Mechanisms that limit the in vitro proliferative potential of human CD8+ T lymphocytes

Human T lymphocytes can be numerically expanded in vitro only to a limited extent. The cyclin-dependent kinase inhibitor p16(INK4a) is essential in the control of cellular proliferation, and its expression, in epithelial cells, is associated with irreversible growth arrest. Using long-term cultured CD8+ T lymphocytes, we have investigated the role of the p16/pRb pathway in the regulation of T cell proliferation and senescence. In this study, we describe at least two mechanisms that cause replicative growth arrest in cultured lymphocytes. The first one depends on the expression of p16(INK4a) and is directly responsible for the exit of a significant proportion of CD8+ T cells from the proliferative population. This induced p16 expression pattern is observed during each round of mitogen stimulation and is not related to activation-induced cell death. Importantly, knocking down p16(INK4a) expression allows increased proliferation of T cells. The second one is a phenomenon that resembles human fibroblast senescence, but is independent of p16(INK4a) and of telomere attrition. Interestingly, virtually all pRb proteins in the senescent population are found in the active form. Our data indicate that newly synthesized p16(INK4a) limits the proliferation of T lymphocytes that respond to mitogen, but is not required for the loss of mitogen responsiveness called senescence.     

CD8+ T-cell responses to different HIV proteins have discordant associations with viral load

Selection of T-cell vaccine antigens for chronic persistent viral infections has been largely empirical. To define the relationship, at the population level, between the specificity of the cellular immune response and viral control for a relevant human pathogen, we performed a comprehensive analysis of the 160 dominant CD8(+) T-cell responses in 578 untreated HIV-infected individuals from KwaZulu-Natal, South Africa. Of the HIV proteins targeted, only Gag-specific responses were associated with lowering viremia. Env-specific and Accessory/Regulatory protein-specific responses were associated with higher viremia. Increasing breadth of Gag-specific responses was associated with decreasing viremia and increasing Env breadth with increasing viremia. Association of the specific CD8(+) T-cell response with low viremia was independent of HLA type and unrelated to epitope sequence conservation. These population-based data, suggesting the existence of both effective immune responses and responses lacking demonstrable biological impact in chronic HIV infection, are of relevance to HIV vaccine design and evaluation.     

FoxP3+ CD25+ CD8+ T-cell induction during primary simian immunodeficiency virus infection in cynomolgus macaques correlates with low CD4+ T-cell activation and high viral load

The early immune response fails to prevent the establishment of chronic human immunodeficiency virus (HIV) infection but may influence viremia during primary infection, thereby possibly affecting long-term disease progression. CD25⁺ FoxP3⁺ regulatory T cells may contribute to HIV/simian immunodeficiency virus (SIV) pathogenesis by suppressing efficient antiviral responses during primary infection, favoring high levels of viral replication and the establishment of chronic infection. In contrast, they may decrease immune activation during chronic infection. CD4⁺ regulatory T cells have been studied in the most detail, but CD8⁺ CD25⁺ FoxP3⁺ T cells also have regulatory properties. We monitored the dynamics of CD25⁺ FoxP3⁺ T cells during primary and chronic SIVmac251 infection in cynomolgus macaques. The number of peripheral CD4⁺ CD25⁺ FoxP3⁺ T cells paralleled that of memory CD4⁺ T cells, with a rapid decline during primary infection followed by a rebound to levels just below baseline and gradual depletion during the course of infection. No change in the proportion of CD25⁺ FoxP3⁺ T cells was observed in peripheral lymph nodes. A small number of CD4⁺ CD25⁺ FoxP3⁺ T cells at set point was associated with a high plasma viral load. In contrast, peripheral CD8⁺ CD25⁺ FoxP3⁺ T cells were induced a few days after peak plasma viral load during primary infection. The number of these cells was positively correlated with viral load and negatively correlated with CD4⁺ T-cell activation, SIV antigen-specific proliferative responses during primary infection, and plasma viral load at set point, with large numbers of CD8⁺ CD25⁺ FoxP3⁺ T cells being indicative of a poor prognosis.     

Detailed analysis of the CD8+ T-cell response following adenovirus vaccination

We examined CD8(+) T-cell expansion and function following intramuscular immunization with a recombinant adenovirus. This study has identified a number of properties which may explain the strong immunogenicity of adenovirus vectors: (i) the ability to deliver large amounts of antigen into the lymphoid tissues, (ii) the ability to induce rapid expansion and migration of CD8(+) T cells throughout the lymphatics, and (iii) the ability to produce a sustained, high-level CD8(+) T-cell response.     

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.     

Ablation of CD8 and CD4 T cell responses by high viral loads

To evaluate the impact of sustained viral loads on anti-viral T cell responses we compared responses that cleared acute lymphocytic choriomeningitis virus infection with those that were elicited but could not resolve chronic infection. During acute infection, as replicating virus was cleared, CD8 T cell responses were down-regulated, and a pool of resting memory cells developed. In chronically infected hosts, the failure to control the infection was associated with pronounced and prolonged activation of virus-specific CD8 T cells. Nevertheless, there was a progressive diminution of their effector activities as their capacity to produce first IL-2, then TNF-alpha, and finally IFN-gamma was lost. Chronic lymphocytic choriomeningitis virus infection was also associated with differential contraction of certain CD8 T cell responses, resulting in altered immunodominance. However, this altered immunodominance was not due to selective expansion of T cells expressing particular TCR Vbeta segments during chronic infection. High viral loads were not only associated with the ablation of CD8 T cell responses, but also with impaired production of IL-2 by virus-specific CD4 T cells. Taken together, our data show that sustained exposure to high viral loads results in the progressive functional inactivation of virus-specific T cell responses, which may further promote virus persistence.     

The level of viral antigen presented by hepatocytes influences CD8 T-cell function

CD8 T cells exert their antiviral function through cytokines and lysis of infected cells. Because hepatocytes are susceptible to noncytolytic mechanisms of viral clearance, CD8 T-cell antiviral efficiency against hepatotropic viruses has been linked to their capacity to produce gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). On the other hand, intrahepatic cytokine production triggers the recruitment of mononuclear cells, which sustain acute and chronic liver damage. Using virus-specific CD8 T cells and human hepatocytes, we analyzed the modulation of virus-specific CD8 T-cell function after recognition peptide-pulsed or virally infected hepatocytes. We observed that hepatocyte antigen presentation was generally inefficient, and the quantity of viral antigen strongly influenced CD8 T-cell antiviral function. High levels of hepatitis B virus production induced robust IFN-gamma and TNF-alpha production in virus-specific CD8 T cells, while limiting amounts of viral antigen, both in hepatocyte-like cells and naturally infected human hepatocytes, preferentially stimulated CD8 T-cell degranulation. Our data document a mechanism where virus-specific CD8 T-cell function is influenced by the quantity of virus produced within hepatocytes.     

Understanding the failure of CD8+ T-cell vaccination against simian/human immunodeficiency virus

Although CD8+ T cells play an important role in controlling viral infections, boosting specific CD8+ T cells by prophylactic vaccination with simian immunodeficiency virus (SIV) epitopes fails to provide sterilizing immunity. Viral replication rates and viral contraction rates after the peak viremia hardly depend on the presence of memory CD8+ T cells. To study these paradoxical findings, we parameterize novel mathematical models for acute SIV and human immunodeficiency virus infection. These models explain that failure of vaccination is due to the fact that effector/target ratios are too low during the viral expansion phase. Because CD8+ T cells require cell-to-cell contacts, immune protection requires high effector/target ratios at the primary site of infection. Effector/target ratios become favorable for immune control at the time of the peak in the viral load when the virus becomes limited by other factors, such as the availability of uninfected target cells. At the viral set point, effector/target ratios are much higher, and perturbations of the number of CD8+ effector cells have a large impact on the viral load. Such protective effector/target ratios are difficult to achieve with nucleic acid- or protein-based vaccines.     

Pathogenic potential of borna disease virus lacking the immunodominant CD8 T-cell epitope

Borna disease virus (BDV) is a highly neurotropic, noncytolytic virus. Experimentally infected B10.BR mice remain healthy unless specific antiviral T cells that infiltrate the infected brain are triggered by immunization. In contrast, infected MRL mice spontaneously mount an antiviral T-cell response that can result in meningoencephalitis and neurological disease. The antiviral T cells may, alternatively, eliminate the virus without inducing disease if they are present in sufficient numbers before the virus replicates to high titers. Since the immune response of H-2(k) mice is directed mainly against the epitope TELEISSI located in the viral nucleoprotein N, we generated BDV mutants that feature TQLEISSI in place of TELEISSI. We show that adoptive transfer of BDV N-specific CD8 T cells induced neurological disease in B10.BR mice persistently infected with wild-type BDV but not with the mutant virus expressing TQLEISSI. Surprisingly, the mutant virus replicated less well in adult MRL wild-type mice than in mutant mice lacking mature CD8 T cells. Furthermore, when MRL mice were infected with the TQLEISSI-expressing BDV mutant as newborns, neurological disease was observed, although at a lower rate and with slower kinetics than in mice infected with wild-type virus. These results confirm that TELEISSI is the major CD8 T-cell epitope in H-2(k) mice and suggest that unidentified minor epitopes are present in the BDV proteome which are recognized rather efficiently by antiviral T cells if the dominant epitope is absent.     

Proliferative capacity of epitope-specific CD8 T-cell responses is inversely related to viral load in chronic human immunodeficiency virus type 1 infection

The relationship between the function of human immunodeficiency virus (HIV)-specific CD8 T-cell responses and viral load has not been defined. In this study, we used a panel of major histocompatibility complex class I tetramers to examine responses to frequently targeted CD8 T-cell epitopes in a large cohort of antiretroviral-therapy-na?ve HIV type 1 clade C virus-infected persons in KwaZulu Natal, South Africa. In terms of effector functions of proliferation, cytokine production, and degranulation, only proliferation showed a significant correlation with viral load. This robust inverse relationship provides an important functional correlate of viral control relevant to both vaccine design and evaluation.     

Role of Bim in regulating CD8+ T-cell responses during chronic viral infection

Apoptosis is critical for the development and maintenance of the immune system. The proapoptotic Bcl-2 family member Bim is important for normal immune system homeostasis. Although previous experiments have shown that Bim is critical for the apoptosis of antigen-specific CD8(+) T cells during acute viral infection, the role of Bim during chronic viral infection is unclear. Using lymphocytic choriomeningitis virus clone 13 infection of mice, we demonstrate a role for Bim in CD8(+) T-cell apoptosis during chronic viral infection. Enumeration of antigen-specific CD8(+) T cells by major histocompatibility complex class I tetramer staining revealed that CD8(+) D(b)NP396-404(+) T cells, which undergo extensive deletion in wild-type mice, exhibited almost no decrease in Bim mutant mice. This contrasts with CD8(+) D(b)GP33-41(+) and CD8(+) D(b)GP276-286(+) T cells that underwent similar decreases in numbers in both Bim mutant and wild-type mice. Increased numbers of CD8(+) D(b)NP396-404(+) T cells in Bim mutant mice were due to lack of apoptosis and could not be explained by altered proliferation, differential homing to tissues, or increased help from CD4(+) T cells. When viral titers were examined, high levels were initially observed in both groups, but in Bim mutant mice, clearance from the spleen and sera was slightly accelerated. These experiments demonstrate the critical role of Bim during chronic viral infection to down-regulate CD8(+) T-cell responses and have implications for designing strategies for optimizing immunotherapies during situations where antigen persists, such as chronic infection, autoimmune syndromes, and cancer.     

Differential antigen presentation kinetics of CD8+ T-cell epitopes derived from the same viral protein

The kinetics of peptide presentation by major histocompatibility complex class I (MHC-I) molecules may contribute to the efficacy of CD8+ T cells. Whether all CD8+ T-cell epitopes from a protein are presented by the same MHC-I molecule with similar kinetics is unknown. Here we show that CD8+ T-cell epitopes derived from SIVmac239 Gag are presented with markedly different kinetics. We demonstrate that this discrepancy in presentation is not related to immunodominance but instead is due to differential requirements for epitope generation. These results illustrate that significant differences in presentation kinetics can exist among CD8+ T-cell epitopes derived from the same viral protein.     

Analysis of total human immunodeficiency virus (HIV)-specific CD4(+) and CD8(+) T-cell responses: relationship to viral load in untreated HIV infection

Human immunodeficiency virus (HIV)-specific T-cell responses are thought to play a key role in viral load decline during primary infection and in determining the subsequent viral load set point. The requirements for this effect are unknown, partly because comprehensive analysis of total HIV-specific CD4(+) and CD8(+) T-cell responses to all HIV-encoded epitopes has not been accomplished. To assess these responses, we used cytokine flow cytometry and overlapping peptide pools encompassing all products of the HIV-1 genome to study total HIV-specific T-cell responses in 23 highly active antiretroviral therapy na?ve HIV-infected patients. HIV-specific CD8(+) T-cell responses were detectable in all patients, ranging between 1.6 and 18.4% of total CD8(+) T cells. HIV-specific CD4(+) T-cell responses were present in 21 of 23 patients, although the responses were lower (0.2 to 2.94%). Contrary to previous reports, a positive correlation was identified between the plasma viral load and the total HIV-, Env-, and Nef-specific CD8(+) T-cell frequency. No correlation was found either between viral load and total or Gag-specific CD4(+) T-cell response or between the frequency of HIV-specific CD4(+) and CD8(+) T cells. These results suggest that overall frequencies of HIV-specific T cells are not the sole determinant of immune-mediated protection in HIV-infection.     

Magnitude of functional CD8+ T-cell responses to the gag protein of human immunodeficiency virus type 1 correlates inversely with viral load in plasma

The importance of CD8+ T-cell responses in the control of human immunodeficiency virus type 1 (HIV-1) infection has been demonstrated, yet few studies have been able to correlate these responses with markers of HIV-1 disease progression. This study measured cell-mediated immune responses using peripheral blood mononuclear cells (PBMC) obtained from 27 patients with chronic HIV-1 infection, the majority of whom were off antiretroviral therapy. The ELISPOT assay was used to detect gamma interferon-secreting PBMC after stimulation with overlapping HIV-1 peptides spanning the Gag, Pol, Env, and Nef proteins in addition to the baculovirus-derived p24 and gp160 proteins. All volunteers had responses to at least one HIV-1-specific peptide. All but one of the subjects (96%) responded to the Gag peptide pool, and 86% responded to the Pol and/or Nef peptide pools. The magnitude and the breadth of T-cell responses directed to either the Gag or p24 peptide pools correlated inversely with viral load in plasma (r = -0.60, P < 0.001 and r = -0.52, P < 0.005, respectively) and directly with absolute CD4+ T-cell counts (r = 0.54, P < 0.01 and r = 0.39, P < 0.05, respectively) using the Spearman rank correlation test. Responses to the Pol and integrase peptide pools also correlated with absolute CD4+ T-cell counts (r = 0.45, P < 0.05 and r = 0.49, P < 0.01, respectively). No correlation with markers of disease progression was seen with specific T-cell responses directed toward the Env or Nef peptides. These data serve as strong evidence that major histocompatibility complex class I presentation of Gag peptides is an essential feature for any HIV-1 vaccine designed to elicit optimal CD8+ T-cell responses.     

Longitudinal comparison of virus load parameters and CD8 T-cell suppressive capacity in two SIVcpz-infected chimpanzees

In a longitudinal study we address the hypothesis that resis tance to disease progression in lentivirus-infected chimpanzees is related to potent non-cytotoxic suppression of virus replication. In a long-term follow-up, the viral suppressive capacity in two simian immunodeficiency virus (SIV)cpz-infected chimpanzees was correlated with two polymerase chain reaction (PCR)- and two culture-based virus load measurements. In both animals, quantitative virus isolation (QVI) tended to decline slowly, whereas in vitro virus suppression was sustained or increased over time. In general, plasma virus loads in SIVcpz-infected animals were maintained for extended periods of time. Based on current assays that measure virus suppressive capacity in peripheral blood, it was not possible to conclude that virus suppression played a major role in the maintenance of the disease-free state in lentivirus-infected chimpanzees.     

Role of thymic output in regulating CD8 T-cell homeostasis during acute and chronic viral infection

Although it is well documented that CD8 T cells play a critical role in controlling chronic viral infections, the mechanisms underlying the regulation of CD8 T-cell responses are not well understood. Using the mouse model of an acute and chronic lymphocytic choriomeningitis virus (LCMV) infection, we have examined the relative importance of peripheral T cells and thymic emigrants in the elicitation and maintenance of CD8 T-cell responses. Virus-specific CD8 T-cell responses were compared between mice that were either sham thymectomized or thymectomized (Thx) at approximately 6 weeks of age. In an acute LCMV infection, thymic deficiency did not affect either the primary expansion of CD8 T cells or the proliferative renewal and maintenance of virus-specific lymphoid and nonlymphoid memory CD8 T cells. Following a chronic LCMV infection, in Thx mice, although the initial expansion of CD8 T cells was normal, the contraction phase of the CD8 T-cell response was exaggerated, which led to a transient but striking CD8 T-cell deficit on day 30 postinfection. However, the virus-specific CD8 T-cell response in Thx mice rebounded quickly and was maintained at normal levels thereafter, which indicated that the peripheral T-cell repertoire is quite robust and capable of sustaining an effective CD8 T-cell response in the absence of thymic output during a chronic LCMV infection. Taken together, these findings should further our understanding of the regulation of CD8 T-cell homeostasis in acute and chronic viral infections and might have implications in the development of immunotherapy.