. Persistent infection led to a disruption of the normal immunodominance hierarchy of CD8 T-cell responses seen following acute infection and dramatically altered the tissue distribution of LCMV-specific CD8 T cells in lymphoid and nonlymphoid tissues. Most importantly, CD8 T-cell functional impairment occurred in a hierarchical fashion in chronically infected mice. Production of interleukin 2 and the ability to lyse target cells in vitro were the first functions compromised, followed by the ability to make tumor necrosis factor alpha, while gamma interferon production was most resistant to functional exhaustion. Antigen appeared to be the driving force for this loss of function, since a strong correlation existed between the viral load and the level of exhaustion. Further, epitopes presented at higher levels in vivo resulted in physical deletion, while those presented at lower levels induced functional exhaustion. A model is proposed in which antigen levels drive the hierarchical loss of different CD8 T-cell effector functions during chronic infection, leading to distinct stages of functional impairment and eventually to physical deletion of virus-specific T cells. These results have implications for the study of human chronic infections, where similar T-cell deletion and functional dysregulation has been observed.     

Effect of chronic viral infection on epitope selection, cytokine production, and surface phenotype of CD8 T cells and the role of IFN-gamma receptor in immune regulation

Regulation of CD8 T cell responses in chronic viral infections is not well understood. In this study, we have compared the CD8 T cell responses to immunodominant and subdominant epitopes during an acute and a chronic lymphocytic choriomeningitis virus (LCMV) infection in mice. The epitope hierarchy of the primary CD8 T cell response was similar in acute and chronic LCMV infections. However, strikingly, the epitope hierarchy of the primary CD8 T cell response was conserved in the T cell memory only in an acute but not in a chronic LCMV infection. Interestingly, in an acute infection, increasing the viral dose caused significant changes in the epitope hierarchy of the LCMV-specific memory CD8 T cell pool, with no effect on the primary CD8 T cell response. Functional and phenotypic analyses revealed that exposure of CD8 T cells to extended periods of antigenic stimulation could lead to long-term defects in cytokine production and alteration in expression of cell surface L-selectin (CD62L). Whereas expression of CD44 was minimally altered, a greater proportion of LCMV-specific memory CD8 T cells were CD62L(low) in mice that have recovered from a chronic LCMV infection, compared with acutely infected mice. Mechanistic studies showed that IFN-gammaR deficiency altered the epitope hierarchy of the pool of LCMV-specific memory CD8 T cells without significantly affecting the immunodominance of the primary CD8 T cell response in an acute infection. Taken together, these findings should further our understanding about the regulation of T cell responses in human chronic viral infections.     

Dynamics of CD8+ T cell responses during acute and chronic lymphocytic choriomeningitis virus infection

Infection of mice with lymphocytic choriomeningitis virus (LCMV) is frequently used to study the underlying principles of viral infections and immune responses. We fit a mathematical model to recently published data characterizing Ag-specific CD8+ T cell responses during acute (Armstrong) and chronic (clone 13) LCMV infection. This allows us to analyze the differences in the dynamics of CD8+ T cell responses against different types of LCMV infections. For the four CD8+ T cell responses studied, we find that, compared with the responses against acute infection, responses against chronic infection are generally characterized by an earlier peak and a faster contraction phase thereafter. Furthermore, the model allows us to give a new interpretation of the effect of thymectomy on the dynamics of CD8+ T cell responses during chronic LCMV infection: a smaller number of naive precursor cells is sufficient to account for the observed differences in the responses in thymectomized mice. Finally, we compare data characterizing LCMV-specific CD8+ T cell responses from different laboratories. Although the data were derived from the same experimental model, we find quantitative differences that can be solved by introducing a scaling factor. Also, we find kinetic differences that are at least partly due to the infrequent measurements of CD8+ T cells in the different laboratories.     

CD4+ T-Cell Help Is Required for Effective CD8+ T Cell-Mediated Resolution of Acute Viral Hepatitis in Mice

Cytotoxic CD8+ T cells are essential for the control of viral liver infections, such as those caused by HBV or HCV. It is not entirely clear whether CD4+ T-cell help is necessary for establishing anti-viral CD8+ T cell responses that successfully control liver infection. To address the role of CD4+ T cells in acute viral hepatitis, we infected mice with Lymphocytic Choriomeningitis Virus (LCMV) of the strain WE; LCMV-WE causes acute hepatitis in mice and is cleared from the liver by CD8+ T cells within about two weeks. The role of CD4+ T-cell help was studied in CD4+ T cell-lymphopenic mice, which were either induced by genetic deficiency of the major histocompatibility (MHC) class II transactivator (CIITA) in CIITA−/− mice, or by antibody-mediated CD4+ cell depletion. We found that CD4+ T cell-lymphopenic mice developed protracted viral liver infection, which seemed to be a consequence of reduced virus-specific CD8+ T-cell numbers in the liver. Moreover, the anti-viral effector functions of the liver-infiltrating CD8+ T cells in response to stimulation with LCMV peptide, notably the IFN-γ production and degranulation capacity were impaired in CIITA−/− mice. The impaired CD8+ T-cell function in CIITA−/− mice was not associated with increased expression of the exhaustion marker PD-1. Our findings indicate that CD4+ T-cell help is required to establish an effective antiviral CD8+ T-cell response in the liver during acute viral infection. Insufficient virus control and protracted viral hepatitis may be consequences of impaired initial CD4+ T-cell help.     

Critical role for alpha/beta and gamma interferons in persistence of lymphocytic choriomeningitis virus by clonal exhaustion of cytotoxic T cells

Under conditions of high antigenic load during infection with invasive lymphocytic choriomeningitis virus (LCMV) strains, virus can persist by selective clonal exhaustion of antigen-specific CD8(+) T cells. In this work we studied the down-regulation of the virus-specific CD8(+)-T-cell response during a persistent infection of adult mice, with particular emphasis on the contribution of the interferon response in promoting host defense. Studies were conducted by infecting mice deficient in receptors for type I (alpha/beta interferon [IFN-alpha/beta]), type II (IFN-gamma), and both type I and II IFNs with LCMV isolates that vary in their capacity to induce T-cell exhaustion. The main conclusions of this study are as follows. (i) IFNs play a critical role in LCMV infection by reducing viral loads in the initial stages of infection and thus modifying both the extent of CD8(+)-T-cell exhaustion and the course of infection. The importance of IFNs in this context varies with the biological properties of the LCMV strain. (ii) An inverse correlation exists between antigen persistence and responsiveness of virus-specific CD8(+) T cells. This results in distinct programs of activation or tolerance (functional unresponsiveness and/or physical elimination of antigen-specific cells) during acute and chronic virus infections, respectively. (iii) A successful immune response associated with definitive viral clearance requires an appropriate balance between cellular and humoral components of the immune system. We discuss the role of IFNs in influencing virus-specific T cells that determine the outcome of persistent infections.     

Attrition of bystander CD8 T cells during virus-induced T-cell and interferon responses

Experiments designed to distinguish virus-specific from non-virus-specific T cells showed that bystander T cells underwent apoptosis and substantial attrition in the wake of a strong T-cell response. Memory CD8 T cells (CD8(+) CD44(hi)) were most affected. During acute viral infection, transgenic T cells that were clearly defined as non-virus specific decreased in number and showed an increase in apoptosis. Also, use of lymphocytic choriomeningitis virus (LCMV) carrier mice, which lack LCMV-specific T cells, showed a significant decline in non-virus-specific memory CD8 T cells that correlated to an increase in apoptosis in response to the proliferation of adoptively transferred virus-specific T cells. Attrition of T cells early during infection correlated with the alpha/beta interferon (IFN-alpha/beta) peak, and the IFN inducer poly(I:C) caused apoptosis and attrition of CD8(+) CD44(hi) T cells in normal mice but not in IFN-alpha/beta receptor-deficient mice. Apoptotic attrition of bystander T cells may make room for the antigen-specific expansion of T cells during infection and may, in part, account for the loss of T-cell memory that occurs when the host undergoes subsequent infections.     

Low CD8 T-cell proliferative potential and high viral load limit the effectiveness of therapeutic vaccination

Therapeutic vaccination has the potential to boost immune responses and enhance viral control during chronic infections. However, many therapeutic vaccination approaches have fallen short of expectations, and effective boosting of antiviral T-cell responses is not always observed. To examine these issues, we studied the impact of therapeutic vaccination, using a murine model of chronic infection with lymphocytic choriomeningitis virus (LCMV). Our results demonstrate that therapeutic vaccination using a recombinant vaccinia virus expressing the LCMV GP33 CD8 T-cell epitope can be effective at accelerating viral control. However, mice with lower viral loads at the time of vaccination responded better to therapeutic vaccination than did those with high viral loads. Also, the proliferative potential of GP33-specific CD8 T cells from chronically infected mice was substantially lower than that of GP33-specific memory CD8 T cells from mice with immunity to LCMV, suggesting that poor T-cell expansion may be an important reason for suboptimal responses to therapeutic vaccination. Thus, our results highlight the potential positive effects of therapeutic vaccination on viral control during chronic infection but also provide evidence that a high viral load at the time of vaccination and the low proliferative potential of responding T cells are likely to limit the effectiveness of therapeutic vaccination.     

Critical Role for Perforin-, Fas/FasL-, and TNFR1-Mediated Cytotoxic Pathways in Down-Regulation of Antigen-Specific T Cells during Persistent Viral Infection

Viral persistence following infection with invasive strains of lymphocytic choriomeningitis virus (LCMV) can be achieved by selective down-regulation of virus-specific T lymphocytes. High viral burden in the onset of infection drives responding cells into functional unresponsiveness (anergy) that can be followed by their physical elimination. In this report, we studied down-regulation of the virus-specific CD8(+)-T-cell response during persistent infection of adult mice with LCMV, with emphasis on the role of perforin-, Fas/FasL-, or tumor necrosis factor receptor 1 (TNFR1)-mediated cytolysis in regulating T-cell homeostasis. The results reveal that the absence of perforin, Fas-ligand, or TNFR1 has no significant effect on the kinetics of proliferation and functional inactivation of virus-specific CD8(+) T cells in the onset of chronic LCMV infection. However, these molecules play a critical role in the homeostatic regulation of T cells, influencing the longevity of the virus-specific CD8(+)-T-cell population once it has become anergic. Thus, CD8(+) T cells specific to the dominant LCMV NP(396-404) epitope persist in an anergic state for at least 70 days in perforin-, FasL-, or TNFR1-deficient mice, but they were eliminated by day 30 in C57BL/6 controls. These effects were additive as shown by a deficit of apoptotic death of NP(396-404) peptide-specific CD8(+) T cells in mice lacking both perforin and TNFR1. This suggests a role for perforin-, FasL-, and TNFR1-mediated pathways in down-regulation of the antiviral T cell response during persistent viral infection by determining the fate of antigen-specific T cells. Moreover, virus-specific anergic CD8(+) T cells in persistently infected C57BL/6 mice contain higher levels of Bcl-2 and Bcl-XL than functionally intact T cells generated during acute LCMV infection. In the case of proapoptotic factors, Bax expression did not differ between T-cell populations and Bad was below the limit of detection in all samples. As expression of the Bcl-2 family members controls susceptibility to apoptosis, this finding may provide a molecular basis for the survival of anergic cells under conditions of prolonged antigen stimulation.     

Suppression of viral specific primary T-cell response following intense physical exercise in young but not old mice.

Intense exercise to exhaustion leads to increased susceptibility and severity of infections. T cells play an essential role in control of viral infections. Whereas immune suppression is considered as a likely mechanism for exhaustive exercise-induced susceptibility to infection, we know little about viral-specific T-cell response following exhaustive exercise in young or old mice. In this study, one group of female young (10-12 wk) and old (22-24 mo) C57BL/6 mice was exposed to a single bout of intense exercise to exhaustion and immediately infected with lymphocytic choriomeningitis virus (LCMV). Eight days later, at the peak of expansion phase of T-cell response, we used tetramers of MHC class I molecules containing viral peptides to directly visualize antigen-specific CD8 T cells and a sensitive functional assay measuring interferon-gamma production at the single-cell level to quantitate the CD8 and CD4 T-cell response. To evaluate the impact of intense exercise during both the initiation and evolution of the expansion phase of the T-cell response, a second group of young and old mice continued their daily bouts of intense exercise to exhaustion over the next 8 days. Our data show that, in young mice, LCMV infection following exhaustive exercise leads to suppression of LCMV-specific CD8 and CD4 T-cell responses, and this suppression effect occurs at the initiation of the expansion phase of viral-specific T cells. However, in old mice, unlike young mice, exhaustive exercise does not cause suppression of LCMV-specific T-cell responses.     

Quantitating the magnitude of the lymphocytic choriomeningitis virus-specific CD8 T-cell response: it is even bigger than we thought

Measuring the magnitudes and specificities of antiviral CD8 T-cell responses is critical for understanding the dynamics and regulation of adaptive immunity. Despite many excellent studies, the accurate measurement of the total CD8 T-cell response directed against a particular infection has been hampered by an incomplete knowledge of all CD8 T-cell epitopes and also by potential contributions of bystander expansion among CD8 T cells of irrelevant specificities. Here, we use several techniques to provide a more complete accounting of the CD8 T-cell response generated upon infection of C57BL/6 mice with lymphocytic choriomeningitis virus (LCMV). Eight days following infection, we found that 85 to 95% of CD8 T cells exhibit an effector phenotype as indicated by granzyme B, 1B11, CD62L, CD11a, and CD127 expression. We demonstrate that CD8 T-cell expansion is due to cells that divide >7 times, whereas heterologous viral infections only elicited <3 divisions among bystander memory CD8 T cells. Furthermore, we found that approximately 80% of CD8 T cells in spleen were specific for ten different LCMV-derived epitopes at the peak of primary infection. These data suggest that following a single LCMV infection, effector CD8 T cells divide > or =15 times and account for at least 80%, and possibly as much as 95%, of the CD8 T-cell pool. Moreover, the response targeted a very broad array of peptide major histocompatibility complexes (MHCs), even though we examined epitopes derived from only two of the four proteins encoded by the LCMV genome and C57BL/6 mice only have two MHC class I alleles. These data illustrate the potential enormity, specificity, and breadth of CD8 T-cell responses to viral infection and demonstrate that bystander activation does not contribute to CD8 T-cell expansion.     

The early kinetics of cytomegalovirus-specific CD8+ T-cell responses are not affected by antigen load or the absence of perforin or gamma interferon

Both innate and adaptive immune responses participate in the control of murine cytomegalovirus (mCMV) infection. In some mouse strains, like BALB/c, the control of infection relies on the activities of CD8(+) T cells. mCMV-specific CD8(+) T-cell responses are unusual in that, even after mCMV has been controlled in the periphery, the numbers of circulating virus-specific CD8(+) T cells remain high compared to those observed in other viral infections. To better understand the generation and maintenance of mCMV-specific CD8(+) T-cell responses, we evaluated how antigen load and effector molecules, such as perforin (Prf) and gamma interferon (IFN-gamma), influence these responses during acute infection in vivo. Viral burden affected the magnitude, but not the early kinetics, of antigen-specific CD8(+) T-cell responses. Similarly, the magnitude of virus-specific CD8(+) T-cell expansion was affected by Prf and IFN-gamma, but contraction of antigen-specific responses occurred normally in both Prf- and IFN-gamma-deficient mice. These data indicate that control of mCMV-specific CD8(+) T-cell expansion and contraction is more complex than anticipated and, despite the role of Prf or IFN-gamma in controlling viral replication, a full program of T-cell expansion and contraction can occur in their absence.     

Enhanced CD8 T Cell Responses through GITR-Mediated Costimulation Resolve Chronic Viral Infection

Chronic infections are characterized by the inability to eliminate the persisting pathogen and often associated with functional impairment of virus-specific T-cell responses. Costimulation through Glucocorticoid-induced TNFR-related protein (GITR) can increase survival and function of effector T cells. Here, we report that constitutive expression of GITR-ligand (GITRL) confers protection against chronic lymphocytic choriomeningitis virus (LCMV) infection, accelerating recovery without increasing pathology. Rapid viral clearance in GITRL transgenic mice coincided with increased numbers of poly-functional, virus-specific effector CD8+ T cells that expressed more T-bet and reduced levels of the rheostat marker PD-1. GITR triggering also boosted the helper function of virus-specific CD4 T cells already early in the infection, as was evidenced by increased IL-2 and IFNγ production, and more expression of CD40L and T-bet. Importantly, CD4-depletion experiments revealed that the expanded pool of virus-specific effector CD8 T cells and the ensuing viral clearance in LCMV-infected GITRL tg mice was entirely dependent on CD4 T cells. We found no major differences for NK cell and regulatory T cell responses, whereas the humoral response to the virus was increased in GITRL tg mice, but only in the late phase of the infection when the virus was almost eradicated. Based on these findings, we conclude that enhanced GITR-triggering mediates its protective, anti-viral effect on the CD8 T cell compartment by boosting CD4 T cell help. As such, increasing costimulation through GITR may be an attractive strategy to increase anti-viral CTL responses without exacerbating pathology, in particular to persistent viruses such as HIV and HCV.     

Control of virus-specific CD8+ T-cell exhaustion and immune-mediated pathology by E3 ubiquitin ligase Cbl-b during chronic viral infection

A characteristic feature in the immune response to many persistent viral infections is the dysfunction or deletion of antigen-specific T cells (exhaustion). This down-regulation of virus-specific T-cell response represents a critical control mechanism that exists within T-cell activation pathways to prevent lethal disease by inappropriate responses against disseminating virus infections. However, the molecular mechanisms by which the immune system determines whether to mount a full response to such infections remain largely unexplored. Here, we have established that in the murine lymphocytic choriomeningitis virus (LCMV) model, induction of the T-cell receptor signaling inhibitor molecule E3 ligase Cbl-b is critically involved in this decision. In particular, our data revealed that Cbl-b controls the program responsible for T-cell tolerance (exhaustion) induction during a chronic viral infection. Thus, Cbl-b(-/-) mice infected with a low dose of LCMV Docile mount a strong CD8(+) T-cell response that rapidly clears the infection, and the animals remain healthy; in contrast, down-regulation of the epitope-specific CD8(+) T-cell population in persistently infected Cbl-b(-/-) mice, compared to that in chronically infected B6 mice, was significantly delayed, and this was associated with increased morbidity and eventual death in nearly 20% of the animals. Interestingly, infection of Cbl-b(-/-) mice with a moderate virus dose resulted in rapid death with 100% mortality by 7 to 8 days after infection, caused by a dysregulated antiviral T-cell response, whereas the infected B6 mice survived and remained healthy. In conclusion, our results suggest that Cbl-b is critically involved in T-cell exhaustion and prevention of lethal disease.     

Intrinsic functional dysregulation of CD4 T cells occurs rapidly following persistent viral infection

Effective T-cell responses are critical to eradicate acute viral infections and prevent viral persistence. Emerging evidence indicates that robust, early CD4 T-cell responses are important in effectively sustaining CD8 T-cell activity. Herein, we illustrate that virus-specific CD4 T cells are functionally inactivated early during the transition into viral persistence and fail to produce effector cytokines (i.e., interleukin-2 and tumor necrosis factor alpha), thereby compromising an efficient and effective antiviral immune response. Mechanistically, the inactivation occurs at the cellular level and is not an active process maintained by regulatory T cells or antigen-presenting cells. Importantly, a small subpopulation of cells is able to resist inactivation and persist into the chronic phase of infection. However, the virus-specific CD4 T-cell population ultimately undergoes a second round of inactivation, and the cells that had retained functional capacity fail to respond to rechallenge in an acute time frame. Based on these results we propose a biological mechanism whereby early CD4 T-cell inactivation leads to a subsequent inability to sustain cytotoxic T-lymphocyte function, which in turn facilitates viral persistence. Moreover, these studies are likely relevant to chronic/persistent infections of humans (e.g., human immunodeficiency virus, hepatitis C virus, and hepatitis B virus) by providing evidence that a reservoir of virus-specific CD4 T cells can remain functional during chronic infection and represent a potential therapeutic target to stimulate the immune response and establish control of infection.     

Negative Regulation of Type I IFN Expression by OASL1 Permits Chronic Viral Infection and CD8+ T-Cell Exhaustion

The type I interferons (IFN-Is) are critical not only in early viral control but also in prolonged T-cell immune responses. However, chronic viral infections such as those of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) in humans and lymphocytic choriomeningitis virus (LCMV) in mice overcome this early IFN-I barrier and induce viral persistence and exhaustion of T-cell function. Although various T-cell-intrinsic and -extrinsic factors are known to contribute to induction of chronic conditions, the roles of IFN-I negative regulators in chronic viral infections have been largely unexplored. Herein, we explored whether 2′–5′ oligoadenylate synthetase-like 1 (OASL1), a recently defined IFN-I negative regulator, plays a key role in the virus-specific T-cell response and viral defense against chronic LCMV. To this end, we infected Oasl1 knockout and wild-type mice with LCMV CL-13 (a chronic virus) and monitored T-cell responses, serum cytokine levels, and viral titers. LCMV CL-13-infected Oasl1 KO mice displayed a sustained level of serum IFN-I, which was primarily produced by splenic plasmacytoid dendritic cells, during the very early phase of infection (2–3 days post-infection). Oasl1 deficiency also led to the accelerated elimination of viremia and induction of a functional antiviral CD8 T-cell response, which critically depended on IFN-I receptor signaling. Together, these results demonstrate that OASL1-mediated negative regulation of IFN-I production at an early phase of infection permits viral persistence and suppresses T-cell function, suggesting that IFN-I negative regulators, including OASL1, could be exciting new targets for preventing chronic viral infection.