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.     

Cutting edge: rapid in vivo CTL activity by polyoma virus-specific effector and memory CD8+ T cells

For viruses that establish persistent infection, continuous immunosurveillance by effector-competent antiviral CD8(+) T cells is likely essential for limiting viral replication. Although it is well documented that virus-specific memory CD8(+) T cells synthesize cytokines after short term in vitro stimulation, there is limited evidence that these T cells exhibit cytotoxicity, the dominant antiviral effector function. Here, we show that antiviral CD8(+) T cells in mice acutely infected by polyoma virus, a persistent mouse pathogen, specifically eliminate viral peptide-pulsed donor spleen cells within minutes after adoptive transfer and do so via a perforin-dependent mechanism. Antiviral memory CD8(+) T cells were similarly capable of rapidly mobilizing potent Ag-specific cytotoxic activity in vivo. These findings strongly support the concept that a cytotoxic effector-memory CD8(+) T cell population operates in vivo to control this persistent viral infection.     

CD8+ T effector memory cells protect against liver-stage malaria

Identification of correlates of protection for infectious diseases including malaria is a major challenge and has become one of the main obstacles in developing effective vaccines. We investigated protection against liver-stage malaria conferred by vaccination with adenoviral (Ad) and modified vaccinia Ankara (MVA) vectors expressing pre-erythrocytic malaria Ags. By classifying CD8(+) T cells into effector, effector memory (T(EM)), and central memory subsets using CD62L and CD127 markers, we found striking differences in T cell memory generation. Although MVA induced accelerated central memory T cell generation, which could be efficiently boosted by subsequent Ad administration, it failed to protect against malaria. In contrast, Ad vectors, which permit persistent Ag delivery, elicit a prolonged effector T cell and T(EM) response that requires long intervals for an efficient boost. A preferential T(EM) phenotype was maintained in liver, blood, and spleen after Ad/MVA prime-boost regimens, and animals were protected against malaria sporozoite challenge. Blood CD8(+) T(EM) cells correlated with protection against malaria liver-stage infection, assessed by estimation of number of parasites emerging from the liver into the blood. The protective ability of Ag-specific T(EM) cells was confirmed by transfer experiments into naive recipient mice. Thus, we identify persistent CD8 T(EM) populations as essential for vaccine-induced pre-erythrocytic protection against malaria, a finding that has important implications for vaccine design.     

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.     

An in vivo cytotoxicity threshold for influenza A virus-specific effector and memory CD8(+) T cells

Influenza A virus infection of C57BL/6 (B6) mice is characterized by prominent CD8(+) T cell responses to H2D(b) complexed with peptides from the viral nucleoprotein (NP(366), ASNENMETM) and acid polymerase (PA(224), SSLENFRAYV). An in vivo cytotoxicity assay that depends on the adoptive transfer of peptide-pulsed, syngeneic targets was used in this study to quantitate the cytotoxic potential of D(b)NP(366)- and D(b)PA(224)-specific acute and memory CD8(+) T cells following primary or secondary virus challenge. Both T cell populations displayed equivalent levels of in vivo effector function when comparable numbers were transferred into naive B6 hosts. Cytotoxic activity following primary infection clearly correlated with the frequency of tetramer-stained CD8(+) T cells. This relationship looked, however, to be less direct following secondary exposure, partly because the numbers of CD8(+)D(b)NP(366)(+) T cells were greatly in excess. However, calculating the in vivo E:T ratios indicated that in vivo lysis, like many other biological functions, is threshold dependent. Furthermore, the capacity to eliminate peptide-pulsed targets was independent of the differentiation state (i.e., primary or secondary effectors) and was comparable for the two T cell specificities that were analyzed. These experiments provide insights that may be of value for adoptive immunotherapy, where careful consideration of both the activation state and the number of effector cells is required.     

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.     

Enhanced expression of cell cycle regulatory genes in virus-specific memory CD8+ T cells

Unlike naive CD8+ T cells, antigen-experienced memory CD8+ T cells persist over time due to their unique ability to homeostatically proliferate. It was hypothesized that memory cells might differentially regulate the expression of genes that control the cell cycle to facilitate homeostatic proliferation. To test this, the expression levels of 96 different cell cycle regulatory genes were compared between transgenic naive and memory CD8+ T cells that specifically recognize the GP33-41 epitope of lymphocytic choriomeningitis virus (LCMV). It was discovered that relative to naive cells, memory cells overexpress several important genes that control the transition between G(1) and S phase. Some of these genes include those encoding cyclins D3, D2, B1, C, and H, cyclin-dependent kinases (cdk's) 4 and 6, the cdk inhibitors p16, p15, and p18, and other genes involved in protein degradation and DNA replication. Importantly, these differences were observed both in total populations of LCMV-specific naive and memory CD8+ cells and in LCMV-specific CD8+ T-cell populations that were in the G(1) phase of the cell cycle only. In addition, the expression differences between naive and memory cells were exaggerated following antigenic stimulation. The fact that memory cells are precharged with several of the major factors that are necessary for the G(1)- to-S-phase transition suggests they may require a lower threshold of stimulation to enter the cell cycle.     

Bystander central memory but not effector memory CD8+ T cells suppress allograft rejection

Memory T cells respond faster and more vigorously than their naive counterparts and are critical for adaptive immunity. However, it is unknown whether and how memory T cells react in the face of irrelevant Ags. It is generally accepted that bystander memory T cells are neutral in immune responsiveness. In this study, we present the first evidence that bystander central memory (TCM), but not effector memory (TEM), CD8+ T cells suppress allograft rejection as well as T cell proliferation in the draining lymph nodes (DLN) of recipient mice. Both bystander TCM and naive T cells, but fewer TEM cells, migrated to DLN, whereas TCM cells exhibited faster turnover than their naive counterparts, suggesting that bystander TCM cells have an advantage over their naive counterparts in suppression. However, bystander TEM cells migrated to inflammatory graft sites, but not DLN, and yet failed to exert their suppression. These findings indicate that bystander memory T cells need to migrate to lymph nodes to exert their suppression by inhibiting responder T cell activation or homeostatic proliferation. Moreover, the suppression mediated by bystander TCM cells was largely dependent on IL-15, as IL-15 was required for their homeostatic proliferation and TCM-mediated suppression of allograft rejection. This suppression also required the presence of TGFbeta1, as TCM cells expressed TGFbeta1 while neutralizing TGFbeta1 abolished their suppression. Thus, bystander TCM, but not TEM, CD8+ T cells are potent suppressors rather than bystanders. This new finding will have an impact on cellular immunology and may have clinic implications for tolerance induction.     

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.     

Human CD4+ T cells displaying viral epitopes elicit a functional virus-specific memory CD8+ T cell response

The Ag-specific cellular recall response to herpes virus infections is characterized by a swift recruitment of virus-specific memory T cells. Rapid activation is achieved through formation of the immunological synapse and supramolecular clustering of signal molecules at the site of contact. During the formation of the immunological synapse, epitope-loaded MHC molecules are transferred via trogocytosis from APCs to T cells, enabling the latter to function as Ag-presenting T cells (T-APCs). The contribution of viral epitope expressing T-APCs in the regulation of the herpes virus-specific CD8+ T cell memory response remains unclear. Comparison of CD4+ T-APCs with professional APCs such as Ag-presenting CD40L-activated B cells (CD40B-APCs) demonstrated reduced levels of costimulatory ligands. Despite the observed differences, CD4+ T-APCs are as potent as CD40B-APCs in stimulating herpes virus-specific CD8+ T cells resulting in a greater than 35-fold expansion of CD8+ T cells specific for dominant and subdominant viral epitopes. Virus-specific CD8+ T cells generated by CD4+ T-APCs or CD40B-APCs showed both comparable effector function such as specific lysis of targets and cytokine production and also did not differ in their phenotype after expansion. These results indicate that viral epitope presentation by Ag-specific CD4+ T cells may contribute to the rapid recruitment of virus-specific memory CD8+ T cells during a viral recall response.     

Dynamic differentiation of activated human peripheral blood CD8+ and CD4+ effector memory T cells

Two functionally different memory T cell subsets were originally defined based on their different CCR7 expression profile, but the lineage relationship between these subsets referred to as central memory T cells (T(CM)) and effector memory T cells (T(EM)), is not resolved. A prevalent model proposes a linear progressive differentiation from T(CM) to T(EM). Our results demonstrate that on activation, human CCR7-CD62L- peripheral blood CD8+ and CD4+ T(EM) cells exhibit a dynamic differentiation, involving transient as well as stable changes to T(CM) phenotype and properties. Whereas the larger fraction of T(EM) cells increases expression of effector molecules, such as perforin or IFN-gamma, a smaller fraction first acquires CCR7 expression. We demonstrate that this acquisition of lymph node homing potential is associated with strong proliferation similar to that of activated T(CM) cells. After proliferation, most of these cells lose CCR7 expression again and acquire effector functions (e.g., perforin production). A small proportion (approximately 6%), however, maintain phenotypic and functional T(CM) properties over a long time interval. These results suggest that T(EM) cells provide immediate effector function by a fraction of cells as well as self-renewal by others through up-regulation of CCR7 followed by either secondary peripheral effector function or long term maintenance of T(CM)-like properties.     

Decreased effector memory CD45RA+ CD62L- CD8+ T cells and increased central memory CD45RA- CD62L+ CD8+ T cells in peripheral blood of rheumatoid arthritis patients

Although a role for CD8+ T cells in the pathogenesis of rheumatoid arthritis (RA) has been suggested, the precise nature of their involvement is not fully understood. In the present study we examined the central and effector memory phenotypes of CD4+ and CD8+ T cells in the peripheral blood of patients with RA and systemic lupus erythematosus. Terminally differentiated effector memory CD45RA+CD62L-CD8+ T cells were significantly decreased in RA patients, whereas the central memory CD45RA-CD62L+ CD8+ T-cell population was increased as compared with levels in healthy control individuals. Na?ve and preterminally differentiated effector memory CD45RA-CD62L- CD8+ T cells did not differ between RA patients and control individuals. The CD45RA-CD62L+ central memory CD4+ T-cell subpopulation was increased in RA patients, whereas the na?ve and effector memory phenotype of CD4+ T cells did not differ between RA patients and control individuals. In patients with systemic lupus erythematosus the distribution of na?ve/memory CD4+ and CD8+ T cells did not differ from that in age- and sex-matched control individuals. These findings show that peripheral blood CD8+ T cells from RA patients exhibit a skewed maturation phenotype that suggests a perturbation in the homeostasis of these cells. The central memory CD45RA-CD62L+ CD4+ and CD8+ T-cell numbers were increased in RA, suggesting an accelerated maturation of na?ve T cells. The decreased numbers of terminally differentiated CD45RA+CD62L- effector memory CD8+ T cells in peripheral blood of RA patients may reflect increased apoptosis of these cells or enhanced migration of these cells to sites of inflammation, which may play a role in the pathogenesis of RA.     

CD40L co-stimulation from CD8+ to CD4+ effector memory T cells supports CD4+ expansion

Effector memory T cells (T(EM)) have an important role in immunity against infection. However, little is known about the factors regulating T(EM) maintenance and proliferation. In this study, we investigated the role of direct interactions between CD4(+) and CD8(+) T cells (TC) for human T(EM) expansion. Proliferation of separated or mixed CD4(+) and CD8(+)T(EM) populations was analyzed after polyclonal stimulation in vitro. Compared to each isolated subset mixed T(EM) populations showed increased proliferation and expansion of both CD4(+) and CD8(+)T(EM) subpopulations. Combined activation of CD4(+) and CD8(+) memory T cells (Tmem) induced an increased expression of CD40L and CD40 on both populations. Subsequently, CD40/CD40L caused a bi-directional stimulation of CD40(+)CD4(+)T(EM) by CD40L(+)CD8(+)T(EM) and of CD40(+)CD8(+)T(EM) by CD40L(+)CD4(+)T(EM). Blocking of CD40L on activated CD8(+)T(EM) selectively inhibited proliferation of CD4(+)T(EM), while blocking of CD40L on CD4(+)T(EM) abrogated proliferation of CD8(+)T(EM). Taken together, we demonstrate for the first time that the expression of CD40L is exploited on the one hand by CD8(+)T(EM) to increase the proliferation of activated CD4(+)T(EM) and on the other hand by CD4(+)T(EM) to support the expansion of activated CD8(+)T(EM). Thus, efficient T(EM) expansion requires bi-directional interactions between CD4(+) and CD8(+)T(EM) cells.     

Differential microenvironment localization of effector and memory CD8 T cells

CD8 T cells are critical for the clearance of intracellular pathogens. Upon infection, naive CD8 T cells differentiate into effector cells that target and eliminate infected cells. Following clearance of the pathogen, most effector cells die, although a small fraction survives to establish a memory population. Subsequent exposure to the same pathogen induces a rapid response of memory T cells and efficient elimination of the pathogen. Although much is known about the CD8 T cell response, the precise microenvironment location of effector and memory CD8 T cells in secondary lymphoid organs is not well characterized. In this study, we present an in situ analysis of the localization of effector and memory CD8 T cells during the murine immune response to lymphocytic choriomenginits virus. We identified the location of these cells using a transgenic mouse model system in which CD8 T cells are irreversibly tagged with yellow fluorescent protein (YFP) after activation. After infection, YFP+ CD8 T cells were initially observed within T cell zones. Later, these cells were found in the red pulp and a disruption of all CD8 T cell zones was observed. After resolution of the immune response, YFP+ memory CD8 T cells were observed primarily in T cells zones. Thus, in the spleens of mice, effector CD8 T cells localize to the red pulp and memory CD8 T cells localize to the T cell zones. Upon rechallenge, memory CD8 T cells rapidly proliferate and the secondary effector CD8 T cells are found in the red pulp.     

Epitope cross-reactivity frequently differs between central and effector memory HIV-specific CD8+ T cells

HIV diversity may limit the breadth of vaccine coverage due to epitope sequence differences between strains. Although amino acid substitutions within CD8(+) T cell HIV epitopes can result in complete or partial abrogation of responses, this has primarily been demonstrated in effector CD8(+) T cells. In an HIV-infected Kenyan cohort, we demonstrate that the cross-reactivity of HIV epitope variants differs dramatically between overnight IFN-gamma and longer-term proliferation assays. For most epitopes, particular variants (not the index peptide) were preferred in proliferation in the absence of corresponding overnight IFN-gamma responses and in the absence of the variant in the HIV quasispecies. Most proliferating CD8(+) T cells were polyfunctional via cytokine analyses. A trend to positive correlation was observed between proliferation (but not IFN-gamma) and CD4 counts. We present findings relevant to the assessment of HIV vaccine candidates and toward a better understanding of how viral diversity is tolerated by central and effector memory CD8(+) T cells.     

Genetic manipulation of telomerase in HIV-specific CD8+ T cells: enhanced antiviral functions accompany the increased proliferative potential and telomere length stabilization

A large proportion of the CD8(+) T cell pool in persons chronically infected with HIV consists of cells that show features of replicative senescence, an end stage characterized by irreversible cell cycle arrest, multiple genetic and functional changes, and shortened telomeres. The objective of our research was to determine whether constitutive expression of the gene for the human telomerase (hTERT) can prevent senescence-induced impairments in human virus-specific CD8(+) T cells, particularly in the context of HIV-1 disease. Our results indicate that hTERT-expressing HIV-specific CD8(+) lymphocytes show both an enhanced and sustained capacity to inhibit HIV-1 replication in in vitro coculture experiments, as well as prolonged ability to produce IFN-gamma and TNF-alpha in response to stimulation with HIV-1-derived peptides, as compared with vector-transduced controls. Loss of CD28 expression, the signature change of replicative senescence in cell culture, was retarded in those CD8(+) T cell cultures that had high levels of CD28 at the time of hTERT transduction. These findings suggest that telomere shortening may be the primary driving force behind several aspects of CD8(+) T cell dysfunction associated with replicative senescence. We also demonstrate reduced accumulation of the p16(INK4a) and p21(WAF1) cell cycle inhibitors in hTERT-transduced lymphocytes, providing a possible mechanism by which stable hTERT expression is able to circumvent the senescence barrier in CD8(+) T cells. Given the key role of CD8(+) T cell function in controlling a variety of acute and latent viral infections, approaches to retard the functional decrements associated with replicative senescence may lead to novel types of immunotherapy.     

Derivation and maintenance of virtual memory CD8 T cells

Memory CD8(+) T cells are an important component of the adaptive immune response against many infections, and understanding how Ag-specific memory CD8(+) T cells are generated and maintained is crucial for the development of vaccines. We recently reported the existence of memory-phenotype, Ag-specific CD8(+) T cells in unimmunized mice (virtual memory or VM cells). However, it was not clear when and where these cells are generated during normal development, nor the factors required for their production and maintenance. This issue is especially pertinent given recent data showing that memory-like CD8 T cells can be generated in the thymus, in a bystander response to IL-4. In this study, we show that the size of the VM population is reduced in IL-4R-deficient animals. However, the VM population appears first in the periphery and not the thymus of normal animals, suggesting this role of IL-4 is manifest following thymic egress. We also show that the VM pool is durable, showing basal proliferation and long-term maintenance in normal animals, and also being retained during responses to unrelated infection.     

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.     

IL-15 activates telomerase and minimizes telomere loss and may preserve the replicative life span of memory CD8+ T cells in vitro

The preservation of the replicative life span of memory CD8(+) T cells is vital for long-term immune protection. Although IL-15 plays a key role in the homeostasis of memory CD8(+) T cells, it is unknown whether IL-15 regulates the replicative life span of memory CD8(+) T cells. In this study, we report an analysis of telomerase expression and telomere length in human memory phenotype CD8(+) T cells maintained by IL-15 in vitro. We demonstrate that IL-15 is capable of activating telomerase in memory CD8(+) T cells via Jak3 and PI3K signaling pathways. Furthermore, IL-15 induces a sustained level of telomerase activity over long periods of time, and in turn minimizes telomere loss in memory CD8(+) T cells after substantial cell divisions. These findings suggest that IL-15 activates stable telomerase expression and compensates telomere loss in memory phenotype CD8(+) T cells, and that telomerase may play an important role in memory CD8(+) T cell homeostasis.     

OX40 costimulatory signals potentiate the memory commitment of effector CD8+ T cells

A T cell costimulatory molecule, OX40, contributes to T cell expansion, survival, and cytokine production. Although several roles for OX40 in CD8(+) T cell responses to tumors and viral infection have been shown, the precise function of these signals in the generation of memory CD8(+) T cells remains to be elucidated. To address this, we examined the generation and maintenance of memory CD8(+) T cells during infection with Listeria monocytogenes in the presence and absence of OX40 signaling. We used the expression of killer cell lectin-like receptor G1 (KLRG1), a recently reported marker, to distinguish between short-lived effector and memory precursor effector T cells (MPECs). Although OX40 was dispensable for the generation of effector T cells in general, the lack of OX40 signals significantly reduced the number and proportion of KLRG1(low) MPECs, and, subsequently, markedly impaired the generation of memory CD8(+) T cells. Moreover, memory T cells that were generated in the absence of OX40 signals in a host animal did not show self-renewal in a second host, suggesting that OX40 is important for the maintenance of memory T cells. Additional experiments making use of an inhibitory mAb against the OX40 ligand demonstrated that OX40 signals are essential during priming, not only for the survival of KLRG1(low) MPECs, but also for their self-renewing ability, both of which contribute to the homeostasis of memory CD8(+) T cells.