Recall proliferation potential of memory CD8+ T cells and antiviral protection

Memory CD8+ T cells play a crucial role in mediating protection from infection with viruses and other intracellular pathogens. Memory T cells are not a homogenous cellular population and may be separated into central memory T cells with substantial recall proliferation capacity and effector memory T cells with limited recall proliferation capacity. It has been suggested that the protective capacity of effector memory T cells is more limited than that of central memory T cells in viral infections. Here, we show that pronounced recall proliferation potential is indeed key for protection against lymphocytic choriomeningitis virus, which replicates in central lymphoid organs and is controlled by contact-dependent lysis of infected cells. In contrast, recall proliferation competence is not sufficient for protection against vaccinia virus, which is replicating in peripheral solid organs and is controlled by cytokines. To protect against vaccinia virus, high numbers of effector-like T cells were required to be present in peripheral tissue before viral challenge. These data indicate that the protective capacity of different subpopulations of memory T cells may vary dependent on the nature and the route of the challenge infection, which must be considered in T cell-based vaccine design.     

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.     

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

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

Shaping and reshaping CD8+ T-cell memory

The ability to develop and sustain populations of memory T cells after infection or immunization is a hallmark of the adaptive immune response and a basis for protective vaccination against infectious disease. Technical advances that allow direct ex vivo identification and characterization of antigen-specific CD8+ T cells at various stages of the response to infection or vaccination in mouse models have fuelled efforts to characterize the factors that control memory CD8+ T-cell generation. Here, we dissect the input signals that shape the characteristics of the memory CD8+ T-cell response and discuss how manipulation of these signals has the potential to reshape CD8+ T-cell memory and improve the efficacy of vaccination.     

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.     

The role of models in understanding CD8+ T-cell memory

Immunological memory - the ability to 'remember' previously encountered pathogens and respond faster on re-exposure - is a central feature of the immune response of vertebrates. We outline how mathematical models have contributed to our understanding of CD8(+) T-cell memory. Together with experimental data, models have helped to quantitatively describe and to further our understanding of both the generation of memory after infection with a pathogen and the maintenance of this memory throughout the life of an individual.     

Accelerated CD8+ T-cell memory and prime-boost response after dendritic-cell vaccination

Efficient boosting of memory T-cell numbers to protective levels generally requires a relatively long interval between immunizations. Decreasing this interval could be crucial in biodefense and cancer immunotherapy, in which rapid protective responses are essential. Here, we show that vaccination with peptide-coated dendritic cells (DCs) generated CD8+ T cells with the phenotype and function of memory cells within 4-6 d. These early memory CD8+ T cells underwent vigorous secondary expansion in response to a variety of booster immunizations, leading to elevated numbers of effector and memory T cells and enhanced protective immunity. Coinjection of CpG oligodeoxynucleotides, potent inducers of inflammation that did not alter the duration of DC antigen display, prevented the rapid generation of memory T cells in wild-type mice but not in mice lacking the interferon (IFN)-gamma receptor. These data show that DC vaccination stimulates a pathway of accelerated generation of memory T cells, and suggest that events of inflammation, including the action of IFN-gamma on the responding T cells, control the rate of development of memory CD8+ T cells.     

Cathepsin B controls the persistence of memory CD8+ T lymphocytes

The persistence of memory T lymphocytes confers lifelong protection from pathogens. Memory T cells survive and undergo homeostatic proliferation (HSP) in the absence of Ag, although the cell-intrinsic mechanisms by which cytokines drive the HSP of memory T cells are not well understood. In this study we report that lysosome stability limits the long-term maintenance of memory CD8(+) T cell populations. Serine protease inhibitor (Spi) 2A, an anti-apoptotic cytosolic cathepsin inhibitor, is induced by both IL-15 and IL-7. Mice deficient in Spi2A developed fewer memory phenotype CD44(hi)CD8(+) T cells with age, which underwent reduced HSP in the bone marrow. Spi2A was also required for the maintenance of central memory CD8(+) T cell populations after acute infection with lymphocytic choriomeningitis virus. Spi2A-deficient Ag-specific CD8(+) T cell populations declined more than wild-type competitors after viral infection, and they were eroded further after successive infections. Spi2A protected memory cells from lysosomal breakdown by inhibiting cathepsin B. The impaired maintenance of Spi2A-deficient memory CD8(+) T cells was rescued by concomitant cathepsin B deficiency, demonstrating that cathepsin B was a physiological target of Spi2A in memory CD8(+) T cell survival. Our findings support a model in which protection from lysosomal rupture through cytokine-induced expression of Spi2A determines the long-term persistence of memory CD8(+) T cells.     

CD8 T-cell memory: the other half of the story

Historically, most immune response studies have been limited to analyses of lymphoid tissue. However, peripheral sites of infection are likely to represent important sites of cell-mediated immune surveillance and effector function. Recent debates have centered on the persistence, trafficking patterns, effector activity, and protective role of non-lymphoid memory T cells.     

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.     

Direct interferon-gamma signaling dramatically enhances CD4+ and CD8+ T cell memory

Studies in IFN-gamma-deficient mice suggest that the delivery of IFN-gamma to CD8(+) T cells early in virus infection programs their eventual contraction, thereby reducing the abundance of CD8(+) memory T cells. In this study, we show that such mice fail to completely eliminate virus infection and that, when evaluated without the confounding factor of persisting Ag, both CD4(+) and CD8(+) T cells undergo profound contraction when they are unable to receive IFN-gamma signals. Furthermore, the abundance of CD4(+) and CD8(+) memory cells that express the IFN-gamma receptor is approximately 100-fold higher than cells lacking this molecule. Thus, direct IFN-gamma signaling is not required for T cell contraction during virus infection, and it enhances, rather than suppresses, the development of virus-specific CD4(+) and CD8(+) T cell memory.     

Respiratory syncytial virus infection suppresses lung CD8+ T-cell effector activity and peripheral CD8+ T-cell memory in the respiratory tract.

Respiratory syncytial virus (RSV) is a major cause of morbidity from respiratory infection in infants, young children and the elderly. No effective vaccine against RSV is currently available and studies of the natural history of RSV infection suggest repeated infections with antigenically related virus strains are common throughout an individual's lifetime. We have studied the CD8+ T-cell response during experimental murine RSV infection and found that RSV inhibits the expression of effector activity by activated RSV-specific CD8+ T cells infiltrating the lung parenchyma and the development of pulmonary CD8+ T-cell memory by interfering with TCR-mediated signaling. These data suggest a possible mechanism to explain the limited duration of protective immunity in RSV infection.     

Chlamydia trachomatis infection alters the development of memory CD8+ T cells

The obligate intracellular bacterium Chlamydia trachomatis is the most common cause of bacterial sexually transmitted disease in the United States and the leading cause of preventable blindness worldwide. Prior exposure to C. trachomatis has been shown to provide incomplete protection against subsequent infection. One possible explanation for the limited immunity afforded by prior C. trachomatis infection is poor activation of Chlamydia-specific memory CD8+ T cells. In this study, we examined the development of CD8+ memory T cell responses specific for the Chlamydia Ag CrpA. The percentage of CrpA63-71-specific T cells expressing an effector memory T cell phenotype (IL-7R+ CD62low) was dramatically diminished in mice immunized with C. trachomatis, compared with mice immunized with vaccinia virus expressing the CrpA protein. These alterations in memory T cell development were correlated with a significant reduction in the capacity of convalescent mice to mount an enhanced recall response to Chlamydia Ags, compared with the primary response. CrpA-specific memory T cells primed during VacCrpA infection also failed to respond to a challenge with Chlamydia. We therefore investigated whether C. trachomatis infection might have a global inhibitory effect on CD8+ T cell activation by coinfecting mice with C. trachomatis and Listeria monocytogenes and we found that the activation of Listeria-specific naive and memory CD8+ T cells was reduced in the presence of C. trachomatis. Together, these results suggest that Chlamydia is able to alter the development of CD8+ T cell responses during both primary and secondary infection, perhaps accounting for the incomplete protection provided by prior Chlamydia infection.     

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.     

Subdominant CD8 T-cell epitopes account for protection against cytomegalovirus independent of immunodomination

Cytomegalovirus (CMV) infection continues to be a complication in recipients of hematopoietic stem cell transplantation (HSCT). Preexisting donor immunity is recognized as a favorable prognostic factor for the reconstitution of protective antiviral immunity mediated primarily by CD8 T cells. Furthermore, adoptive transfer of CMV-specific memory CD8 T (CD8-T(M)) cells is a therapeutic option for preventing CMV disease in HSCT recipients. Given the different CMV infection histories of donor and recipient, a problem may arise from an antigenic mismatch between the CMV variant that has primed donor immunity and the CMV variant acquired by the recipient. Here, we have used the BALB/c mouse model of CMV infection in the immunocompromised host to evaluate the importance of donor-recipient CMV matching in immundominant epitopes (IDEs). For this, we generated the murine CMV (mCMV) recombinant virus mCMV-DeltaIDE, in which the two memory repertoire IDEs, the IE1-derived peptide 168-YPHFMPTNL-176 presented by the major histocompatibility complex class I (MHC-I) molecule L(d) and the m164-derived peptide 257-AGPPRYSRI-265 presented by the MHC-I molecule D(d), are both functionally deleted. Upon adoptive transfer, polyclonal donor CD8-T(M) cells primed by mCMV-DeltaIDE and the corresponding revertant virus mCMV-revDeltaIDE controlled infection of immunocompromised recipients with comparable efficacy and regardless of whether or not IDEs were presented in the recipients. Importantly, CD8-T(M) cells primed under conditions of immunodomination by IDEs protected recipients in which IDEs were absent. This shows that protection does not depend on compensatory expansion of non-IDE-specific CD8-T(M) cells liberated from immunodomination by the deletion of IDEs. We conclude that protection is, rather, based on the collective antiviral potential of non-IDEs independent of the presence or absence of IDE-mediated immunodomination.     

Loss of the orphan nuclear receptor NR2F6 enhances CD8+ T-cell memory via IFN-γ

Memory formation is a hallmark of T cell-mediated immunity, but how differentiation into either short-lived effector cells (SLECs, CD127⁻KLRG1⁺) or memory precursors cells (MPECs, CD127⁺KLRG1⁻) and subsequent regulation of long-term memory is adjusted is incompletely understood. Here, we show that loss of the nuclear orphan receptor NR2F6 in germ-line Nr2f6-deficient mice enhances antigen-specific CD8⁺ memory formation up to 70 days after bacterial infection with Listeria monocytogenes (LmOVA) and boosts inflammatory IFN-γ, TNFα, and IL-2 cytokine recall responses. Adoptive transfer experiments using Nr2f6^−/− OT-I T-cells showed that the augmented memory formation is CD8⁺ T-cell intrinsic. Although the relative difference between the Nr2f6^+/+ and Nr2f6^−/− OT-I memory compartment declines over time, Nr2f6-deficient OT-I memory T cells mount significantly enhanced IFN-γ responses upon reinfection with increased clonal expansion and improved host antigen-specific CD8⁺ T-cell responses. Following a secondary adoptive transfer into naïve congenic mice, Nr2f6-deficient OT-I memory T cells are superior in clearing LmOVA infection. Finally, we show that the commitment to enhanced memory within Nr2f6-deficient OT-I T cells is established in the early phases of the antibacterial immune response and is IFN-γ mediated. IFN-γ blocking normalized MPEC formation of Nr2f6-deficient OT-I T cells. Thus, deletion or pharmacological inhibition of NR2F6 in antigen-specific CD8⁺ T cells may have therapeutic potential for enhancing early IFN-γ production and consequently the functionality of memory CD8⁺ T cells in vivo.Subject terms: Interferons, Bacterial infection     

Insights into human CD8(+) T-cell memory using the yellow fever and smallpox vaccines

Live virus vaccines provide a unique opportunity to study human CD8(+) T-cell memory in the context of a controlled, primary acute viral infection. Yellow fever virus-17D and Dryvax are two such live-virus vaccines that are highly efficacious, used worldwide and provide long-term immunity against yellow fever and smallpox respectively. In this review, we describe the properties of virus-specific memory CD8(+) T cells generated in smallpox and yellow fever vaccinees. We address fundamental questions regarding magnitude, functional quality and longevity of the CD8(+) T-cell response, which are otherwise challenging to address in humans. These findings provide insights into the attributes of the human immune system as well as provide a benchmark for the optimal quality of a CD8(+) T-cell response that can be used to evaluate novel candidate vaccines.     

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

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

Reduced protection from simian immunodeficiency virus SIVmac251 infection afforded by memory CD8+ T cells induced by vaccination during CD4+ T-cell deficiency

Adaptive CD4⁺ and CD8⁺ T-cell responses have been associated with control of human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) replication. Here, we have designed a study with Indian rhesus macaques to more directly assess the role of CD8 SIV-specific responses in control of viral replication. Macaques were immunized with a DNA prime-modified vaccinia virus Ankara (MVA)-SIV boost regimen under normal conditions or under conditions of antibody-induced CD4⁺ T-cell deficiency. Depletion of CD4⁺ cells was performed in the immunized macaques at the peak of SIV-specific CD4⁺ T-cell responses following the DNA prime dose. A group of naïve macaques was also treated with the anti-CD4 depleting antibody as a control, and an additional group of macaques immunized under normal conditions was depleted of CD8⁺ T cells prior to challenge exposure to SIV_mac251. Analysis of the quality and quantity of vaccine-induced CD8⁺ T cells demonstrated that SIV-specific CD8⁺ T cells generated under conditions of CD4⁺ T-cell deficiency expressed low levels of Bcl-2 and interleukin-2 (IL-2), and plasma virus levels increased over time. Depletion of CD8⁺ T cells prior to challenge exposure abrogated vaccine-induced protection as previously shown. These data support the notion that adaptive CD4⁺ T cells are critical for the generation of effective CD8⁺ T-cell responses to SIV that, in turn, contribute to protection from AIDS. Importantly, they also suggest that long-term protection from disease will be afforded only by T-cell vaccines for HIV that provide a balanced induction of CD4⁺ and CD8⁺ T-cell responses and protect against early depletion of CD4⁺ T cells postinfection.