Differential tissue-specific regulation of antiviral CD8+ T-cell immune responses during chronic viral infection

The hallmarks of the immune response to viral infections are the expansion of antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs) after they encounter antigen-presenting cells in the lymphoid tissues and their subsequent redistribution to nonlymphoid tissues to deal with the pathogen. Control mechanisms exist within CTL activation pathways to prevent inappropriate CTL responses against disseminating infections with a broad distribution of pathogen in host tissues. This is demonstrated during overwhelming infection with the noncytolytic murine lymphocytic choriomeningitis virus, in which clonal exhaustion (anergy and/or deletion) of CTLs prevents immune-mediated pathology but allows persistence of the virus. The mechanism by which the immune system determines whether or not to mount a full response to such infections is unknown. Here we present data showing that the initial encounter of specific CTLs with infected cells in lymphoid tissues is critical for this decision. Whether the course of the viral infection is acute or persistent for life primarily depends on the degree and kinetics of CTL exhaustion in infected lymphoid tissues. Virus-driven CTL expansion in lymphoid tissues resulted in the migration of large quantities of CTLs to nonlymphoid tissues, where they persisted at stable levels. Surprisingly, although virus-specific CTLs were rapidly clonally exhausted in lymphoid tissues under conditions of chronic infection, a substantial number of them migrated to nonlymphoid tissues, where they retained an effector phenotype for a long time. However, these cells were unable to control the infection and progressively lost their antiviral capacities (cytotoxicity and cytokine secretion) in a hierarchical manner before their eventual physical elimination. These results illustrate the differential tissue-specific regulation of antiviral T-cell responses during chronic infections and may help us to understand the dynamic relationship between antigen and T-cell populations in many persistent infections in humans.     

Granzyme B regulates antiviral CD8+ T cell responses

CTLs and NK cells use the perforin/granzyme cytotoxic pathway to kill virally infected cells and tumors. Human regulatory T cells also express functional granzymes and perforin and can induce autologous target cell death in vitro. Perforin-deficient mice die of excessive immune responses after viral challenges, implicating a potential role for this pathway in immune regulation. To further investigate the role of granzyme B in immune regulation in response to viral infections, we characterized the immune response in wild-type, granzyme B-deficient, and perforin-deficient mice infected with Sendai virus. Interestingly, granzyme B-deficient mice, and to a lesser extent perforin-deficient mice, exhibited a significant increase in the number of Ag-specific CD8(+) T cells in the lungs and draining lymph nodes of virally infected animals. This increase was not the result of failure in viral clearance because viral titers in granzyme B-deficient mice were similar to wild-type mice and significantly less than perforin-deficient mice. Regulatory T cells from WT mice expressed high levels of granzyme B in response to infection, and depletion of regulatory T cells from these mice resulted in an increase in the number of Ag-specific CD8(+) T cells, similar to that observed in granzyme B-deficient mice. Furthermore, granzyme B-deficient regulatory T cells displayed defective suppression of CD8(+) T cell proliferation in vitro. Taken together, these results suggest a role for granzyme B in the regulatory T cell compartment in immune regulation to viral infections.     

Gammaherpesvirus persistence alters key CD8 T-cell memory characteristics and enhances antiviral protection

In herpesvirus infections, the virus persists for life but is contained through T-cell-mediated immune surveillance. How this immune surveillance operates is poorly understood. Recent studies of other persistent infections have indicated that virus persistence is associated with functional deficits in the CD8(+) T-cell response. To test whether this is the case in a herpesvirus infection, we used a mutant murine gammaherpesvirus that is defective in its ability to persist in the host. By comparing the immune response to this virus with a revertant virus that can persist, we were able to dissect the changes in the antiviral CD8(+) T-cell response that are induced by virus persistence. Surprisingly, persistently infected mice controlled a secondary challenge infection more rapidly than nonpersistently infected mice, indicating enhanced rather than diminished effector functions. Consistent with this, virus-specific CD8 T cells from these mice exhibited faster upregulation of the cytotoxic mediator granzyme B. Another unexpected finding was that CD8(+) T cells from neither infection responded efficiently to homeostatic cytokines. The unresponsiveness of the memory cells from the nonpersistently infected mice appears to be linked to the prolonged replication of virus within the lungs. Other changes seen in different chronic infection models were also observed, such as changes in Bcl-2 levels, interleukin-2 production, and the immunodominance hierarchy. These data show persistence of gammaherpesvirus type 68 alters the properties of CD8(+) T cells and illustrates that immune surveillance does not require CD8 T cells with the same attributes as "classical" memory CD8(+) T cells.     

Differential regulation of antiviral T-cell immunity results in stable CD8+ but declining CD4+ T-cell memory

Emerging evidence indicates that CD8+ and CD4+ T-cell immunity is differentially regulated. Here we have delineated differences and commonalities among antiviral T-cell responses by enumeration and functional profiling of eight specific CD8+ and CD4+ T-cell populations during primary, memory and recall responses. A high degree of coordinate regulation among all specific T-cell populations stood out against an approximately 20-fold lower peak expansion and prolonged contraction phase of specific CD4+ T-cell populations. Surprisingly, although CD8+ T-cell memory was stably maintained for life, levels of specific CD4+ memory T cells gradually declined. However, this decay, which seemed to result from less efficient rescue from apoptosis, did not affect functionality of surviving virus-specific CD4+ T cells. Our results indicate that CD4+ T-cell memory might become limiting under physiological conditions and that conditions precipitating CD4+ T-cell loss might compromise protective immunity even in the presence of unimpaired CD8+ T-cell responses.     

Mcl-1 antagonizes Bax/Bak to promote effector CD4+ and CD8+ T-cell responses

Members of the Bcl-2 family have critical roles in regulating tissue homeostasis by modulating apoptosis. Anti-apoptotic molecules physically interact and restrain pro-apoptotic family members preventing the induction of cell death. However, the specificity of the functional interactions between pro- and anti-apoptotic Bcl-2 family members remains unclear. The pro-apoptotic Bcl-2 family member Bcl-2 interacting mediator of death (Bim) has a critical role in promoting the death of activated, effector T cells following viral infections. Although Bcl-2 is an important Bim antagonist in effector T cells, and Bcl-xL is not required for effector T-cell survival, the roles of other anti-apoptotic Bcl-2 family members remain unclear. Here, we investigated the role of myeloid cell leukemia sequence 1 (Mcl-1) in regulating effector T-cell responses in vivo. We found, at the peak of the response to lymphocytic choriomeningitis virus (LCMV) infection, that Mcl-1 expression was increased in activated CD4⁺ and CD8⁺ T cells. Retroviral overexpression of Mcl-1-protected activated T cells from death, whereas deletion of Mcl-1 during the course of infection led to a massive loss of LCMV-specific CD4⁺ and CD8⁺ T cells. Interestingly, the co-deletion of Bim failed to prevent the loss of Mcl-1-deficient T cells. Furthermore, lck-driven overexpression of a Bcl-xL transgene only partially rescued Mcl-1-deficient effector T cells suggesting a lack of redundancy between the family members. In contrast, additional loss of Bax and Bak completely rescued Mcl-1-deficient effector T-cell number and function, without enhancing T-cell proliferation. These data suggest that Mcl-1 is critical for promoting effector T-cell responses, but does so by combating pro-apoptotic molecules beyond Bim.     

CD80 and CD86 control antiviral CD8+ T-cell function and immune surveillance of murine gammaherpesvirus 68

The interactions between CD80 and CD86 on antigen-presenting cells and CD28 on T cells serve as an important costimulatory signal in the activation of T cells. Although the simplistic two-signal hypothesis has been challenged in recent years by the identification of different costimulators, this classical pathway has been shown to significantly impact antiviral humoral and cellular immune responses. How the CD80/CD86-CD28 pathway affects the control of chronic or latent infections has been less well characterized. In this study, we investigated its role in antiviral immune responses against murine gammaherpesvirus 68 (MHV-68) and immune surveillance using CD80/CD86(-/-) mice. In the absence of CD80/CD86, primary antiviral CD8(+) T-cell responses and the induction of neutralizing antibodies were severely impaired. During long-term immune surveillance, the virus-specific CD8(+) T cells were impaired in IFN-gamma production and secondary expansion and exhibited an altered phenotype. Surprisingly, a low level of viral reactivation in the lung was observed, and this effect was independent of CD28 and CTLA-4. Thus, CD80 and CD86, signaling through CD28 and possibly another unidentified receptor, are required for optimal immune surveillance and antiviral immune responses to murine gammaherpesvirus.     

Type I interferons and MAVS signaling are necessary for tissue resident memory CD8+ T cell responses to RSV infection

Respiratory syncytial virus (RSV) can cause bronchiolitis and viral pneumonia in young children and the elderly. Lack of vaccines and recurrence of RSV infection indicate the difficulty in eliciting protective memory immune responses. Tissue resident memory T cells (T_RM) can confer protection from pathogen re-infection and, in human experimental RSV infection, the presence of lung CD8⁺ T_RM cells correlates with a better outcome. However, the requirements for generating and maintaining lung T_RM cells during RSV infection are not fully understood. Here, we use mouse models to assess the impact of innate immune response determinants in the generation and subsequent expansion of the T_RM cell pool during RSV infection. We show that CD8⁺ T_RM cells expand independently from systemic CD8⁺ T cells after RSV re-infection. Re-infected MAVS and MyD88/TRIF deficient mice, lacking key components involved in innate immune recognition of RSV and induction of type I interferons (IFN-α/β), display impaired expansion of CD8⁺ T_RM cells and reduction in antigen specific production of granzyme B and IFN-γ. IFN-α treatment of MAVS deficient mice during primary RSV infection restored T_RM cell expansion upon re-challenge but failed to recover T_RM cell functionality. Our data reveal how innate immunity, including the axis controlling type I IFN induction, instructs and regulates CD8⁺ T_RM cell responses to RSV infection, suggesting possible mechanisms for therapeutic intervention.     

Type 1 interferons and myositis

Recent studies suggest a mechanistic role for molecules induced by type 1 interferons in the pathogenesis of some forms of myositis. For dermatomyositis, evidence that these molecules injure myofibers seems especially strong. In the group of disorders known as polymyositis, the study of blood samples suggests a potential role. It is unknown what drives the sustained presence of type 1 interferon-inducible molecules in these diseases, as the type 1 interferons themselves have not been specifically detected along with their downstream biomarkers. Therapeutic development for blockade of IFNα is in progress aided by the identification of blood genomic biomarkers.     

Differences in the regulation of CD4 and CD8 T-cell clones during immune responses

The functional units of immune response are lymphocyte clones. Analysis of lymphocyte life span in vivo shows that the overall turnover of CD4 and CD8 lymphocytes does not differ greatly. Recently, molecular methods have been developed which allow a global analysis of T-cell clones responding to an antigen in vivo. We have used a sensitive, modified heteroduplex analysis to follow T-cell clones responding to Epstein-Barr virus in acute infectious mononucleosis (AIM). Strikingly, all the many large clones detected in freshly isolated AIM blood were found within the CD8 fraction. CD4 clonal populations responding to the soluble recall antigen tetanus toxoid could only be detected after in vitro re-stimulation. These data imply that CD4 responses may be more polyclonal than those of CD8 cells and that the size of CD4 clones is more tightly regulated. Several molecular mechanisms may contribute to this. Up-regulation of telomerase allows very large expansions of CD8 cells to occur without exhaustion of proliferative capacity.     

Lymphocytic choriomeningitis virus infection yields overlapping CD4+ and CD8+ T-cell responses

Activation of CD4⁺ T cells helps establish and sustain other immune responses. We have previously shown that responses against a broad set of nine CD4⁺ T-cell epitopes were present in the setting of lymphocytic choriomeningitis virus (LCMV) Armstrong infection in the context of H-2^d. This is quite disparate to the H-2^b setting, where only two epitopes have been identified. We were interested in determining whether a broad set of responses was unique to H-2^d or whether additional CD4⁺ T-cell epitopes could be identified in the setting of the H-2^b background. To pursue this question, we infected C57BL/6 mice with LCMV Armstrong and determined the repertoire of CD4⁺ T-cell responses using overlapping 15-mer peptides corresponding to the LCMV Armstrong sequence. We confirmed positive responses by intracellular cytokine staining and major histocompatibility complex (MHC)-peptide binding assays. A broad repertoire of responses was identified, consisting of six epitopes. These epitopes originate from the nucleoprotein (NP) and glycoprotein (GP). Out of the six newly identified CD4⁺ epitopes, four of them also stimulate CD8⁺ T cells in a statistically significant manner. Furthermore, we assessed these CD4⁺ T-cell responses during the memory phase of LCMV Armstrong infection and after infection with a chronic strain of LCMV and determined that a subset of the responses could be detected under these different conditions. This is the first example of a broad repertoire of shared epitopes between CD4⁺ and CD8⁺ T cells in the context of viral infection. These findings demonstrate that immunodominance is a complex phenomenon in the context of helper responses.     

Virus-specific CD4+ and CD8+ cytotoxic T-cell responses and long-term T-cell memory in individuals vaccinated against polio

The presence of poliovirus (PV)-specific CD4(+) T cells in individuals vaccinated against polio has been shown, but CD8(+) T-cell responses have not been described. Here, we functionally characterize the CD4(+) T-cell response and show for the first time that dendritic cells and macrophages can stimulate PV-specific CD8(+) T-cell responses in vitro from vaccinees. Both CD4(+) T and CD8(+) T cells secrete gamma interferon in response to PV antigens and are cytotoxic via the perforin/granzyme B-mediated pathway. Furthermore, the T cells also recognize and kill Sabin 1 vaccine-infected targets. The macrophage-stimulated CD4(+) T and CD8(+) T cells most likely represent memory T cells that persist for long periods in vaccinated individuals. Thus, immunity to PV vaccination involves not only an effective neutralizing antibody titer but also long-term CD4(+) and CD8(+) cytotoxic T-cell responses.     

CD8(+) T-cell responses to adeno-associated virus capsid in humans

Hepatic adeno-associated virus (AAV)-serotype 2 mediated gene transfer results in transgene product expression that is sustained in experimental animals but not in human subjects. We hypothesize that this is caused by rejection of transduced hepatocytes by AAV capsid-specific memory CD8(+) T cells reactivated by AAV vectors. Here we show that healthy subjects carry AAV capsid-specific CD8(+) T cells and that AAV-mediated gene transfer results in their expansion. No such expansion occurs in mice after AAV-mediated gene transfer. In addition, we show that AAV-2 induced human T cells proliferate upon exposure to alternate AAV serotypes, indicating that other serotypes are unlikely to evade capsid-specific immune responses.     

Effects of neutralizing antibodies on escape from CD8+ T-cell responses in HIV-1 infection

Despite substantial advances in our knowledge of immune responses against HIV-1 and of its evolution within the host, it remains unclear why control of the virus eventually breaks down. Here, we present a new theoretical framework for the infection dynamics of HIV-1 that combines antibody and CD8⁺ T-cell responses, notably taking into account their different lifespans. Several apparent paradoxes in HIV pathogenesis and genetics of host susceptibility can be reconciled within this framework by assigning a crucial role to antibody responses in the control of viraemia. We argue that, although escape from or progressive loss of quality of CD8⁺ T-cell responses can accelerate disease progression, the underlying cause of the breakdown of virus control is the loss of antibody induction due to depletion of CD4⁺ T cells. Furthermore, strong antibody responses can prevent CD8⁺ T-cell escape from occurring for an extended period, even in the presence of highly efficacious CD8⁺ T-cell responses.     

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.     

Analysis of 'driver' and 'passenger' CD8+ T-cell responses against variable viruses

Variable viruses, such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV), persist despite host immune responses directed against them. Numerous lines of evidence have suggested that antiviral CD8+ T-cell responses are key among these immune responses, but these vary widely in their ability to contain virus. We propose that only a proportion of responses may exert significant antiviral pressure ('driver' responses), leading to control over viral replication (protection) and/or, ultimately, selection of escape mutants. Another set of responses may exert only weak pressure on the virus ('passenger' responses): these neither protect nor select. To examine this we have analysed (using established databases of HIV and HCV sequences and cytotoxic T-lymphocyte (CTL) epitopes, and published experimental datasets) two important features--predicted binding of the epitope to major histocompatibility complex molecule and observed variability of the epitope--that might distinguish such responses. We find that a high predicted binding estimate could only explain a limited set of 'driver' responses associated with protection or selection. There is statistical evidence that readily defined (and non-protective) CTL responses target regions associated with lower levels of viral variability. Taken together, this suggests that a large number of well-documented responses may represent 'passengers' and we propose a mechanism that might explain their presence.     

Activation and inactivation of antiviral CD8 T cell responses during murine pneumovirus infection

Pneumonia virus of mice (PVM) is a natural pathogen of mice and has been proposed as a tractable model for the replication of a pneumovirus in its natural host, which mimics human infection with human respiratory syncytial virus (RSV). PVM infection in mice is highly productive in terms of virus production compared with the situation seen with RSV in mice. Because RSV suppresses CD8 T cell effector function in the lungs of infected mice, we have investigated the nature of PVM-induced CD8 T cell responses to study pneumovirus-induced T cell responses in a natural virus-host setting. PVM infection was associated with a massive influx of activated CD8 T cells into the lungs. After identification of three PVM-specific CD8 T cell epitopes, pulmonary CD8 T cell responses were enumerated. The combined frequency of cytokine-secreting CD8 T cells specific for the three epitopes was much smaller than the total number of activated CD8 T cells. Furthermore, quantitation of the CD8 T cell response against one of these epitopes (residues 261-270 from the phosphoprotein) by MHC class I pentamer staining and by in vitro stimulation followed by intracellular IFN-gamma and TNF-alpha staining indicated that the majority of pulmonary CD8 specific for the P261 epitope were deficient in cytokine production. This deficient phenotype was retained up to 96 days postinfection, similar to the situation in the lungs of human RSV-infected mice. The data suggest that PVM suppresses T cell effector functions in the lungs.     

Purification of a modified form of bovine antithrombin III as an HIV-1 CD8+ T-cell antiviral factor

CD8(+) T-cells secrete soluble factor(s) capable of inhibiting both R5- and X4-tropic strains of human immunodeficiency virus type 1 (HIV-1). CCR5 chemokine ligands, released from activated CD8(+) T-cells, contribute to the antiviral activity of these cells. These CC-chemokines, however, do not account for all CD8(+) T-cell antiviral factor(s) (CAF) released from these cells, particularly because the elusive CAF can inhibit the replication of X4 HIV-1 strains that use CXCR4 and not CCR5 as a coreceptor. Here we demonstrate that activated CD8(+) T-cells of HIV-1-seropositive individuals modify serum bovine antithrombin III into an HIV-1 inhibitory factor capable of suppressing the replication of X4 HIV-1. These data indicate that antithrombin III may play a role in the progression of HIV-1 disease.     

Influenza A virus-specific CD8 T-cell responses: from induction to function

Seasonal influenza virus infection is a leading cause of illness and mortality in young children and the elderly each year. Current influenza vaccines generate protective antibody responses; however, these must be given annually to provide protection against serologically distinct viruses. By contrast, CD8(+) T cells are capable of recognizing conserved antigenic determinants within the influenza virion and, as such, may provide protection against a number of variant strains of the virus. CD8(+) T cells play a critical key role in controlling and resolving influenza virus infections via the production of cytokines and cytolytic mediators. This article focuses on the induction of the influenza-specific CD8(+) T-cell response and how these cells acquire and maintain effector function after induction. Moreover, we discuss how cytotoxic T-lymphocyte function correlates with protection following vaccination.     

Expansion of protective CD8+ T-cell responses driven by recombinant cytomegaloviruses

CD8(+) T cells are critical for the control of many persistent viral infections, such as human immunodeficiency virus, hepatitis C virus, Epstein-Barr virus, and cytomegalovirus (CMV). In most infections, large CD8(+)-T-cell populations are induced early but then contract and are maintained thereafter at lower levels. In contrast, CD8(+) T cells specific for murine CMV (MCMV) have been shown to gradually accumulate after resolution of primary infection. This unique behavior is restricted to certain epitopes, including an immunodominant epitope derived from the immediate-early 1 (IE1) gene product. To explore the mechanism behind this further, we measured CD8(+)-T-cell-mediated immunity induced by recombinant MCMV-expressing epitopes derived from influenza A virus or lymphocytic choriomeningitis virus placed under the control of an IE promoter. We observed that virus-specific CD8(+)-T-cell populations were induced and that these expanded gradually over time. Importantly, these CD8(+) T cells provided long-term protection against challenge without boosting. These results demonstrate a unique pattern of accumulating T cells, which provide long-lasting immune protection, that is independent of the initial immunodominance of the epitope and indicates the potential of T-cell-inducing vaccines based on persistent vectors.