Nonspecific recruitment of memory CD8+ T cells to the lung airways during respiratory virus infections

Previous studies have shown that heterologous viral infections have a significant impact on pre-existing memory T cell populations in secondary lymphoid organs through a combination of cross-reactive and bystander effects. However, the impact of heterologous viral infections on effector/memory T cells in peripheral sites is not well understood. In this study, we have analyzed the impact of a heterologous influenza virus infection on Sendai virus-specific CD8(+) effector/memory cells present in the lung airways. The data show a transient increase in the numbers of Sendai virus nucleoprotein 324-332/K(b)-specific CD8(+) memory T cells in the airways of the influenza-infected mice peaking around day 4 postinfection. Intratracheal transfer studies and 5-bromo-2'-deoxyuridine incorporation demonstrate that this increase is due to the recruitment of resting memory cells into the airways. In addition, the data show that these immigrating memory cells are phenotypically distinct from the resident memory T cells of the lung airways. A similar influx of nonproliferating Sendai virus nucleoprotein 324-332/K(b)-specific CD8(+) memory T cells is also induced by a secondary (homologous) infection with Sendai virus. Together, these data suggest that inflammation can accelerate memory T cell migration to nonlymphoid tissues and is a part of the normal recall response during respiratory infections.     

Renewal of peripheral CD8+ memory T cells during secondary viral infection of antibody-sufficient mice

Kinetic studies and short pulses of injected 5-bromo-2-deoxyuridine have been used to analyze the development and renewal of peripheral CD8(+) memory T cells in the lungs during primary and secondary respiratory virus infections. We show that developing peripheral CD8(+) memory T cells proliferate during acute viral infection with kinetics that are indistinguishable from those of lymphoid CD8(+) memory T cells. Secondary exposure to the same virus induces a new round of T cell proliferation and extensive renewal of the peripheral and lymphoid CD8(+) memory T cell pools in both B cell-deficient mice and mice with immune Abs. In mice with virus-specific Abs, CD8(+) T cell proliferation takes place with minimal inflammation or effector cell recruitment to the lungs. The delayed arrival of CD8(+) memory T cells to the lungs of these animals suggests that developing memory cells do not require the same inflammatory signals as effector cells to reach the lung airways. These studies provide important new insight into mechanisms that control the maintenance and renewal of peripheral memory T cell populations during natural infections.     

Cutting edge: Antigen is not required for the activation and maintenance of virus-specific memory CD8+ T cells in the lung airways

Respiratory virus infections establish a population of memory CD8(+) T cells in the lung airways that persist for months after infection. However, the relationship between Ag-specific memory T cells in the lung airways and the systemic memory T cell pool is not well understood. The majority of lung airway memory T cells express a highly activated phenotype (CD69(+)/CD127(-)), suggesting that recent Ag stimulation is required to drive T cell activation and recruitment to the lung airways. In this study, we demonstrate that the lung airway environment itself in the absence of cognate Ag alters the expression of acute activation markers such as CD69 and CD127 on memory CD8(+) T cells. Furthermore, the steady-state recruitment of virus-specific memory CD8(+) T cells to the lung airways from the circulation can occur without recent Ag stimulation. These findings alter the current perceptions concerning the contribution of Ag to the maintenance of peripheral T cell memory.     

Long-term maintenance of virus-specific effector memory CD8+ T cells in the lung airways depends on proliferation

Recent studies have shown that virus-specific effector memory T cells can be recovered from the lung airways long after clearance of a respiratory virus infection. These cells are thought to play an important role in the recall response to secondary viral infection. It is currently unclear whether these cells actually persist at this site or are maintained by continual proliferation and recruitment. In this study, we have analyzed the mechanisms underlying the persistence of memory CD8(+) T cells in the lung airway lumina following recovery from a respiratory virus infection. The data identify two distinct populations of memory cells. First, a large population Ag-specific CD8(+) T cells is deposited in the airways during the acute response to the virus. These cells persist in a functional state for several weeks with minimal further division. Second, a smaller population of Ag-specific CD8(+) T cells is maintained in the lung airways by homeostatic proliferation and migration to lung airways after viral clearance. This rate of proliferation is identical to that observed in the spleen, suggesting that these cells may be recent immigrants from the lymphoid organs. These data have significant implications for vaccines designed to promote cellular immunity at mucosal sites such as the lung.     

Antibody-independent antiviral function of memory CD4+ T cells in vivo requires regulatory signals from CD8+ effector T cells

Previous studies have shown that vaccine-primed CD4(+) T cells can mediate accelerated clearance of respiratory virus infection. However, the relative contributions of Ab and CD8(+) T cells, and the mechanism of viral clearance, are poorly understood. Here we show that control of a Sendai virus infection by primed CD4(+) T cells is mediated through the production of IFN-gamma and does not depend on Ab. This effect is critically dependent on CD8(+) cells for the expansion of CD4(+) T cells in the lymph nodes and the recruitment of memory CD4(+) T cells to the lungs. Passive transfer of a CD8(+) T cell supernatant into CD8(+) T cell-depleted, hemagglutinin-neuraminidase (HN)(421-436)-immune muMT mice substantially restored the virus-specific memory CD4(+) response and enhanced viral control in the lung. Together, the data demonstrate for the first time that in vivo primed CD4(+) T cells have the capacity to control a respiratory virus infection in the lung by an Ab-independent mechanism, provided that CD8(+) T cell "help" in the form of soluble factor(s) is available during the virus infection. These studies highlight the importance of synergistic interactions between CD4(+) and CD8(+) T cell subsets in the generation of optimal antiviral immunity.     

Activated antigen-specific CD8+ T cells persist in the lungs following recovery from respiratory virus infections

The poor correlation between cellular immunity to respiratory virus infections and the numbers of memory CD8(+) T cells in the secondary lymphoid organs suggests that there may be additional reservoirs of T cell memory to this class of infection. Here we identify a substantial population of Ag-specific T cells in the lung that persist for several months after recovery from an influenza or Sendai virus infection. These cells are present in high numbers in both the airways and lung parenchyma and can be distinguished from memory cell populations in the spleen and peripheral lymph nodes in terms of the relative frequencies among CD8(+) T cells, activation status, and kinetics of persistence. In addition, these cells are functional in terms of their ability to proliferate, express cytolytic activity, and secrete cytokines, although they do not express constitutive cytolytic activity. Adoptive transfer experiments demonstrated that the long-term establishment of activated T cells in the lung did not require infection in the lung by a pathogen carrying the inducing Ag. The kinetics of persistence of Ag-specific CD8(+) T cells in the lung suggests that they play a key role in protective cellular immunity to respiratory virus infections.     

Cutting edge: effector memory CD8+ T cells in the lung airways retain the potential to mediate recall responses

Previous studies have shown that long-lived memory CD8(+) T cells persist in the lung airways following the resolution of a murine Sendai virus infection. These cells are CD11a(low), noncytolytic, and do not proliferate in the lung airways raising the possibility that they are "end stage" or terminally differentiated memory cells. In this current report, we investigated the functional characteristics of these cells by analyzing their capacity to respond to secondary viral infection outside of the lung environment. We show that, after transfer into the bloodstream, CD11a(low) memory T cells from the lung airways can return to the secondary lymphoid tissue and respond to a secondary viral challenge. Furthermore, these cells re-express CD11a, which may contribute to their migratory and proliferative capacity. These data demonstrate that lung airway memory CD8(+) T cells are not terminally differentiated cells and retain the capacity to mediate recall responses to infection.     

Dynamics of human respiratory virus-specific CD8+ T cell responses in blood and airways during episodes of common cold

We determined the dynamics of CD8(+) T cells specific for influenza virus and respiratory syncytial virus in blood and tracheostoma aspirates of children during the course of respiratory infections. We showed that during localized respiratory infections the ratio of activated effector CD8(+) T cells to resting memory/naive CD8(+) T cells in peripheral blood increased significantly. Furthermore, the number of effector/memory T cells specific for respiratory viruses declined in blood and increased in the airways, suggesting that these T cells redistributed from blood to airways. T cells specific for the infecting virus were present in the airways for longer periods at increased levels than nonspecifically recruited bystander T cells. After clearance of the infection, the ratio of resting memory and naive CD8(+) T cells normalized in peripheral blood and also memory T cell numbers specific for unrelated viruses that declined during the infection due to bystander recruitment were restored. Taken together, these results showed a significant systemic T cell response during relatively mild secondary infections and extensive dynamics of virus-specific and nonspecific Ag-experienced T cells.     

Cellular immune responses associated with occult hepatitis C virus infection of the liver

Occult hepatitis C virus (HCV) infection is a type of recently identified chronic infection that is evidenced only by detection of HCV RNA in liver; patients consistently test negative for antibodies to HCV and HCV RNA in serum. Using ex vivo and in vitro measures of T-cell responses, we have identified functional virus-specific memory CD4(+) and CD8(+) T cells in the peripheral blood of patients with occult HCV infection. The features of the virus-specific T cells were consistent with immune surveillance functions, supporting previous exposure to HCV. In addition, the magnitudes of CD4(+) and CD8(+) T-cell responses were in parallel and correlated inversely with the extent of liver HCV infection. The detection of HCV-specific T cells in individuals in whom HCV RNA can persist in the liver despite the absence of viremia and antibodies indicates that HCV replication is prolonged in the face of virus-specific CD4(+) and CD8(+) T-cell responses. These findings demonstrate that HCV-specific cellular immune responses are markers not only of previous exposure to and recovery from HCV but also of ongoing occult HCV infection.     

Quantifying antigen-specific CD4 T cells during a viral infection: CD4 T cell responses are larger than we think

The number of virus-specific CD8 T cells increases substantially during an acute infection. Up to 90% of CD8 T cells are virus specific following lymphocytic choriomeningitis virus (LCMV) infection. In contrast, studies identifying virus-specific CD4 T cell epitopes have indicated that CD4 T cells often recognize a broader array of Ags than CD8 T cells, consequently making it difficult to accurately quantify the total magnitude of pathogen-specific CD4 T cell responses. In this study, we show that CD4 T cells become CD11a(hi)CD49d(+) after LCMV infection and retain this expression pattern into memory. During the effector phase, all the LCMV-specific IFN-γ(+) CD4 T cells display a CD11a(hi)CD49d(+) cell surface expression phenotype. In addition, only memory CD11a(hi)CD49d(+) CD4 T cells make IFN-γ after stimulation. Furthermore, upon secondary LCMV challenge, only CD11a(hi)CD49d(+) memory CD4 T cells from LCMV-immune mice undergo proliferative expansion, demonstrating that CD11a(hi)CD49d(+) CD4 T cells are truly Ag specific. Using the combination of CD11a and CD49d, we demonstrate that up to 50% of the CD4 T cells are virus specific during the peak of the LCMV response. Our results indicate that the magnitude of the virus-specific CD4 T cell response is much greater than previously recognized.     

Bone marrow of persistently hepatitis C virus-infected individuals accumulates memory CD8+ T cells specific for current and historical viral antigens: a study in patients with benign hematological disorders

The role of virus-specific T cells in hepatitis C virus (HCV) pathogenesis is not clear. Existing knowledge on the frequency, phenotype, and behavior of these cells comes from analyses of blood and liver, but other lymphoid compartments that may be important sites for functionally mature T cells have not yet been analyzed. We studied HCV-specific T cells from bone marrow, in comparison to those from peripheral blood and liver biopsy tissue, from 20 persistently HCV-infected patients with benign hematological disorders. Bone marrow contained a sizeable pool of CD8(+) T cells specific for epitopes from structural and nonstructural HCV proteins. These cells displayed the same effector memory phenotype as liver-derived equivalents and the same proliferative potential as blood-derived equivalents but had greater antiviral effector functions such as Ag-specific cytotoxicity and IFN-gamma production. These features were not shared by influenza virus-specific CD8(+) T cells in the same bone marrow samples. Despite their highly differentiated phenotype and activated status, some bone marrow-resident HCV-specific CD8(+) T cells were not directed against the infecting virus but, instead, against historical HCV Ags (i.e., viral species of a previous infection or minor viral species of the current infection). These findings provide a snapshot view of the distribution, differentiation, and functioning of virus-specific memory T cells in patients with persistent HCV infection.     

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.     

CD4+ T cell priming accelerates the clearance of Sendai virus in mice, but has a negative effect on CD8+ T cell memory

Current vaccines designed to promote humoral immunity to respiratory virus infections also induce potent CD4+ T cell memory. However, little is known about the impact of primed CD4+ T cells on the immune response to heterologous viruses that are serologically distinct, but that share CD4+ T cell epitopes. In addition, the protective capacity of primed CD4+ T cells has not been fully evaluated. In the present study, we addressed these two issues using a murine Sendai virus model. Mice were primed with an HN421-436 peptide that represents the dominant CD4+ T cell epitope on the hemagglutinin-neuraminidase (HN) of Sendai virus. This vaccination strategy induced strong CD4+ T cell memory to the peptide, but did not induce Abs specific for the Sendai virus virion. Subsequent Sendai virus infection of primed mice resulted in 1) a substantially accelerated virus-specific CD4+ T cell response in the pneumonic lung; 2) enhanced primary antiviral Ab-forming cell response in the mediastinal lymph nodes; and 3) accelerated viral clearance. Interestingly, the virus-specific CD8+ T cell response in the lung and the development of long-term memory CD8+ T cells in the spleen were significantly reduced. Taken together, our data demonstrate that primed CD4+ T cells, in the absence of pre-existing Ab, can have a significant effect on the subsequent immune responses to a respiratory virus infection.     

CC chemokine receptor 7 expression by effector/memory CD4+ T cells depends on antigen specificity and tissue localization during influenza A virus infection

The lung is an important entry site for respiratory pathogens such as influenza A virus. In order to combat such invading infectious agents, effector/memory T cells home to the lung and other peripheral tissues as well as lymphoid organs. In this process, chemokines and their receptors fulfill important roles in the guidance of T cells into such organs and specialized microenvironments within tissues. In this study, we determined if CD4(+) T cells residing in different lung compartments and draining lymph nodes of influenza A virus-infected and na?ve mice express receptors allowing their recirculation into secondary lymphoid tissues. We found high levels of l-selectin and CC chemokine receptor 7 (CCR7) expression in lung-derived CD4(+) T cells, similar to that detected on T cells in secondary lymphoid organs. Upon influenza A virus infection, the bulk of gamma interferon-positive (IFN-gamma(+)) and IFN-gamma(-) CD4(+) T cells recovered from lung parenchyma retained functional CCR7, whereas virus-specific IFN-gamma-producing T cells were CCR7(-). In contrast, a majority of virus-specific IFN-gamma(+) T cells in the lung draining lymph node were CCR7(+). Independent of infection, CD4(+) T cells obtained from the lung airways exhibited the lowest expression level of l-selectin and CCR7, indicating that T cells at this anatomical site represent the most differentiated effector cell type, lacking the ability to recirculate. Our results suggest that effector/memory T cells that enter inflammatory sites retain functional CCR7 expression, which is lost only upon response to viral antigen and after localization to the final effector site.     

Functionally Heterogeneous CD8+ T-Cell Memory Is Induced by Sendai Virus Infection of Mice

It has recently been established that memory CD8(+) T cells induced by viral infection are maintained at unexpectedly high frequencies in the spleen. While it has been established that these memory cells are phenotypically heterogeneous, relatively little is known about the functional status of these cells. Here we investigated the proliferative potential of CD8(+) memory T cells induced by Sendai virus infection. High frequencies of CD8(+) T cells specific for both dominant and subdominant Sendai virus epitopes persisted for many weeks after primary infection, and these cells were heterogeneous with respect to CD62L expression (approximately 20% CD62L(hi) and 80% CD62L(lo)). Reactivation of these cells with the antigenic peptide in vitro induced strong proliferation of antigen-specific CD8(+) T cells. However, approximately 20% of the cells failed to proliferate in vitro in response to a cognate peptide but nevertheless differentiated into effector cells and acquired full cytotoxic potential. These cells also expressed high levels of CD62L (in marked contrast to the CD62L(lo) status of the proliferating cells in the culture). Direct isolation of CD62L(hi) and CD62L(lo) CD8(+) T cells from memory mice confirmed the correlation of this marker with proliferative potential. Taken together, these data demonstrate that Sendai virus infection induces high frequencies of memory CD8(+) T cells that are highly heterogeneous in terms of both their phenotype and their proliferative potential.     

Independent regulation of lymphocytic choriomeningitis virus-specific T cell memory pools: relative stability of CD4 memory under conditions of CD8 memory T cell loss

Infection of mice with a series of heterologous viruses causes a reduction of memory CD8(+) T cells specific to viruses from earlier infections, but the fate of the virus-specific memory CD4(+) T cell pool following multiple virus infections has been unknown. We have previously reported that the virus-specific CD4(+) Th precursor (Thp) frequency remains stable into long-term immunity following lymphocytic choriomeningitis virus (LCMV) infection. In this study, we questioned whether heterologous virus infections or injection with soluble protein CD4 Ags would impact this stable LCMV-specific CD4(+) Thp memory pool. Limiting dilution analyses for IL-2-producing cells and intracellular cytokine staining for IFN-gamma revealed that the LCMV-specific CD4(+) Thp frequency remains relatively stable following multiple heterologous virus infections or protein Ag immunizations, even under conditions that dramatically reduce the LCMV-specific CD8(+) CTL precursor frequency. These data indicate that the CD4(+) and CD8(+) memory T cell pools are regulated independently and that the loss in CD8(+) T cell memory following heterologous virus infections is not a consequence of a parallel loss in the memory CD4(+) T cell population.     

Delayed clearance of Sendai virus in mice lacking class I MHC-restricted CD8+ T cells.

The role and interdependence of CD8+ and CD4+ alpha beta-T cells in the acute response after respiratory infection with the murine parainfluenza type 1 virus, Sendai virus, has been analyzed for H-2b mice. Enrichment of CD8+ virus-specific CTL effectors in the lungs of immunologically intact C57BL/6 animals coincided with the clearance of the virus from this site by day 10 after infection. Removal of the CD4+ T cells by in vivo mAb treatment did not affect appreciably either the recruitment of CD8+ T cells to the infected lung, or their development into virus-specific cytotoxic effectors. In contrast, depletion of the CD8+ subset delayed virus clearance, although most mice survived the infection. Transgenic H-2b F3 mice homozygous (-/-) for a beta 2 microglobulin (beta 2-m) gene disruption, which lack both class I MHC glycoproteins and mature CD8+ alpha beta-T cells, showed a comparable, delayed clearance of Sendai virus from the lung. Virus-specific, class II MHC-restricted CTL were demonstrated in both freshly isolated bronchoalveolar lavage populations and cultured lymph node and spleen tissue from the beta 2-m (-/-) transgenics. Treatment of the beta 2-m (-/-) mice with the mAb to CD4 led to delayed virus clearance and death, which was also the case for normal mice that were depleted simultaneously of the CD4+ and CD8+ subsets. These results indicate that, although classical class I MHC-restricted CD8+ cytotoxic T cells normally play a dominant role in the recovery of mice acutely infected with Sendai virus, alternative mechanisms involving CD4+ T cells exist and can compensate, in time, for the loss of CD8+ T cell function.     

Memory T cell populations in the lung airways are maintained by continual recruitment

Effector memory T cell populations in the periphery play a key role in cellular immune responses to secondary infections. However, it is unclear how these populations are maintained under steady-state conditions in nonlymphoid peripheral sites, such as the lung airways. In this study, we show that LFA-1 expression is selectively down-regulated following entry of memory T cells into the lung airways. Using Sendai virus as a mouse model of respiratory virus infection, we use LFA-1 expression levels to demonstrate that effector memory T cell populations in the lung airways are maintained by continual recruitment of new cells from the circulation. The rate of memory cell recruitment is surprisingly rapid, resulting in replacement of 90% of the population every 10 days, and is maintained for well over 1 year following viral clearance. These data indicate that peripheral T cell memory is dynamic and depends on a systemic source of T cells.     

Vaccinia virus-specific CD8+ cytotoxic T lymphocytes in humans.

Stimulation of human vaccinia virus immune peripheral blood mononuclear cells in vitro from vaccinia virus-immune donors with live vaccinia virus-infected autologous cells generated vaccinia virus-specific cytotoxic T lymphocytes (CTL) capable of lysing vaccinia virus-infected cells. We generated vaccinia virus-specific CD8+ clones and CD4+ CTL lines by limiting dilution from two donors by using peripheral blood mononuclear cells obtained 2 months or 4 years postrevaccination with vaccinia virus. These results demonstrate that vaccinia virus-specific CTL are generated as a result of immunization of humans with vaccinia virus and that both CD8(+)- and CD4(+)-specific T cells are maintained as memory cells.     

Impaired T cell immunity in B cell-deficient mice following viral central nervous system infection

CD8(+) T cells are required to control acute viral replication in the CNS following infection with neurotropic coronavirus. By contrast, studies in B cell-deficient (muMT) mice revealed Abs as key effectors in suppressing virus recrudescence. The apparent loss of initial T cell-mediated immune control in the absence of B cells was investigated by comparing T cell populations in CNS mononuclear cells from infected muMT and wild-type mice. Following viral recrudescence in muMT mice, total CD8(+) T cell numbers were similar to those of wild-type mice that had cleared infectious virus; however, virus-specific T cells were reduced at least 3-fold by class I tetramer and IFN-gamma ELISPOT analysis. Although overall T cell recruitment into the CNS of muMT mice was not impaired, discrepancies in frequencies of virus-specific CD8(+) T cells were most severe during acute infection. Impaired ex vivo cytolytic activity of muMT CNS mononuclear cells, concomitant with reduced frequencies, implicated IFN-gamma as the primary anti viral factor early in infection. Reduced virus-specific CD8(+) T cell responses in the CNS coincided with poor peripheral expansion and diminished CD4(+) T cell help. Thus, in addition to the lack of Ab, limited CD8(+) and CD4(+) T cell responses in muMT mice contribute to the ultimate loss of control of CNS infection. Using a model of virus infection restricted to the CNS, the results provide novel evidence for a role of B cells in regulating T cell expansion and differentiation into effector cells.