4-1BBL induces TNF receptor-associated factor 1-dependent Bim modulation in human T cells and is a critical component in the costimulation-dependent rescue of functionally impaired HIV-specific CD8 T cells

During chronic infection, HIV-specific CD8 T cells exhibit progressive signs of functional impairment, attributed to persistent antigenic stimulation, up-regulation of the inhibitory receptor PD-1, and declining T cell help. Strategies that directly improve CD8 T cell function offer the potential of restoring immune control of HIV. Although PD-1 expression has been identified as a cause of functional impairment in HIV, in this study, PD-1 expression was observed on only a subfraction of HIV-specific CD8 T cells in a subfraction of donors, whereas HIV-specific CTL from all donors exhibited a limited repertoire of effector functions. CD137L (4-1BBL) is emerging as an important stimulator of antiviral CD8 T cell responses. Regardless of the PD-1 status of the donors, here we show that 4-1BBL, when combined with CD80 or CD70, expands a population of Ag-specific CD8 T cells expressing multiple markers of effector function, from the functionally impaired starting population. In contrast, CD70 in combination with CD80 was insufficient for these effects and the related TNF family ligand, LIGHT, had negligible activity. The unique contribution of 4-1BBL correlated with down-regulation of the proapoptotic molecule Bim in activated CD8 T cells. Decreasing the level of TNFR-associated factor 1 in T cells using small interfering RNA resulted in increased levels of Bim in the 4-1BBL-stimulated T cells. Thus, costimulation via 4-1BBL leads to TNFR-associated factor 1-dependent Bim down-modulation in T cells, resulting in increased T cell expansion. These studies identify 4-1BBL as a critical component in therapeutic strategies aimed at improving CD8 T cell function.     

Large TCR diversity of virus-specific CD8 T cells provides the mechanistic basis for massive TCR renewal after antigen exposure

Ex vivo analysis of virus-specific CD8 T cell populations by anchored PCR has shown that the CD8 TCR repertoire was less oligoclonal (seven to nine clonotypes per individual epitope) than previously thought. In the current study, TCR diversity was investigated by assessing both the overall TCR β-chain variable regions usage as well as the CDR3 regions in ex vivo-isolated CMV- and EBV-specific CD8 T cells from 27 healthy donors. The average number of clonotypes specific to most single viral epitopes comprised between 14 and 77. Changes in the CD8 TCR repertoire were also longitudinally assessed under conditions of HIV-1 chronic infection (i.e., in patients with suppressed virus replication and after treatment interruption and Ag re-exposure). The results showed that a large renewal (≤ 80%) of the TRB repertoire occurred after Ag re-exposure and was eventually associated with an increased T cell recognition functional avidity. These results demonstrate that the global CD8 TCR repertoire is much more diverse (≤ 9-fold) than previously estimated and provide the mechanistic basis for supporting massive repertoire renewal during chronic virus infection and Ag re-exposure.     

Maintenance of large numbers of virus-specific CD8+ T cells in HIV-infected progressors and long-term nonprogressors

The virus-specific CD8+ T cell responses of 21 HIV-infected patients were studied including a unique cohort of long-term nonprogressors with low levels of plasma viral RNA and strong proliferative responses to HIV Ags. HIV-specific CD8+ T cell responses were studied by a combination of standard cytotoxic T cell (CTL) assays, MHC tetramers, and TCR repertoire analysis. The frequencies of CD8+ T cells specific to the majority of HIV gene products were measured by flow cytometric detection of intracellular IFN-gamma in response to HIV-vaccinia recombinant-infected autologous B cells. Very high frequencies (0.8-18.0%) of circulating CD8+ T cells were found to be HIV specific. High frequencies of HIV-specific CD8+ T cells were not limited to long-term nonprogressors with restriction of plasma virus. No correlation was found between the frequency of HIV-specific CD8+ T cells and levels of plasma viremia. In each case, the vast majority of cells (up to 17.2%) responded to gag-pol. Repertoire analysis showed these large numbers of Ag-specific cells were scattered throughout the repertoire and in the majority of cases not contained within large monoclonal expansions. These data demonstrate that high numbers of HIV-specific CD8+ T cells exist even in patients with high-level viremia and progressive disease. Further, they suggest that other qualitative parameters of the CD8+ T cell response may differentiate some patients with very low levels of plasma virus and nonprogressive disease.     

Programmed death 1 expression on HIV-specific CD4+ T cells is driven by viral replication and associated with T cell dysfunction

Functional impairment of HIV-specific CD4(+) T cells during chronic HIV infection is closely linked to viral replication and thought to be due to T cell exhaustion. Programmed death 1 (PD-1) has been linked to T cell dysfunction in chronic viral infections, and blockade of the PD-1 pathway restores HIV-specific CD4(+) and CD8(+) T cell function in HIV infection. This study extends those findings by directly examining PD-1 expression on virus-specific CD4(+) T cells. To investigate the role of PD-1 in HIV-associated CD4(+) T cell dysfunction, we measured PD-1 expression on blood and lymph node T cells from HIV-infected subjects with chronic disease. PD-1 expression was significantly higher on IFN-gamma-producing HIV-specific CD4(+) T cells compared with total or CMV-specific CD4(+) T cells in untreated HIV-infected subjects (p = 0.0001 and p < 0.0001, respectively). PD-1 expression on HIV-specific CD4(+) T cells from subjects receiving antiretroviral therapy was significantly reduced (p = 0.007), and there was a direct correlation between PD-1 expression on HIV-specific CD4(+) T cells and plasma viral load (r = 0.71; p = 0.005). PD-1 expression was significantly higher on HIV-specific T cells in the lymph node, the main site of HIV replication, compared with those in the blood (p = 0.0078). Thus, PD-1 expression on HIV-specific CD4(+) T cells is driven by persistent HIV replication, providing a potential target for enhancing the functional capacity of HIV-specific CD4(+) T cells.     

Upregulation of PD-1 expression on HIV-specific CD8+ T cells leads to reversible immune dysfunction

The engagement of programmed death 1 (PD-1) to its ligands, PD-L1 and PD-L2, inhibits proliferation and cytokine production mediated by antibodies to CD3 (refs. 5,6,7). Blocking the PD-1-PD-L1 pathway in mice chronically infected with lymphocytic choriomeningitis virus restores the capacity of exhausted CD8(+) T cells to undergo proliferation, cytokine production and cytotoxic activity and, consequently, results in reduced viral load. During chronic HIV infection, HIV-specific CD8(+) T cells are functionally impaired, showing a reduced capacity to produce cytokines and effector molecules as well as an impaired capacity to proliferate. Here, we found that PD-1 was upregulated on HIV-specific CD8(+) T cells; PD-1 expression levels were significantly correlated both with viral load and with the reduced capacity for cytokine production and proliferation of HIV-specific CD8(+) T cells. Notably, cytomegalovirus (CMV)-specific CD8(+) T cells from the same donors did not upregulate PD-1 and maintained the production of high levels of cytokines. Blocking PD-1 engagement to its ligand (PD-L1) enhanced the capacity of HIV-specific CD8(+) T cells to survive and proliferate and led to an increased production of cytokines and cytotoxic molecules in response to cognate antigen. The accumulation of HIV-specific dysfunctional CD8(+) T cells in the infected host could prevent the renewal of a functionally competent HIV-specific CD8(+) repertoire.     

High-functional-avidity cytotoxic T lymphocyte responses to HLA-B-restricted Gag-derived epitopes associated with relative HIV control

Virus-specific cytotoxic T lymphocytes (CTL) with high levels of functional avidity have been associated with viral clearance in hepatitis C virus infection and with enhanced antiviral protective immunity in animal models. However, the role of functional avidity as a determinant of HIV-specific CTL efficacy remains to be assessed. Here we measured the functional avidities of HIV-specific CTL responses targeting 20 different, optimally defined CTL epitopes restricted by 13 different HLA class I alleles in a cohort comprising 44 HIV controllers and 68 HIV noncontrollers. Responses restricted by HLA-B alleles and responses targeting epitopes located in HIV Gag exhibited significantly higher functional avidities than responses restricted by HLA-A or HLA-C molecules (P = 0.0003) or responses targeting epitopes outside Gag (P < 0.0001). The functional avidities of Gag-specific and HLA-B-restricted responses were higher in HIV controllers than in noncontrollers (P = 0.014 and P = 0.018) and were not restored in HIV noncontrollers initiating antiretroviral therapy. T-cell receptor (TCR) analyses revealed narrower TCR repertoires in higher-avidity CTL populations, which were dominated by public TCR sequences in HIV controllers. Together, these data link the presence of high-avidity Gag-specific and HLA-B-restricted CTL responses with viral suppression in vivo and provide new insights into the immune parameters that mediate spontaneous control of HIV infection.     

Late priming and variability of epitope-specific CD8+ T cell responses during a persistent virus infection

Control of persistently infecting viruses requires that antiviral CD8(+) T cells sustain their numbers and effector function. In this study, we monitored epitope-specific CD8(+) T cells during acute and persistent phases of infection by polyoma virus, a mouse pathogen that is capable of potent oncogenicity. We identified several novel polyoma-specific CD8(+) T cell epitopes in C57BL/6 mice, a mouse strain highly resistant to polyoma virus-induced tumors. Each of these epitopes is derived from the viral T proteins, nonstructural proteins produced by both productively and nonproductively (and potentially transformed) infected cells. In contrast to CD8(+) T cell responses described in other microbial infection mouse models, we found substantial variability between epitope-specific CD8(+) T cell responses in their kinetics of expansion and contraction during acute infection, maintenance during persistent infection, as well as their expression of cytokine receptors and cytokine profiles. This epitope-dependent variability also extended to differences in maturation of functional avidity from acute to persistent infection, despite a narrowing in TCR repertoire across all three specificities. Using a novel minimal myeloablation-bone marrow chimera approach, we visualized priming of epitope-specific CD8(+) T cells during persistent virus infection. Interestingly, epitope-specific CD8(+) T cells differed in CD62L-selectin expression profiles when primed in acute or persistent phases of infection, indicating that the context of priming affects CD8(+) T cell heterogeneity. In summary, persistent polyoma virus infection both quantitatively and qualitatively shapes the antiviral CD8(+) T cell response.     

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.     

Altering effects of antigenic variations in HIV-1 on antiviral effectiveness of HIV-specific CTLs

The mutational escape of HIV-1 from established CTL responses is becoming evident. However, it is not yet clear whether antigenic variations of HIV-1 may have an additional effect on the differential antiviral effectiveness of HIV-specific CTLs. Herein, we characterized HIV-specific CTL responses toward Pol, Env, and Nef optimal epitopes presented by HLA-B*35 during a chronic phase of HIV-1 infection. We found CTL escape variants within Pol and Nef epitopes that affected recognition by TCRs, although there was no mutation within the Env epitope. An analysis of peptide-HLA tetrameric complexes revealed that CD8 T cells exclusively specific for the Nef variant were generated following domination by the variant viruses. The variant-specific cells were capable of killing target cells and producing antiviral cytokines but showed impaired Ag-specific proliferation ex vivo, whereas wild-type specific cells had potent activities. Moreover, clonotypic CD8 T cells specific for the Pol variant showed diminished proliferation, whereas Env-specific ones had no functional heterogeneity. Taken together, our data indicate that antigenic variations that abolished TCR recognition not only resulted in escape from established CTL responses but also eventually generated another subset of variant-specific CTLs having decreased antiviral activity, causing an additional negative effect on antiviral immune responses during a chronic HIV infection.     

Antiretroviral therapy reduces the magnitude and T cell receptor repertoire diversity of HIV-specific T cell responses without changing T cell clonotype dominance

After initiation of antiretroviral therapy (ART), HIV loads and frequencies of HIV epitope-specific immune responses decrease. A diverse virus-specific T cell receptor (TCR) repertoire allows the host to respond to viral epitope diversity, but the effect of antigen reduction as a result of ART on the TCR repertoire of epitope-specific CD8(+) T cell populations has not been well defined. We determined the TCR repertoires of 14 HIV-specific CD8(+) T cell responses from 8 HIV-positive individuals before and after initiation of ART. We used multiparameter flow cytometry to measure the distribution of memory T cell subsets and the surface expression of PD-1 on T cell populations and T cell clonotypes within epitope-specific responses from these individuals. Post-ART, we noted decreases in the frequency of circulating epitope-specific T cells (P = 0.02), decreases in the number of T-cell clonotypes found within epitope-specific T cell receptor repertoires (P = 0.024), and an overall reduction in the amino acid diversity within these responses (P < 0.0001). Despite this narrowing of the T cell response to HIV, the overall hierarchy of dominant T cell receptor clonotypes remained stable compared to that pre-ART. CD8(+) T cells underwent redistributions in memory phenotypes and a reduction in CD38 and PD-1 expression post-ART. Despite extensive remodeling at the structural and phenotypic levels, PD-1 was expressed at higher levels on dominant clonotypes within epitope-specific responses before and after initiation of ART. These data suggest that the antigen burden may maintain TCR diversity and that dominant clonotypes are sensitive to antigen even after dramatic reductions after initiation of ART.     

Dominant clonotypes within HIV-specific T cell responses are programmed death-1high and CD127low and display reduced variant cross-reactivity

HIV epitope-specific T cell responses are often comprised of clonotypic expansions with distinct functional properties. In HIV(+) individuals, we measured programmed death-1 (PD-1) and IL-7Rα expression, MHC class I tetramer binding, cytokine production, and proliferation profiles of dominant and subdominant TCR clonotypes to evaluate the relationship between the composition of the HIV-specific T cell repertoire and clonotypic phenotype and function. Dominant clonotypes are characterized by higher PD-1 expression and lower C127 expression compared with subdominant clonotypes, and TCR avidity positively correlates with PD-1 expression. At low peptide concentrations, dominant clonotypes fail to survive in culture. In response to stimulation with peptides representing variant epitopes, subdominant clonotypes produce higher relative levels of cytokines and display greater capacity for cross-recognition compared with dominant clonotypes. These data indicate that dominant clonotypes within HIV-specific T cell responses display a phenotype consistent with ongoing exposure to cognate viral epitopes and suggest that cross-reactive, subdominant clonotypes may retain greater capacity to suppress replication of viral variants as well as to survive in the absence of strong antigenic signaling.     

Early Skewed Distribution of Total and HIV-Specific CD8+ T-Cell Memory Phenotypes during Primary HIV Infection Is Related to Reduced Antiviral Activity and Faster Disease Progression

The important role of the CD8⁺ T-cells on HIV control is well established. However, correlates of immune protection remain elusive. Although the importance of CD8⁺ T-cell specificity and functionality in virus control has been underscored, further unraveling the link between CD8⁺ T-cell differentiation and viral control is needed. Here, an immunophenotypic analysis (in terms of memory markers and Programmed cell death 1 (PD-1) expression) of the CD8⁺ T-cell subset found in primary HIV infection (PHI) was performed. The aim was to seek for associations with functional properties of the CD8⁺ T-cell subsets, viral control and subsequent disease progression. Also, results were compared with samples from Chronics and Elite Controllers. It was found that normal maturation of total and HIV-specific CD8⁺ T-cells into memory subsets is skewed in PHI, but not at the dramatic level observed in Chronics. Within the HIV-specific compartment, this alteration was evidenced by an accumulation of effector memory CD8⁺ T (T_EM) cells over fully differentiated terminal effector CD8⁺ T (T_TE) cells. Furthermore, higher proportions of total and HIV-specific CD8⁺ T_EM cells and higher HIV-specific T_EM/(T_EM+T_TE) ratio correlated with markers of faster progression. Analysis of PD-1 expression on total and HIV-specific CD8⁺ T-cells from PHI subjects revealed not only an association with disease progression but also with skewed memory CD8⁺ T-cell differentiation. Most notably, significant direct correlations were obtained between the functional capacity of CD8⁺ T-cells to inhibit viral replication in vitro with higher proportions of fully-differentiated HIV-specific CD8⁺ T_TE cells, both at baseline and at 12 months post-infection. Thus, a relationship between preservation of CD8⁺ T-cell differentiation pathway and cell functionality was established. This report presents evidence concerning the link among CD8⁺ T-cell function, phenotype and virus control, hence supporting the instauration of early interventions to prevent irreversible immune damage.     

Programmed death 1 regulates development of central memory CD8 T cells after acute viral infection

The T cell response possesses a number of inhibitory receptors to regulate the extent of the antiviral response and prevent immune pathology. These receptors are generally transiently upregulated during an effector response and then downregulated during memory. Some inhibitory receptors, such as programmed death 1 (PD-1) and LAG-3, were shown to be aberrantly upregulated during memory to chronic lymphocytic choriomeningitis virus infection, limiting functional capabilities. However, little is known about the impact of inhibitory receptors on memory development during a normal CD8 T cell response to acute virus infection. Our previous data showed that PD-1 is aberrantly upregulated during a secondary response by memory CD8 T cells that were generated without CD4 T cell help. Therefore, we examined the role of PD-1 in memory differentiation during acute vaccinia virus infection in intact mice. In the absence of PD-1, the primary and memory CD8 T cell responses were enhanced. Moreover, there were distinct phenotypic and functional changes in the memory PD-1(-/-) CD8 T cells. Higher levels of CD62L, CD27, and CCR7 were detected; cells produced more IL-2 and made an enhanced secondary response. These changes indicate a skewing of the memory population toward the central memory phenotype in the absence of PD-1 signaling.     

Broad TCR usage in functional HIV-1-specific CD8+ T cell expansions driven by vaccination during highly active antiretroviral therapy

During chronic HIV-1 infection, continuing viral replication is associated with impaired proliferative capacity of virus-specific CD8+ T cells and with the expansion and persistence of oligoclonal T cell populations. TCR usage may significantly influence CD8+ T cell-mediated control of AIDS viruses; however, the potential to modulate the repertoire of functional virus-specific T cells by immunotherapy has not been explored. To investigate this, we analyzed the TCR Vbeta usage of CD8+ T cells populations which were expanded following vaccination with modified vaccinia virus Ankara expressing a HIV-1 gag/multiepitope immunogen (MVA.HIVA) in HIV-1-infected patients receiving highly active antiretroviral therapy. Vaccinations induced the re-expansion of HIV-1-specific CD8+ T cells and these showed broad TCR Vbeta usage which was maintained for at least 1 year in some individuals. By contrast, virus-specific CD8+ T cell populations in the same donors which failed to expand after vaccination and in unvaccinated controls were oligoclonal. Simultaneously, we observed that CD8+ T cells recognizing vaccine-derived HIV-1 epitopes displayed enhanced capacity to proliferate and to inhibit HIV-1 replication in vitro, following MVA.HIVA immunizations. Taken together, these data indicate that an attenuated viral-vectored vaccine can modulate adaptive CD8+ T cell responses to HIV-1 and improve their antiviral functional capacity. The potential therapeutic benefit of this vaccination approach warrants further investigation.     

Early Gag Immunodominance of the HIV-Specific T-Cell Response during Acute/Early Infection Is Associated with Higher CD8+ T-Cell Antiviral Activity and Correlates with Preservation of the CD4+ T-Cell Compartment

The important role of the CD8⁺ T-cell response on HIV control is well established. Moreover, the acute phase of infection represents a proper scenario to delineate the antiviral cellular functions that best correlate with control. Here, multiple functional aspects (specificity, ex vivo viral inhibitory activity [VIA] and polyfunctionality) of the HIV-specific CD8⁺ T-cell subset arising early after infection, and their association with disease progression markers, were examined. Blood samples from 44 subjects recruited within 6 months from infection (primary HIV infection [PHI] group), 16 chronically infected subjects, 11 elite controllers (EC), and 10 healthy donors were obtained. Results indicated that, although Nef dominated the anti-HIV response during acute/early infection, a higher proportion of early anti-Gag T cells correlated with delayed progression. Polyfunctional HIV-specific CD8⁺ T cells were detected at early time points but did not associate with virus control. Conversely, higher CD4⁺ T-cell set points were observed in PHI subjects with higher HIV-specific CD8⁺ T-cell VIA at baseline. Importantly, VIA levels correlated with the magnitude of the anti-Gag cellular response. The advantage of Gag-specific cells may result from their enhanced ability to mediate lysis of infected cells (evidenced by a higher capacity to degranulate and to mediate VIA) and to simultaneously produce IFN-γ. Finally, Gag immunodominance was associated with elevated plasma levels of interleukin 2 (IL-2) and macrophage inflammatory protein 1β (MIP-1β). All together, this study underscores the importance of CD8⁺ T-cell specificity in the improved control of disease progression, which was related to the capacity of Gag-specific cells to mediate both lytic and nonlytic antiviral mechanisms at early time points postinfection.     

Upregulation of the Tim-3/Galectin-9 Pathway of T Cell Exhaustion in Chronic Hepatitis B Virus Infection

The S-type lectin galectin-9 binds to the negative regulatory molecule Tim-3 on T cells and induces their apoptotic deletion or functional inactivation. We investigated whether galectin-9/Tim-3 interactions contribute to the deletion and exhaustion of the antiviral T cell response in chronic hepatitis B virus infection (CHB). We found Tim-3 to be expressed on a higher percentage of CD4 and CD8 T cells from patients with CHB than healthy controls (p<0.0001) and to be enriched on activated T cells and those infiltrating the HBV-infected liver. Direct ex vivo examination of virus-specific CD8 T cells binding HLA-A2/peptide multimers revealed that Tim-3 was more highly upregulated on HBV-specific CD8 T cells than CMV-specific CD8 T cells or the global CD8 T cell population in patients with CHB (p<0.001) or than on HBV-specific CD8 after resolution of infection. T cells expressing Tim-3 had an impaired ability to produce IFN-γ and TNF-α upon recognition of HBV-peptides and were susceptible to galectin-9-triggered cell death in vitro. Galectin-9 was detectable at increased concentrations in the sera of patients with active CHB-related liver inflammation (p = 0.02) and was strongly expressed by Kupffer cells within the liver sinusoidal network. Tim-3 blockade resulted in enhanced expansion of HBV-specific CD8 T cells able to produce cytokines and mediate cytotoxicity in vitro. Blocking PD-1 in combination with Tim-3 enhanced the number of patients from whom functional antiviral responses could be recovered and/or the strength of responses, indicating that these co-inhibitory molecules play a non-redundant role in driving T cell exhaustion in CHB. Patients taking antivirals able to potently suppress HBV viraemia continued to express Tim-3 on their T cells and respond to Tim-3 blockade. In summary, both Tim-3 and galectin-9 are increased in CHB and may contribute to the inhibition and deletion of T cells as they infiltrate the HBV-infected liver.