Mirabelle Dagarag

The role of CD8+ T-cell replicative senescence in human aging

Summary:  The strict limit in proliferative potential of normal human somatic cells – a process known as replicative senescence – is highly relevant to the immune system, because clonal expansion is fundamental to adaptive immunity. CD8+ T cells that undergo extensive rounds of antigen‐driven proliferation in cell culture invariably reach the end stage of replicative senescence, characterized by irreversible cell‐cycle arrest and a critically short telomere length. Cultures of senescent CD8+ T cells also show resistance to apoptosis, permanent loss of CD28 expression, altered cytokine profiles, reduced ability to respond to stress, and various functional changes. Cells with similar characteristics accumulate during normal aging as well as in younger persons infected with human immunodeficiency virus, suggesting that the process of replicative senescence is not an artifact of cell culture but is also occurring in vivo. Interestingly, in elderly persons, the presence of high proportions of CD8+ T cells with characteristics of replicative senescence is correlated with reduced antibody responses to vaccines as well as with osteoporotic fractures. CD8+CD28– T cells also accumulate in patients with certain types of cancer. The emerging picture is that senescent CD8+ T cells may modulate both immune and non‐immune functions, contributing not only to reduced anti‐viral immunity but also to diverse age‐related pathologies.

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-γ and TNF-α 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 p16INK4a and p21WAF1 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.

Differential Impairment of Lytic and Cytokine Functions in Senescent Human Immunodeficiency Virus Type 1-Specific Cytotoxic T Lymphocytes

ABSTRACT Telomere length is abnormally short in the CD8+ T-cell compartment of human immunodeficiency virus type 1 (HIV-1)-infected persons, likely because of chronic cell turnover. Although clonal exhaustion of CD8+ cytotoxic T lymphocytes (CTL) has been proposed as a mechanism for loss of antigen-specific responses, the functional consequences of exhaustion are poorly understood. Here we used telomerase transduction to evaluate the impact of senescence on CTL effector functions. Constitutive expression of telomerase in an HIV-1-specific CTL clone results in enhanced proliferative capacity, in agreement with prior studies of other human cell types. Whereas the CTL remain phenotypically normal in terms of antigenic specificity and requirements for proliferation, their cytolytic and antiviral capabilities are superior to those of control CTL. In contrast, their ability to produce gamma interferon and RANTES is essentially unchanged. The selective enhancement of cytolytic function in memory CTL by ectopic telomerase expression implies that loss of this function (but not cytokine production) is a specific consequence of replicative senescence. These data suggest a unifying mechanism for the in vivo observations that telomere lengths are shortened in the CD8+ cells of HIV-1-infected persons and that HIV-1-specific CTL are deficient in perforin. Telomerase transduction could therefore be a tool with which to explore a potential therapeutic approach to an important pathophysiologic process of immune dysfunction in chronic viral infection.

Epitope-Dependent Avidity Thresholds for Cytotoxic T-Lymphocyte Clearance of Virus-Infected Cells

ABSTRACT Cytotoxic T lymphocytes (CTLs) are crucial for immune control of viral infections. “Functional avidity,” defined by the sensitizing dose of exogenously added epitope yielding half-maximal CTL triggering against uninfected target cells (SD50), has been utilized extensively as a measure of antiviral efficiency. However, CTLs recognize infected cells via endogenously produced epitopes, and the relationship of SD50 to antiviral activity has never been directly revealed. We elucidate this relationship by comparing CTL killing of cells infected with panels of epitope-variant viruses to the corresponding SD50 for the variant epitopes. This reveals a steeply sigmoid relationship between avidity and infected cell killing, with avidity thresholds (defined as the SD50 required for CTL to achieve 50% efficiency of infected cell killing [KE50]), below which infected cell killing rapidly drops to none and above which killing efficiency rapidly plateaus. Three CTL clones recognizing the same viral epitope show the same KE50 despite differential recognition of individual epitope variants, while CTLs recognizing another epitope show a 10-fold-higher KE50, demonstrating epitope dependence of KE50. Finally, the ability of CTLs to suppress viral replication depends on the same threshold KE50. Thus, defining KE50 values is required to interpret the significance of functional avidity measurements and predict CTL efficacy against virus-infected cells in pathogenesis and vaccine studies.

In vitro senescence of immune cells

Immune cells are eminently suitable model systems in which to address the possible role of replicative senescence during in vivo aging. Since there are more than 10(8) unique antigen specificities present within the total T lymphocyte population of each individual, the immune response to any single antigen requires massive clonal expansion of the small proportion of T cells whose receptors recognize that antigen. The Hayflick Limit may, therefore, constitute a barrier to effective immune function, at least for those T cells that encounter their specific antigen more than once over the life course. Application of the fibroblast replicative senescence model to the so-called cytotoxic or CD8 T cell, the class of T cells that controls viral infection and cancer, has revealed certain features in common with other cell types as well as several characteristics that are unique to T cells. One senescence-associated change that is T cell-specific is the complete loss of expression of the activation signaling surface molecule, CD28, an alteration that enabled the documentation of high proportions of senescent T cells in vivo. The T cell model has also provided the unique opportunity to analyze telomere dynamics in a cell type that has the ability to upregulate telomerase yet nevertheless undergoes senescence. The intimate involvement of the immune system in the control of pathogens and cancer as well as in modulation of bone homeostasis suggests that more extensive analysis of the full range of characteristics of senescent T cells may help elucidate a broad spectrum of age-associated physiological changes.

Evasion of cytotoxic T lymphocytes is a functional constraint maintaining HIV-1 Nef expression.

Nef expression is not required for HIV‐1 replication and is highly targeted by CD8+ CTL, raising the question of why Nef expression is not lost in order to evade immunity in vivo. We explore whether MHC class I (MHC‐I) down‐regulation to evade CTL in general is a selective pressure maintaining Nef. HIV‐1 with functional Nef (wild type, WT) is compared to virus containing a Nef point mutation (M20A) that selectively ablates MHC‐I down‐regulation. WT‐infected cells are relatively resistant to cytolysis and less suppressed for viral replication by Gag‐ and RT‐specific CTL compared to M20A. These viruses grow similarly in vitro in the absence of CTL, but the presence of Gag‐ or RT‐specific CTL strongly favors WT overgrowth of M20A. Finally, while in vitro selection by Nef‐specific CTL readily drives disruption of the nef reading frame, the addition of Gag‐ or RT‐specific CTL markedly limits such escape. These data indicate that MHC‐I down‐regulation is an important function favoring Nef maintenance due to a net selective advantage in the setting of the general CTL response.

Partial Escape of HIV-1 From Cytotoxic T Lymphocytes During Chronic Infection

ABSTRACT Viral mutational escape from CD8+ cytotoxic T lymphocytes (CTLs) is typically considered to be a dichotomous process and uncommon during chronic HIV-1 infection. Ex vivo passaging of HIV-1 from persons with chronic infection, however, revealed the evolution of many fixed substitutions within and around CTL-targeted regions, with an associated increase in replicative capacity. This indicates an evolution of mutations during chronic HIV-1 infection that trade replicative fitness for incomplete evasion of CTLs, or “partial escape.”