Activation, differentiation, and migration of naive virus-specific CD8+ T cells during pulmonary influenza virus infection

The low precursor frequency of individual virus-specific CD8(+) T cells in a naive host makes the early events of CD8(+) T cell activation, proliferation, and differentiation in response to viral infection a challenge to identify. We have therefore examined the response of naive CD8(+) T cells to pulmonary influenza virus infection with a murine adoptive transfer model using hemagglutinin-specific TCR transgenic CD8(+) T cells. Initial activation of CD8(+) T cells occurs during the first 3 days postinfection exclusively within the draining lymph nodes. Acquisition of CTL effector functions, including effector cytokine and granule-associated protease expression, occurs in the draining lymph nodes and differentially correlates with cell division. Division of activated CD8(+) T cells within the draining lymph nodes occurs in an asynchronous manner between days 3 and 4 postinfection. Despite the presence of Ag for several days within the draining lymph nodes, dividing T cells do not appear to maintain contact with residual Ag. After multiple cell divisions, CD8(+) T cells exit the draining lymph nodes and migrate to the infected lung. Activated CD8(+) T cells also disseminate throughout lymphoid tissue including the spleen and distal lymph nodes following their emigration from draining lymph nodes. These results demonstrate an important role for draining lymph nodes in orchestrating T cell responses during a local infection of a discrete organ to generate effector CD8(+) T cells capable of responding to infection and seeding peripheral lymphoid tissues.     

Induction of autoreactive CD8+ cytotoxic T cells during Theiler's murine encephalomyelitis virus infection: implications for autoimmunity

Theiler's murine encephalomyelitis virus (TMEV) belongs to the family Picornaviridae and causes demyelinating disease in the spinal cords of infected mice. Although immune responses have been shown to play an important role in demyelination, the precise effector mechanism(s) is unknown. Potentially autoreactive cytotoxic cells could contribute to the destruction. We tested whether an autoreactive cell induced by TMEV infection mediated cytotoxicity by using a 5-h (51)Cr release assay in SJL/J mice. Spleen cells from TMEV-infected mice were stimulated with irradiated TMEV antigen-presenting cells and used as effector cells. The effector cells differed from conventional cytotoxic T cells since these cells could kill both TMEV-infected and uninfected syngeneic or semisyngenic cell lines (PSJLSV and BxSF11gSV) but could not kill an allogeneic cell line (C57SV). The TMEV-induced autoreactive cells were also different from conventional natural killer (NK) cells or lymphokine-activated killer (LAK) cells, because they could kill neither NK cell-sensitive YAC-1 nor NK cell-resistant P815 and EL4 cells. Induction of autoreactive cells was not detected in vaccinia virus infection. The autoreactive killing required direct cell-to-cell contact and was mediated by a Fas-FasL pathway but not by a perforin pathway. The phenotype of the killer cells was CD3(+) CD4(-) CD8(+). Intracerebral inoculation of the effector cells into naive mice caused meningitis and perivascular cuffing not only in the brain parenchyma but also in the spinal cord, with no evidence of viral antigen-positive cells. This is the first report demonstrating that TMEV can induce autoreactive cytotoxic cells that induce central nervous system pathology.     

Regulatory T cells suppress in vitro proliferation of virus-specific CD8+ T cells during persistent hepatitis C virus infection

The basis of chronic infection following exposure to hepatitis C virus (HCV) infection is unexplained. One factor may be the low frequency and immature phenotype of virus-specific CD8(+) T cells. The role of CD4(+)CD25(+) T regulatory (T(reg)) cells in priming and expanding virus-specific CD8(+) T cells was investigated. Twenty HLA-A2-positive patients with persistent HCV infection and 46 healthy controls were studied. Virus-specific CD8(+) T-cell proliferation and gamma interferon (IFN-gamma) frequency were analyzed with/without depletion of T(reg) cells, using peptides derived from HCV, Epstein-Barr virus (EBV), and cytomegalovirus (CMV). CD4(+)CD25(+) T(reg) cells inhibited anti-CD3/CD28 CD8(+) T-cell proliferation and perforin expression. Depletion of CD4(+)CD25(+) T(reg) cells from chronic HCV patients in vitro increased HCV and EBV peptide-driven expansion (P = 0.0005 and P = 0.002, respectively) and also the number of HCV- and EBV-specific IFN-gamma-expressing CD8(+) T cells. Although stimulated CD8(+) T cells expressed receptors for transforming growth factor beta and interleukin-10, the presence of antibody to transforming growth factor beta and interleukin-10 had no effect on the suppressive effect of CD4(+)CD25(+) regulatory T cells on CD8(+) T-cell proliferation. In conclusion, marked CD4(+)CD25(+) regulatory T-cell activity is present in patients with chronic HCV infection, which may contribute to weak HCV-specific CD8(+) T-cell responses and viral persistence.     

Kinetics of virus-specific CD8+ T cells and the control of human immunodeficiency virus infection

Several primate models indicate that cytotoxic T lymphocyte-inducing vaccines may be unable to prevent human immunodeficiency virus infection but may have a long-term benefit in controlling viral replication and delaying disease progression. Here we show that analysis of the kinetics of antigen-specific CD8+ T-cell expansion suggests a delay in activation following infection that allows unimpeded early viral replication. Viral kinetics do not differ between controls and vaccinees during this delay phase. An increase in virus-specific CD8+ T-cell numbers around day 10 postinfection coincides with a slowing in viral replication in vaccinees and reduces peak viral loads by around 1 log. However, this response is too little too late to prevent establishment of persistent infection.     

T cells with a CD4+CD25+ regulatory phenotype suppress in vitro proliferation of virus-specific CD8+ T cells during chronic hepatitis C virus infection

Chronic hepatitis C virus (HCV) infection is associated with impaired proliferative, cytokine, and cytotoxic effector functions of HCV-specific CD8(+) T cells that probably contribute significantly to viral persistence. Here, we investigated the potential role of T cells with a CD4(+)CD25(+) regulatory phenotype in suppressing virus-specific CD8(+) T-cell proliferation during chronic HCV infection. In vitro depletion studies and coculture experiments revealed that peptide specific proliferation as well as gamma interferon production of HCV-specific CD8(+) T cells were inhibited by CD4(+)CD25(+) T cells. This inhibition was dose dependent, required direct cell-cell contact, and was independent of interleukin-10 and transforming growth factor beta. Interestingly, the T-cell-mediated suppression in chronically HCV-infected patients was not restricted to HCV-specific CD8(+) T cells but also to influenza virus-specific CD8(+) T cells. Importantly, CD4(+)CD25(+) T cells from persons recovered from HCV infection and from healthy blood donors exhibited significantly less suppressor activity. Thus, the inhibition of virus-specific CD8(+) T-cell proliferation was enhanced in chronically HCV-infected patients. This was associated with a higher frequency of circulating CD4(+)CD25(+) cells observed in this patient group. Taken together, our results suggest that chronic HCV infection leads to the expansion of CD4(+)CD25(+) T cells that are able to suppress CD8(+) T-cell responses to different viral antigens. Our results further suggest that CD4(+)CD25(+) T cells may contribute to viral persistence in chronically HCV-infected patients and may be a target for immunotherapy of chronic hepatitis C.     

Memories of virus-specific CD8+ T cells

This brief review focuses on the way that our understanding of virus-specific CD8(+) T-cell-mediated immunity evolved, giving particular attention to the early impact of the program at the Australian National University. The story developed through a sequence of distinct eras, each of which can be defined in the context of the technologies available at that time. The progress has been enormous, but there is a great deal still to be learned. A particular challenge is to use what we know for human benefit.     

Functional impairment of simian immunodeficiency virus-specific CD8+ T cells during the chronic phase of infection

In an attempt to determine why high frequencies of circulating virus-specific CD8+ T cells are unable to control human immunodeficiency virus and simian immunodeficiency virus (SIV) replication, we assessed the functional nature of SIV-specific CD8+ lymphocytes. After vaccination and early after infection, nearly all tetramer-staining CD8+ cells produced gamma interferon in response to their specific stimulus. However, by 4 months postinfection with pathogenic SIVmac239, signs of functional impairment in the CD8+ T-cell compartment were detected which might prevent these T cells from efficiently controlling the infection during the chronic phase.     

Visualization of polyoma virus-specific CD8+ T cells in vivo during infection and tumor rejection.

T cells are critical for clearing infection and preventing tumors induced by polyoma virus, a natural murine papovavirus. We previously identified the immunodominant epitope for polyoma virus-specific CTL in tumor-resistant H-2k mice as the Dk-restricted peptide, MT389-397, derived from the polyoma middle T oncoprotein. In this study, we developed tetrameric Dk complexes containing the MT389-397 peptide to directly visualize and enumerate MT389-397-specific CTL during polyoma virus infection. We found that Dk/MT389 tetramer+CD8+ T cells undergo a massive expansion during primary infection such that by day 7 postinfection these Ag-specific CD8+ T cells constitute approximately 20% of the total and approximately 40% of the activated CD8+ T cells in the spleen. This expansion of Dk/MT389 tetramer+CD8+ T cells parallels the emergence of MT389-397-specific ex vivo cytolytic activity and clearance of polyoma virus. Notably, Dk/MT389 tetramer+CD8+ T cells are maintained in memory at very high levels. The frequencies of Dk/MT389 tetramer+CD8+ effector and memory T cells in vivo match those of CD8+ T cells producing intracellular IFN-gamma after 6-h in vitro stimulation by MT389-397 peptide. Consistent with preferential Vbeta6 expression by MT389-397-specific CD8+CTL lines and clones, Dk/MT389 tetramer+CD8+ T cells exhibit biased expression of this Vbeta gene segment. Finally, we show that Dk/MT389 tetramer+CD8+ T cells efficiently infiltrate a polyoma tumor challenge to virus-immune mice. Taken together, these findings strongly implicate virus-induced MT389-397-specific CD8+ T cells as essential effectors in eliminating polyoma-infected and polyoma-transformed cells in vivo.     

CD4+ and CD8+ T cells exhibit differential requirements for CCR7-mediated antigen transport during influenza infection

Upon encounter of viral Ags in an inflammatory environment, dendritic cells up-regulate costimulatory molecules and the chemokine receptor CCR7, with the latter being pivotal for their migration to the lymph node. By utilizing mice deficient in CCR7, we have examined the requirement of dendritic cell-mediated Ag transport from the lung to the draining lymph node for the induction of anti-influenza immune responses in vivo. We found that CCR7-mediated migration of dendritic cells was more crucial for CD8(+) T cell than CD4(+) T cell responses. While no specific CD8(+) T cell response could be detected in the airways or lymphoid tissues during the primary infection, prolonged infection in CCR7-deficient mice did result in a sustained inflammatory chemokine profile, which led to nonspecific CD8(+) T cell recruitment to the airways. The recruitment of influenza-specific CD4(+) T cells to the airways was also below levels of detection in the absence of CCR7 signaling, although a small influenza-specific CD4(+) T cell population was detectable in the draining lymph node, which was sufficient for the generation of class-switched anti-influenza Abs and a normal CD4(+) T cell memory population. Overall, our data show that CCR7-mediated active Ag transport is differentially required for CD4(+) and CD8(+) T cell expansion during influenza infection.     

Costimulation requirements for antiviral CD8+ T cells differ for acute and persistent phases of polyoma virus infection

The requirement for costimulation in antiviral CD8+ T cell responses has been actively investigated for acutely resolved viral infections, but it is less defined for CD8+ T cell responses to persistent virus infection. Using mouse polyoma virus (PyV) as a model of low-level persistent virus infection, we asked whether blockade of the CD40 ligand (CD40L) and CD28 costimulatory pathways impacts the magnitude and function of the PyV-specific CD8+ T response, as well as the humoral response and viral control during acute and persistent phases of infection. Costimulation blockade or gene knockout of either CD28 or CD40L substantially dampened the magnitude of the acute CD8+ T cell response; simultaneous CD28 and CD40L blockade severely depressed the acute T cell response, altered the cell surface phenotype of PyV-specific CD8+ T cells, decreased PyV VP1-specific serum IgG titers, and resulted in an increase in viral DNA levels in multiple organs. CD28 and CD40L costimulation blockade during acute infection also diminished the memory PyV-specific CD8+ T cell response and serum IgG titer, but control of viral persistence varied between mouse strains and among organs. Interestingly, we found that CD28 and CD40L costimulation is dispensable for generating and/or maintaining PyV-specific CD8+ T cells during persistent infection; however, blockade of CD27 and CD28 costimulation in persistently infected mice caused a reduction in PyV-specific CD8+ T cells. Taken together, these data indicate that CD8+ T cells primed within the distinct microenvironments of acute vs persistent virus infection differ in their costimulation requirements.     

Impaired effector function of hepatitis C virus-specific CD8+ T cells in chronic hepatitis C virus infection

The cellular immune response contributes to clearance of hepatitis C virus (HCV) and persists for decades after recovery from infection. The immunological basis for the inefficiency of the cellular immune response in chronically infected persons is not known. Here, we used four HLA-A2 tetramers, specific for two HCV core and two HCV NS3 epitopes, to investigate at the single-cell level effector function and phenotype of HCV-specific CD8+ T cells in 20 chronically infected and 12 long-term recovered patients. Overall, HCV-specific, tetramer+ T cells were more frequently found in PBMCs of chronically infected patients than in those of recovered patients. However, when compared with HCV-tetramer+ T cells of recovered patients, they displayed an impaired proliferative capacity. As a result of the impaired proliferative capacity, HCV-specific T cell lines derived from chronically infected patients displayed less peptide-specific cytotoxicity than those from recovered patients. In addition, proliferation and ex vivo IFN-gamma production of HCV-tetramer+ cells, but not influenza-virus-specific T cells, were defective in chronically infected patients and could not be restored by in vitro stimulation with peptide and IL-2. At least three distinct phenotypes of HCV-specific CD8+ T cells were identified and associated with certain functional characteristics. In addition, impairment of proliferative, cytokine, and cytotoxic effector functions of tetramer+ T cells in viremic patients was associated with weak ex vivo HCV-specific CD4+ T cell responses. Thus, the defective functions of HCV-specific CD8+ T cells might contribute to viral persistence in chronically infected patients, and knowledge on their reversibility may facilitate the development of immunotherapeutic vaccines.     

Complement opsonization enhances friend virus infection of B cells and thereby amplifies the virus-specific CD8+ T cell response

B cells are one of the targets of Friend virus (FV) infection, a well-established mouse model often used to study retroviral infections in vivo. Although B cells may be effective in stimulating cytotoxic T lymphocyte responses, studies involving their role in FV infection have mainly focused on neutralizing antibody production. Here we show that polyclonal activation of B cells promotes their infection with FV both in vitro and in vivo. Furthermore, we demonstrate that complement opsonization of Friend murine leukemia virus (F-MuLV) enhances infection of B cells, which correlates with increased potency of B cells to activate FV-specific CD8(+) T cells.     

Attrition of virus-specific memory CD8+ T cells during reconstitution of lymphopenic environments

Viruses can cause a severe lymphopenia early in infection and a subsequent, lasting loss of pre-existing CD8(+) memory T cells. We therefore questioned how well virus Ag-specific memory CD8(+) T cells could reconstitute mice rendered lymphopenic as a consequence of genetics, irradiation, or viral or poly(I:C)-induced cytokines. In each case, reconstitution of the CD8(+) compartment was associated with limited division of virus-specific memory T cells and a reduction in their proportion. This indicates that foreign Ag-experienced CD44(high)CD8(+) memory T cells may respond differently to homeostatic signals than other CD44(high)CD8(+) cells, and that events inducing lymphopenia may lead to a permanent reduction in T cell memory.     

Differential in vitro activation of CD4+CD8- and CD8+CD4- herpes simplex virus-specific human cytotoxic T cells

In order to clarify the differential activation of CD4+ and CD8+ HSV-specific CTL, we compared the characteristics of CTL generated by different methods of in vitro HSV stimulation by treatment of effectors with anti-CD4 and anti-CD8 mAb and C after the elimination of nonspecific cytotoxic effector cells. Cell-free HSV mainly activated CD4+ CTL precursors, whereas HSV-infected fibroblasts were more effective in activating CD8+ CTL precursors than CD4+ CTL precursors. In addition, limiting dilution analyses with enriched T cells from two HSV-seropositive donors revealed that the frequency of HSV-specific CD4+ CTL precursors responsive to stimulation with free HSV was approximately 1/4,000 to 6,000 CD4+ T cells, whereas that of precursors responsive to stimulation with HSV-infected fibroblasts was approximately 1/19,000 to 22,000 CD4+ T cells. Conversely, the frequency of CD8+ CTL precursors in peripheral blood responsive to stimulation with free HSV was approximately 1/28,000 to 30,000 CD8+ T cells, whereas that of precursors responsive to stimulation with HSV-infected fibroblasts was approximately 1/10,000 to 11,000 CD8+ T cells. The present data suggest that generalized viral infection due to cell-free viruses is fought mainly by CD4+ CTL, which have previously been reported to possess both cytotoxicity and helper function, and that localized viral infection on HLA class II-negative fibroblasts is prevented from spreading to adjacent cells mainly by CD8+ CTL. Such differential activation of CD4+ and CD8+ CTL seems probable when considering the protective mechanisms against viral infection.     

Sequential activation of CD8+ T cells in the draining lymph nodes in response to pulmonary virus infection

We have used a TCR-transgenic CD8+ T cell adoptive transfer model to examine the tempo of T cell activation and proliferation in the draining lymph nodes (DLN) in response to respiratory virus infection. The T cell response in the DLN differed for mice infected with different type A influenza strains with the onset of T cell activation/proliferation to the A/JAPAN virus infection preceding the A/PR8 response by 12-24 h. This difference in T cell activation/proliferation correlated with the tempo of accelerated respiratory DC (RDC) migration from the infected lungs to the DLN in response to influenza virus infection, with the migrant RDC responding to the A/JAPAN infection exhibiting a more rapid accumulation in the lymph nodes (i.e., peak migration for A/JAPAN at 18 h, A/PR8 at 24-36 h). Furthermore, in vivo administration of blocking anti-CD62L Ab at various time points before/after infection revealed that the virus-specific CD8+ T cells entered the DLN and activated in a sequential "conveyor belt"-like fashion. These results indicate that the tempo of CD8+ T cell activation/proliferation after viral infection is dependent on the tempo of RDC migration to the DLN and that T cell activation occurs in an ordered sequential fashion.     

Differential usage of cellular niches by cytomegalovirus versus EBV- and influenza virus-specific CD8+ T cells

Immunological memory provides long-term protection against reinfection or reactivation of pathogens. Murine memory T cell populations may be compressed following infections with new pathogens. Humans have to retain memory T cells directed against a variety of microbes for many decades. Under these circumstances, the effect of pathogens that mount robust T cell reactivity on the pre-existing memory directed against unrelated microbes is unknown. In this study, we studied peripheral blood memory CD8+ T cells directed against different viruses following primary CMV infection in renal transplant recipients. The entrance of CMV-specific CD8+ T cells expanded the Ag-primed CD8+ T cell compartment rather than competing for space with pre-existing memory T cells specific for persistent or cleared viruses. Neither numbers nor phenotype of EBV- or influenza-specific CD8+ T cells was altered by primary CMV infection. CMV-specific CD8+ T cells accumulated over time, resulting in increased total CD8+ T cell numbers. Additionally, they acquired a highly differentiated cytolytic phenotype that was clearly distinct from EBV- or influenza-reactive T cells. Thus, the human immune system appears to be flexible and is able to expand when encountering CMV. In view of the phenotypic differences between virus-specific T cells, this expansion may take place in cellular niches different from those occupied by EBV- or influenza-specific T cells, thereby preserving immunity to these pathogens.     

The role of virus-specific CD4+ T cells in the control of Epstein-Barr virus infection

Epstein-Barr virus (EBV) establishes lifelong persistent infections in humans and has been implicated in the pathogenesis of several human malignancies. Protective immunity against EBV is mediated by T cells, as indicated by an increased incidence of EBV-associated malignancies in immunocompromised patients, and by the successful treatment of EBV-associated post-transplant lymphoproliferative disease (PTLD) in transplant recipients by the infusion of polyclonal EBV-specific T cell lines. To implement this treatment modality as a conventional therapeutic option, and to extend this protocol to other EBV-associated diseases, generic and more direct approaches for the generation of EBV-specific T cell lines enriched in disease-relevant specificities need to be developed. To this aim, we studied the poorly defined EBV-specific CD4+ T cell response during acute and chronic infection.