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.     

Discriminating between different pathways of memory CD8+ T cell differentiation

Despite the rapid accumulation of quantitative data on the dynamics of CD8(+) T cell responses following acute viral or bacterial infections of mice, the pathways of differentiation of naive CD8(+) T cells into memory during an immune response remain controversial. Currently, three models have been proposed. In the "stem cell-associated differentiation" model, following activation, naive T cells differentiate into stem cell-like memory cells, which then convert into terminally differentiated short-lived effector cells. In the "linear differentiation" model, following activation, naive T cells first differentiate into effectors, and after Ag clearance, effectors convert into memory cells. Finally, in the "progressive differentiation" model, naive T cells differentiate into memory or effector cells depending on the amount of specific stimulation received, with weaker stimulation resulting in formation of memory cells. This study investigates whether the mathematical models formulated from these hypotheses are consistent with the data on the dynamics of the CD8(+) T cell response to lymphocytic choriomeningitis virus during acute infection of mice. Findings indicate that two models, the stem cell-associated differentiation model and the progressive differentiation model, in which differentiation of cells is strongly linked to the number of cell divisions, fail to describe the data at biologically reasonable parameter values. This work suggests additional experimental tests that may allow for further discrimination between different models of CD8(+) T cell differentiation in acute infections.     

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.     

Profound protection against respiratory challenge with a lethal H7N7 influenza A virus by increasing the magnitude of CD8(+) T-cell memory

The recall of CD8(+) T-cell memory established by infecting H-2(b) mice with an H1N1 influenza A virus provided a measure of protection against an extremely virulent H7N7 virus. The numbers of CD8(+) effector and memory T cells specific for the shared, immunodominant D(b)NP(366) epitope were greatly increased subsequent to the H7N7 challenge, and though lung titers remained as high as those in naive controls for 5 days or more, the virus was cleared more rapidly. Expanding the CD8(+) memory T-cell pool (<0.5 to >10%) by sequential priming with two different influenza A viruses (H3N2-->H1N1) gave much better protection. Though the H7N7 virus initially grew to equivalent titers in the lungs of naive and double-primed mice, the replicative phase was substantially controlled within 3 days. This tertiary H7N7 challenge caused little increase in the magnitude of the CD8(+) D(b)NP(366)(+) T-cell pool, and only a portion of the memory population in the lymphoid tissue could be shown to proliferate. The great majority of the CD8(+) D(b)NP(366)(+) set that localized to the infected respiratory tract had, however, cycled at least once, though recent cell division was shown not to be a prerequisite for T-cell extravasation. The selective induction of CD8(+) T-cell memory can thus greatly limit the damage caused by a virulent influenza A virus, with the extent of protection being directly related to the number of available responders. Furthermore, a large pool of CD8(+) memory T cells may be only partially utilized to deal with a potentially lethal influenza infection.     

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.     

Dendritic cells and CD28 costimulation are required to sustain virus-specific CD8+ T cell responses during the effector phase in vivo

Although much is known about the initiation of immune responses, much less is known about what controls the effector phase. CD8(+) T cell responses are believed to be programmed in lymph nodes during priming without any further contribution by dendritic cells (DCs) and Ag. In this study, we report the requirement for DCs, Ag, and CD28 costimulation during the effector phase of the CD8(+) T cell response. Depleting DCs or blocking CD28 after day 6 of primary influenza A virus infection decreases the virus-specific CD8(+) T cell response by inducing apoptosis, and this results in decreased viral clearance. Furthermore, effector CD8(+) T cells adoptively transferred during the effector phase fail to expand without DC, CD28 costimulation, and cognate Ag. The absence of costimulation also leads to reduced survival of virus-specific effector cells as they undergo apoptosis mediated by the proapoptotic molecule Bim. Finally, IL-2 treatment restored the effector response in the absence of CD28 costimulation. Thus, in contrast to naive CD8(+) T cells, which undergo an initial Ag-independent proliferation, effector CD8(+) T cells expanding in the lungs during the effector phase require Ag, CD28 costimulation, and DCs for survival and expansion. These requirements would greatly impair effector responses against viruses and tumors that are known to inhibit DC maturation and in chronic infections and aging where CD28(-/-) CD8(+) T cells accumulate.     

Both Memory and CD45RA+/CD62L+ Naive CD4+ T Cells Are Infected in Human Immunodeficiency Virus Type 1-Infected Individuals

Cellular activation is critical for the propagation of human immunodeficiency virus type 1 (HIV-1) infection. It has been suggested that truly naive CD4(+) T cells are resistant to productive HIV-1 infection because of their constitutive resting state. Memory and naive CD4(+) T-cell subsets from 11 HIV-1-infected individuals were isolated ex vivo by a combination of magnetic bead depletion and fluorescence-activated cell sorting techniques with stringent criteria of combined expression of CD45RA and CD62L to identify naive CD4(+) T-cell subsets. In all patients HIV-1 provirus could be detected within naive CD45RA+/CD62L+ CD4(+) T cells; in addition, replication-competent HIV-1 was isolated from these cells upon CD4(+) T-cell stimulation in tissue cultures. Memory CD4(+) T cells had a median of fourfold more replication-competent virus and a median of sixfold more provirus than naive CD4(+) T cells. Overall, there was a median of 16-fold more integrated provirus identified in memory CD4(+) T cells than in naive CD4(+) T cells within a given patient. Interestingly, there was a trend toward equalization of viral loads in memory and naive CD4(+) T-cell subsets in those patients who harbored CXCR4-using (syncytium-inducing) viruses. Within any given patient, there was no selective usage of a particular coreceptor by virus isolated from memory versus naive CD4(+) T cells. Our findings suggest that naive CD4(+) T cells may be a significant viral reservoir for HIV, particularly in those patients harboring CXCR4-using viruses.     

Serum antibodies critically affect virus-specific CD4+/CD8+ T cell balance during respiratory syncytial virus infections

Following infection with respiratory syncytial virus (RSV), reinfection in healthy individuals is common and presumably due to ineffective memory T cell responses. In peripheral blood of healthy adults, a higher CD4(+)/CD8(+) memory T cell ratio was observed compared with the ratio of virus-specific effector CD4(+)/CD8(+) T cells that we had found in earlier work during primary RSV infections. In mice, we show that an enhanced ratio of RSV-specific neutralizing to nonneutralizing Abs profoundly enhanced the CD4(+) T cell response during RSV infection. Moreover, FcγRs and complement factor C1q contributed to this Ab-mediated enhancement. Therefore, the increase in CD4(+) memory T cell response likely occurs through enhanced endosomal Ag processing dependent on FcγRs. The resulting shift in memory T cell response was likely amplified by suppressed T cell proliferation caused by RSV infection of APCs, a route important for Ag presentation via MHC class I molecules leading to CD8(+) T cell activation. Decreasing memory CD8(+) T cell numbers could explain the inadequate immunity during repeated RSV infections. Understanding this interplay of Ab-mediated CD4(+) memory T cell response enhancement and infection mediated CD8(+) memory T cell suppression is likely critical for development of effective RSV vaccines.     

Naive, effector, and memory CD8 T cells in protection against pulmonary influenza virus infection: homing properties rather than initial frequencies are crucial.

The goal of adoptive immunotherapy is to target a high number of persisting effector cells to the site of a virus infection or tumor. In this study, we compared the protective value of hemagglutinin peptide-specific CD8 T cells generated from the clone-4 TCR-transgenic mice, defined by different stages of their differentiation, against lethal pulmonary influenza infection. We show that the adoptive transfer of high numbers of Ag-specific unprimed, naive CD8 T cells failed to clear the pulmonary virus titer and to promote host survival. The same numbers of in vitro generated primary Ag-specific Tc1 effector cells, producing high amounts of IFN-gamma, or resting Tc1 memory cells, generated from these effectors, were protective. Highly activated CD62Llow Tc1 effectors accumulated in the lung with rapid kinetics and most efficiently reduced the pulmonary viral titer early during infection. The resting CD62Lhigh naive and memory populations first increased in cell numbers in the draining lymph nodes. Subsequently, memory cells accumulated more rapidly and to a greater extent in the lung lavage as compared with naive cells. Thus, effector cells are most effective against a localized virus infection, which correlates with their ability to rapidly distribute at the infected tissue site. The finding that similar numbers of naive Ag-specific CD8 T cells are not protective supports the view that qualitative differences between the two resting populations, the naive and the memory population, may play a major role in their protective value against disease.     

Virus-specific and bystander CD8 T cells recruited during virus-induced encephalomyelitis

Neurotropic coronavirus-induced encephalitis was used to evaluate recruitment, functional activation, and retention of peripheral bystander memory CD8+ T cells. Mice were first infected with recombinant vaccinia virus expressing a non-cross-reactive human immunodeficiency virus (HIV) epitope, designated p18. Following establishment of an endogenous p18-specific memory CD8+ T-cell population, mice were challenged with coronavirus to directly compare recruitment, longevity, and activation characteristics of both primary coronavirus-specific and bystander memory populations trafficking into the central nervous system (CNS). HIV-specific memory CD8+ T cells were recruited early into the CNS as components of the innate immune response, preceding CD8+ T cells specific for the dominant coronavirus epitope, designated pN. Although pN-specific T-cell numbers gradually exceeded bystander p18-specific CD8+ T-cell numbers, both populations peaked concurrently within the CNS. Nevertheless, coronavirus-specific CD8+ T cells were preferentially retained. By contrast, bystander CD8+ T-cell numbers declined to background numbers following control of CNS virus replication. Furthermore, in contrast to highly activated pN-specific CD8+ T cells, bystander p18-specific CD8+ T cells recruited to the site of inflammation maintained a nonactivated memory phenotype and did not express ex vivo cytolytic activity. Therefore, analysis of host CD8+ T-cell responses to unrelated infections demonstrates that bystander memory CD8+ T cells can comprise a significant proportion of CNS inflammatory cells during virus-induced encephalitis. However, transient CNS retention and the absence of activation suggest that memory bystander CD8+ T cells may not overtly contribute to pathology in the absence of antigen recognition.     

Cutting edge: effector memory CD8+ T cells play a prominent role in recall responses to secondary viral infection in the lung

The relative contributions of CD62L(high) (central) memory and CD62L(low) (effector) memory T cell populations to recall responses are poorly understood, especially in the respiratory tract. In this study, we took advantage of a dual-adoptive transfer system in the mouse to simultaneously follow the recall response of effector and central memory subpopulations to intranasal parainfluenza virus infection. Using MHC class I and class II multimers, we tracked the responses of Ag-specific CD8(+) and CD4(+) memory T cells in the same animals. The data show that effector memory T cells mounted recall responses that were equal to, or greater than, those mounted by central memory T cells. Moreover, effector memory T cells were more efficient at subsequently establishing a second generation of memory T cells. These data contrast with other studies indicating that central memory CD8(+) T cells are the prominent contributors to systemic virus infections.     

Induction of telomerase activity and maintenance of telomere length in virus-specific effector and memory CD8+ T cells

Acute viral infections induce extensive proliferation and differentiation of virus-specific CD8+ T cells. One mechanism reported to regulate the proliferative capacity of activated lymphocytes is mediated by the effect of telomerase in maintaining the length of telomeres in proliferating cells. We examined the regulation of telomerase activity and telomere length in naive CD8+ T cells and in virus-specific CD8+ T cells isolated from mice infected with lymphocytic choriomeningitis virus. These studies reveal that, compared with naive CD8+ T cells, which express little or no telomerase activity, Ag-specific effector and long-lived memory CD8+ T cells express high levels of telomerase activity. Despite the extensive clonal expansion that occurs during acute lymphocytic choriomeningitis virus infection, telomere length is maintained in both effector and memory CD8+ T cells. These results suggest that induction of telomerase activity in Ag-specific effector and memory CD8+ T cells is important for the extensive clonal expansion of both primary and secondary effector cells and for the maintenance and longevity of the memory CD8+ T cell population.     

Selective bystander proliferation of memory CD4+ and CD8+ T cells upon NK T or T cell activation

Ag-experienced or memory T cells have increased reactivity to recall Ag, and can be distinguished from naive T cells by altered expression of surface markers such as CD44. Memory T cells have a high turnover rate, and CD8(+) memory T cells proliferate upon viral infection, in the presence of IFN-alphabeta and/or IL-15. In this study, we extend these findings by showing that activated NKT cells and superantigen-activated T cells induce extensive bystander proliferation of both CD8(+) and CD4(+) memory T cells. Moreover, proliferation of memory T cells can be induced by an IFN-alphabeta-independent, but IFN-gamma- or IL-12-dependent pathway. In these conditions of bystander activation, proliferating memory (CD44(high)) T cells do not derive from activation of naive (CD44(low)) T cells, but rather from bona fide memory CD44(high) T cells. Together, these data demonstrate that distinct pathways can induce bystander proliferation of memory T cells.     

CD8+ T cells specific for EBV, cytomegalovirus, and influenza virus are activated during primary HIV infection

Primary viral infections, including primary HIV infection, trigger intense activation of the immune system, with marked expansion of CD38(+)CD8(+) T cells. Whether this expansion involves only viral-specific cells or includes a degree of bystander activation remains a matter of debate. We therefore examined the activation status of EBV-, CMV-, and influenza virus (FLU)-specific CD8(+) T cells during primary HIV infection, in comparison to HIV-specific CD8(+) T cells. The activation markers CD38 and HLA-DR were strongly expressed on HIV-specific CD8(+) T cells. Surprisingly, CD38 expression was also up-regulated on CD8(+) T cells specific for other viruses, albeit to a lesser extent. Activation marker expression returned to normal or near-normal values after 1 year of highly active antiretroviral therapy. HIV viral load correlated with CD38 expression on HIV-specific CD8(+) T cells but also on EBV-, CMV-, and FLU-specific CD8(+) T cells. In primary HIV infection, EBV-specific CD8(+) T cells also showed increased Ki67 expression and decreased Bcl-2 expression, compared with values observed in HIV-seronegative control subjects. These results show that bystander activation occurs during primary HIV infection, even though HIV-specific CD8(+) T cells express the highest level of activation. The role of this bystander activation in lymphocyte homeostasis and HIV pathogenesis remains to be determined.     

Differential sensitivity of naive and memory CD8+ T cells to apoptosis in vivo

Apoptosis is a critical regulator of homeostasis in the immune system. In this study we demonstrate that memory CD8(+) T cells are more resistant to apoptosis than naive cells. After whole body irradiation of mice, both naive and memory CD8(+) T cells decreased in number, but the reduction in the number of naive cells was 8-fold greater than that in memory CD8(+) T cells. In addition to examining radiation-induced apoptosis, we analyzed the expansion and contraction of naive and memory CD8(+) T cells in vivo following exposure to Ag. We found that memory CD8(+) T cells not only responded more quickly than naive cells after viral infection, but that secondary effector cells generated from memory cells underwent much less contraction compared with primary effectors generated from naive cells (3- to 5-fold vs 10- to 20-fold decrease). Increased numbers of secondary memory cells were observed in both lymphoid and non-lymphoid tissues. When naive and memory cells were transferred into the same animal, secondary effectors underwent less contraction than primary effector cells. These experiments analyzing apoptosis of primary and secondary effectors in the same animal show unequivocally that decreased downsizing of the secondary response reflects an intrinsic property of the memory T cells and is not simply due to environmental effects. These findings have implications for designing prime/boost vaccine strategies and also for optimizing immunotherapeutic regimens for treatment of chronic infections.     

Stable CD8+ T cell memory during persistent Trypanosoma cruzi infection

CD8(+) T cell responses to persistent infections caused by intracellular pathogens are dominated by resting T effectors and T effector memory cells, with little evidence suggesting that a T central memory (T(CM)) population is generated. Using a model of Trypanosoma cruzi infection, we demonstrate that in contrast to the T effector/T effector memory phenotype of the majority of T. cruzi-specific CD8(+) T cells, a population of cells displaying hallmark characteristics of T(CM) cells is also present during long-term persistent infection. This population expressed the T(CM) marker CD127 and a subset expressed one or more of three other T(CM) markers: CD62L, CCR7, and CD122. Additionally, the majority of CD127(high) cells were KLRG1(low), indicating that they have not been repetitively activated through TCR stimulation. These CD127(high) cells were better maintained than their CD127(low) counterparts following transfer into naive mice, consistent with their observed surface expression of CD127 and CD122, which confer the ability to self-renew in response to IL-7 and IL-15. CD127(high) cells were capable of IFN-gamma production upon peptide restimulation and expanded in response to challenge infection, indicating that these cells are functionally responsive upon Ag re-encounter. These results are in contrast to what is typically observed during many persistent infections and indicate that a stable population of parasite-specific CD8(+) T cells capable of Ag-independent survival is maintained in mice despite the presence of persistent Ag.