Anti-Tat immunity defines CD4+ T-cell dynamics in people living with HIV on long-term cART.

BACKGROUND: Low-level HIV viremia originating from virus reactivation in HIV reservoirs is often present in cART treated individuals and represents a persisting source of immune stimulation associated with sub-optimal recovery of CD4+ T cells. The HIV-1 Tat protein is released in the extracellular milieu and activates immune cells and latent HIV, leading to virus production and release. However, the relation of anti-Tat immunity with residual viremia, persistent immune activation and CD4+ T-cell dynamics has not yet been defined. METHODS: Volunteers enrolled in a 3-year longitudinal observational study were stratified by residual viremia, Tat serostatus and frequency of anti-Tat cellular immune responses. The impact of anti-Tat immunity on low-level viremia, persistent immune activation and CD4+ T-cell recovery was investigated by test for partitions, longitudinal regression analysis for repeated measures and generalized estimating equations. FINDINGS: Anti-Tat immunity is significantly associated with higher nadir CD4+ T-cell numbers, control of low-level viremia and long-lasting CD4+ T-cell recovery, but not with decreased immune activation. In adjusted analysis, the extent of CD4+ T-cell restoration reflects the interplay among Tat immunity, residual viremia and immunological determinants including CD8+ T cells and B cells. Anti-Env immunity was not related to CD4+ T-cell recovery. INTERPRETATION: Therapeutic approaches aiming at reinforcing anti-Tat immunity should be investigated to improve immune reconstitution in people living with HIV on long-term cART. TRIAL REGISTRATION: ISS OBS T-002 ClinicalTrials.gov identifier: NCT01024556 FUNDING: Italian Ministry of Health, special project on the Development of a vaccine against HIV based on the Tat protein and Ricerca Corrente 2019/2020.

Continued Decay of HIV Proviral DNA Upon Vaccination With HIV-1 Tat of Subjects on Long-Term ART: An 8-Year Follow-Up Study.

Introduction: Tat, a key HIV virulence protein, has been targeted for the development of a therapeutic vaccine aimed at cART intensification. Results from phase II clinical trials in Italy (ISS T-002) and South Africa (ISS T-003) indicated that Tat vaccination promotes increases of CD4+ T-cells and return to immune homeostasis while reducing the virus reservoir in chronically cART-treated patients. Here we present data of 92 vaccinees (59% of total vaccinees) enrolled in the ISS T-002 8-year extended follow-up study (ISS T-002 EF-UP, ClinicalTrials.gov NCT02118168). Results: Anti-Tat antibodies (Abs) induced upon vaccination persisted for the entire follow-up in 34/92 (37%) vaccinees, particularly when all 3 Ab classes (A/G/M) were present (66% of vaccinees), as most frequently observed with Tat 30 µg regimens. CD4+ T cells increased above study-entry levels reaching a stable plateau at year 5 post-vaccination, with the highest increase (165 cells/µL) in the Tat 30 µg, 3 × regimen. CD4+ T-cell increase occurred even in subjects with CD4+ nadir ≤ 250 cells/uL and in poor immunological responders and was associated with a concomitant increase of the CD4+/CD8+ T-cell ratio, a prognostic marker of morbidity/mortality inversely related to HIV reservoir size. Proviral DNA load decreased over time, with a half-life of 2 years and an estimated 90% reduction at year 8 in the Tat 30 µg, 3 × group. In multivariate analysis the kinetic and amplitude of both CD4+ T-cell increase and proviral DNA reduction were fastest and highest in subjects with all 3 anti-Tat Ab classes and in the 30 µg, 3 × group, irrespective of drug regimens (NNRTI/NRTI vs. PI). HIV proviral DNA changes from baseline were inversely related to CD4+/CD8+ T-cell ratio and CD4+ T-cell changes, and directly related to the changes of CD8+ T cells. Further, HIV DNA decay kinetics were inversely related to the frequency and levels of intermittent viremia. Finally, Tat vaccination was similarly effective irrespective of the individual immunological status or HIV reservoir size at study entry. Conclusions: Tat immunization induces progressive immune restoration and reduction of virus reservoirs above levels reached with long-term cART, and may represent an optimal vaccine candidate for cART intensification toward HIV reservoirs depletion, functional cure, and eradication strategies.

"cART intensification by the HIV-1 Tat B clade vaccine: progress to phase III efficacy studies".

INTRODUCTION: In spite of its success at suppressing HIV replication, combination antiretroviral therapy (cART) only partially reduces immune dysregulation and loss of immune functions. These cART-unmet needs appear to be due to persistent virus replication and cell-to-cell transmission in reservoirs, and are causes of increased patients' morbidity and mortality. Up to now, therapeutic interventions aimed at cART-intensification by attacking the virus reservoir have failed. AREAS COVERED: We briefly review the rationale and clinical development of Tat therapeutic vaccine in cART-treated subjects in Italy and South Africa (SA). Vaccination with clade-B Tat induced cross-clade neutralizing antibodies, immune restoration, including CD4+ T cell increase particularly in low immunological responders, and reduction of proviral DNA. Phase III efficacy trials in SA are planned both in adult and pediatric populations. EXPERT COMMENTARY: We propose the Tat therapeutic vaccine as a pathogenesis-driven intervention that effectively intensifies cART and may lead to a functional cure and provide new perspectives for prevention and virus eradication strategies.

HIV-Tat immunization induces cross-clade neutralizing antibodies and CD4(+) T cell increases in antiretroviral-treated South African volunteers: a randomized phase II clinical trial.

BACKGROUND: Although combined antiretroviral therapy (cART) has saved millions of lives, it is incapable of full immune reconstitution and virus eradication. The transactivator of transcription (Tat) protein is a key human immunodeficiency virus (HIV) virulence factor required for virus replication and transmission. Tat is expressed and released extracellularly by infected cells also under cART and in this form induces immune dysregulation, and promotes virus reactivation, entry and spreading. Of note, anti-Tat antibodies are rare in natural infection and, when present, correlate with asymptomatic state and reduced disease progression. This suggested that induction of anti-Tat antibodies represents a pathogenesis-driven intervention to block progression and to intensify cART. Indeed Tat-based vaccination was safe, immunogenic and capable of immune restoration in an open-label, randomized phase II clinical trial conducted in 168 cART-treated volunteers in Italy. To assess whether B-clade Tat immunization would be effective also in patients with different genetic background and infecting virus, a phase II trial was conducted in South Africa. METHODS: The ISS T-003 was a 48-week randomised, double-blinded, placebo-controlled trial to evaluate immunogenicity (primary endpoint) and safety (secondary endpoint) of B-clade Tat (30 µg) given intradermally, three times at 4-week intervals, in 200 HIV-infected adults on effective cART (randomised 1:1) with CD4(+) T-cell counts ≥200 cells/µL. Study outcomes also included cross-clade anti-Tat antibodies, neutralization, CD4(+) T-cell counts and therapy compliance. RESULTS: Immunization was safe and well-tolerated and induced durable, high titers anti-Tat B-clade antibodies in 97 % vaccinees. Anti-Tat antibodies were cross-clade (all vaccinees tested) and neutralized Tat-mediated entry of oligomeric B-clade and C-clade envelope in dendritic cells (24 participants tested). Anti-Tat antibody titers correlated positively with neutralization. Tat vaccination increased CD4(+) T-cell numbers (all participants tested), particularly when baseline levels were still low after years of therapy, and this had a positive correlation with HIV neutralization. Finally, in cART non-compliant patients (24 participants), vaccination contained viral load rebound and maintained CD4(+) T-cell numbers over study entry levels as compared to placebo. CONCLUSIONS: The data indicate that Tat vaccination can restore the immune system and induces cross-clade neutralizing anti-Tat antibodies in patients with different genetic backgrounds and infecting viruses, supporting the conduct of phase III studies in South Africa. Trial registration ClinicalTrials.gov NCT01513135, 01/23/2012.

Development of a novel AIDS vaccine: the HIV-1 transactivator of transcription protein vaccine.

INTRODUCTION: Classical approaches aimed at targeting the HIV-1 envelope as well as other structural viral proteins have largely failed. The HIV-1 transactivator of transcription (Tat) is a key HIV virulence factor, which plays pivotal roles in virus gene expression, replication, transmission and disease progression. Notably, anti-Tat Abs are uncommon in natural infection and, when present, correlate with the asymptomatic state and lead to lower or no disease progression. Hence, targeting Tat represents a pathogenesis-driven intervention. AREAS COVERED: Here, we review the rationale and the translational development of a therapeutic vaccine targeting the Tat protein. Preclinical and Phase I studies, Phase II trials with Tat in anti-Tat Ab-negative, virologically suppressed highly active antiretroviral therapy-treated subjects in Italy and South Africa were conducted. The results indicate that Tat-induced immune responses are necessary to restore immune homeostasis, to block the replenishment and to reduce the size of the viral reservoir. Additionally, they may help in establishing key parameters for highly active antiretroviral therapy intensification and a functional cure. EXPERT OPINION: We propose the therapeutic setting as the most feasible to speed up the testing and comparison of preventative vaccine candidates, as the distinction lies in the use of the vaccine in uninfected versus infected subjects and not in the vaccine formulation.

HIV-1 Tat immunization restores immune homeostasis and attacks the HAART-resistant blood HIV DNA: results of a randomized phase II exploratory clinical trial.

BACKGROUND: The phase II multicenter, randomized, open label, therapeutic trial (ISS T-002, Clinicaltrials.gov NCT00751595) was aimed at evaluating the immunogenicity and the safety of the biologically active HIV-1 Tat protein administered at 7.5 or 30 µg, given 3 or 5 times monthly, and at exploring immunological and virological disease biomarkers. The study duration was 48 weeks, however, vaccinees were followed until the last enrolled subject reached the 48 weeks. Reported are final data up to 144 weeks of follow-up. The ISS T-002 trial was conducted in 11 clinical centers in Italy on 168 HIV positive subjects under Highly Active Antiretroviral Therapy (HAART), anti-Tat Antibody (Ab) negative at baseline, with plasma viremia <50 copies/mL in the last 6 months prior to enrollment, and CD4(+) T-cell number ≥200 cells/µL. Subjects from a parallel observational study (ISS OBS T-002, Clinicaltrials.gov NCT0102455) enrolled at the same clinical sites with the same criteria constituted an external reference group to explore biomarkers of disease. RESULTS: The vaccine was safe and well tolerated and induced anti-Tat Abs in most patients (79%), with the highest frequency and durability in the Tat 30 µg groups (89%) particularly when given 3 times (92%). Vaccination promoted a durable and significant restoration of T, B, natural killer (NK) cells, and CD4(+) and CD8(+) central memory subsets. Moreover, a significant reduction of blood proviral DNA was seen after week 72, particularly under PI-based regimens and with Tat 30 µg given 3 times (30 µg, 3x), reaching a predicted 70% decay after 3 years from vaccination with a half-life of 88 weeks. This decay was significantly associated with anti-Tat IgM and IgG Abs and neutralization of Tat-mediated entry of oligomeric Env in dendritic cells, which predicted HIV-1 DNA decay. Finally, the 30 µg, 3x group was the only one showing significant increases of NK cells and CD38(+)HLA-DR(+)/CD8(+) T cells, a phenotype associated with increased killing activity in elite controllers. CONCLUSIONS: Anti-Tat immune responses are needed to restore immune homeostasis and effective anti-viral responses capable of attacking the virus reservoir. Thus, Tat immunization represents a promising pathogenesis-driven intervention to intensify HAART efficacy.

The presence of anti-Tat antibodies in HIV-infected individuals is associated with containment of CD4+ T-cell decay and viral load, and with delay of disease progression: results of a 3-year cohort study.

BACKGROUND: Tat is a key HIV-1 virulence factor, which plays pivotal roles in virus gene expression, replication, transmission and disease progression. After release, extracellular Tat accumulates in tissues and exerts effects on both the virus and the immune system, promoting immune activation and virus spreading while disabling the host immune defense. In particular, Tat binds Env spikes on virus particles forming a virus entry complex, which favors infection of dendritic cells and efficient transmission to T cells via RGD-binding integrins. Tat also shields the CCR5-binding sites of Env rendering ineffective virus neutralization by anti-Env antibodies (Abs). This is reversed by the anti-Tat Abs present in natural infection or induced by vaccination. FINDINGS: Here we present the results of a cohort study, showing that the presence of anti-Tat Abs in asymptomatic and treatment-naïve HIV-infected subjects is associated with containment of CD4+ T-cell loss and viral load and with a delay of disease progression. In fact, no subjects with high anti-Tat Ab titers initiated antiretroviral therapy during the three years of follow-up. In contrast, no significant effects were seen for anti-Env and anti-Gag Abs. The increase of anti-Env Ab titers was associated with a reduced risk of starting therapy only in the presence of anti-Tat Abs, suggesting an effect of combined anti-Tat and anti-Env Abs on the Tat/Env virus entry complex and on virus neutralization. CONCLUSIONS: Anti-Tat immunity may help delay HIV disease progression, thus, targeting Tat may offer a novel therapeutic intervention to postpone antiretroviral treatment or to increase its efficacy.

Therapeutic immunization with HIV-1 Tat reduces immune activation and loss of regulatory T-cells and improves immune function in subjects on HAART.

UNLABELLED: Although HAART suppresses HIV replication, it is often unable to restore immune homeostasis. Consequently, non-AIDS-defining diseases are increasingly seen in treated individuals. This is attributed to persistent virus expression in reservoirs and to cell activation. Of note, in CD4(+) T cells and monocyte-macrophages of virologically-suppressed individuals, there is continued expression of multi-spliced transcripts encoding HIV regulatory proteins. Among them, Tat is essential for virus gene expression and replication, either in primary infection or for virus reactivation during HAART, when Tat is expressed, released extracellularly and exerts, on both the virus and the immune system, effects that contribute to disease maintenance. Here we report results of an ad hoc exploratory interim analysis (up to 48 weeks) on 87 virologically-suppressed HAART-treated individuals enrolled in a phase II randomized open-label multicentric clinical trial of therapeutic immunization with Tat (ISS T-002). Eighty-eight virologically-suppressed HAART-treated individuals, enrolled in a parallel prospective observational study at the same sites (ISS OBS T-002), served for intergroup comparison. Immunization with Tat was safe, induced durable immune responses, and modified the pattern of CD4(+) and CD8(+) cellular activation (CD38 and HLA-DR) together with reduction of biochemical activation markers and persistent increases of regulatory T cells. This was accompanied by a progressive increment of CD4(+) T cells and B cells with reduction of CD8(+) T cells and NK cells, which were independent from the type of antiretroviral regimen. Increase in central and effector memory and reduction in terminally-differentiated effector memory CD4(+) and CD8(+) T cells were accompanied by increases of CD4(+) and CD8(+) T cell responses against Env and recall antigens. Of note, more immune-compromised individuals experienced greater therapeutic effects. In contrast, these changes were opposite, absent or partial in the OBS population. These findings support the use of Tat immunization to intensify HAART efficacy and to restore immune homeostasis. TRIAL REGISTRATION: ClinicalTrials.gov NCT00751595.

The preventive phase I trial with the HIV-1 Tat-based vaccine.

The native HIV-1 Tat protein was chosen as vaccine candidate for phase I clinical trials based on its role in the natural infection and AIDS pathogenesis, on the association of Tat-specific immune response with the asymptomatic stage as well as on its sequence conservation among HIV clades. A randomized, double blind, placebo-controlled phase I study (ISS P-001) was conducted in healthy adult volunteers without identifiable risk of HIV infection. Tat was administered 5 times monthly, subcute in alum or intradermic alone at 7.5 microg, 15 microg or 30 microg, respectively (ClinicalTrials.gov identifier: NCT00529698). Vaccination with Tat resulted to be safe and well tolerated (primary endpoint) both locally and systemically. In addition, Tat induced both Th1 and Th2 type specific immune responses in all subjects (secondary endpoint) with a wide spectrum of functional antibodies that are rarely seen in natural infection, providing key information for further clinical development of the Tat vaccine candidate.

HIV-1 Tat-based vaccines: an overview and perspectives in the field of HIV/AIDS vaccine development.

The HIV epidemic continues to represent one of the major problems worldwide, particularly in the Asia and Sub-Saharan regions of the world, with social and economical devastating effects. Although antiretroviral drugs have had a dramatically beneficial impact on HIV-infected individuals that have access to treatment, it has had a negligible impact on the global epidemic. Hence, the inexorable spreading of the HIV pandemic and the increasing deaths from AIDS, especially in developing countries, underscore the urgency for an effective vaccine against HIV/AIDS. However, the generation of such a vaccine has turned out to be extremely challenging. Here we provide an overview on the rationale for the use of non-structural HIV proteins, such as the Tat protein, alone or in combination with other HIV early and late structural HIV antigens, as novel, promising preventative and therapeutic HIV/AIDS vaccine strategies.

Phase I therapeutic trial of the HIV-1 Tat protein and long term follow-up.

A randomized, double blind, placebo-controlled phase I vaccine trial based on the native Tat protein was conducted in HIV-infected asymptomatic individuals. The vaccine was administered five times subcute with alum or intradermally without adjuvant at 7.5microg, 15microg or 30microg doses, respectively. The Tat vaccine was well tolerated both locally and systemically and induced and/or maintained Tat-specific T helper (Th)-1 T-cell responses and Th-2 responses in all subjects with a wide spectrum of functional anti-Tat antibodies, rarely seen in HIV-infected subjects. The data indicate the achievement of both the primary (safety) and secondary (immunogenicity) endpoints of the study.

PD-L1 negatively regulates CD4+CD25+Foxp3+ Tregs by limiting STAT-5 phosphorylation in patients chronically infected with HCV.

CD4+CD25+Foxp3+ Tregs suppress autoimmune responses. In addition, they limit T cell responses during chronic infection, thereby minimizing T cell-dependent immunopathology. We sought to investigate how Tregs are regulated in the livers of patients chronically infected with HCV, where they control the balance between an adequate protective immune response and suppression of immunopathology. We found that, despite accumulating and proliferating at sites of infection in the livers of patients chronically infected with HCV, Tregs were relatively less expanded than CD4+CD25+Foxp3- effector T cells. The relative lower expansion of intrahepatic Tregs coincided with their upregulation of programmed death-1 (PD-1). PD-1 expression inversely correlated with both Treg proliferation and clinical markers of immune suppression in vivo. Consistent with the possibility that PD-1 controls Tregs, blockade of the interaction between PD-1 and programmed death-1 ligand 1 (PD-L1) enhanced the in vitro expansion and function of Tregs isolated from the livers of patients chronically infected with HCV. Blockade of the interaction between PD-L1 and B7.1 also improved the proliferation of these cells. Interestingly, both PD-1 and phosphorylated STAT-5 were overexpressed in intrahepatic Tregs in a parallel fashion in steady disease conditions, and in an alternate-fluctuating fashion during the course of severe hepatitis reactivation. Notably, PD-L1 blockade upregulated STAT-5 phosphorylation in Tregs ex vivo. These data suggest that PD-L1 negatively regulates Tregs at sites of chronic inflammation by controlling STAT-5 phosphorylation.

HIV-1 Tat addresses dendritic cells to induce a predominant Th1-type adaptive immune response that appears prevalent in the asymptomatic stage of infection.

Tat is an early regulatory protein that plays a major role in human HIV-1 replication and AIDS pathogenesis, and therefore, it represents a key target for the host immune response. In natural infection, however, Abs against Tat are produced only by a small fraction (approximately 20%) of asymptomatic individuals and are rarely seen in progressors, suggesting that Tat may possess properties diverting the adaptive immunity from generating humoral responses. Here we show that a Th1-type T cell response against Tat is predominant over a Th2-type B cell response in natural HIV-1 infection. This is likely due to the capability of Tat to selectively target and very efficiently enter CD1a-expressing monocyte-derived dendritic cells (MDDC), which represent a primary target for the recognition and response to virus Ag. Upon cellular uptake, Tat induces MDDC maturation and Th1-associated cytokines and beta-chemokines production and polarizes the immune response in vitro to the Th1 pattern through the transcriptional activation of TNF-alpha gene expression. This requires the full conservation of Tat transactivation activity since neither MDDC maturation nor TNF-alpha production are found with either an oxidized Tat, which does not enter MDDC, or with a Tat protein mutated in the cysteine-rich region (cys22 Tat), which enters MDDC as the wild-type Tat but is transactivation silent. Consistently with these data, inoculation of monkeys with the native wild-type Tat induced a predominant Th1 response, whereas cys22 Tat generated mostly Th2 responses, therefore providing evidence that Tat induces a predominant Th1 polarized adaptive immune response in the host.

Frequency of naturally-occurring regulatory T cells is reduced in patients with ST-segment elevation myocardial infarction.

BACKGROUND: Naturally-occurring regulatory T cells (Treg) constitute a mature T-cell population characterized phenotypically by co-expression of CD4 and CD25(high) surface molecules. We investigated here the frequency of circulating Treg in patients presenting with STEMI in comparison with subjects without coronary artery disease (CAD). The effect of primary percutaneous coronary intervention (PCI) with implantation of a bare (BS) or paclitaxel-eluting stent (PES) on peripheral Treg distribution was also examined. METHODS: Peripheral blood mononuclear cells were isolated from 30 consecutive patients presenting with STEMI and from 30 age-matched control subjects with angiographically normal coronary arteries. Treg were detected by flow cytometry according to their characteristic CD4+ CD25(high) membrane phenotype, and their frequency was assessed before PCI and at 48 h and at 6 days after PCI. CD27 expression identifying a highly suppressive Treg subset was also analysed. RESULTS: The percentages of both (CD27+)Treg and (CD27-)Treg were significantly lower in patients with STEMI in comparison with controls. In addition, the (CD27+)Treg/(CD27-)Treg ratio was skewed toward the CD27- population. The frequency of both Treg subsets significantly increased 48 h after either BS or PES implantation, remaining elevated for up to at least 6 days after PCI. CONCLUSIONS: Our data suggest that the percentage of circulating Treg is significantly reduced in patients with STEMI, suggesting that this immunosuppressive T-cell subset is compartmentalized within the acutely ischemic myocardium to limit the ongoing inflammation associated with this condition, and that coronary revascularization is associated with partial reconstitution of peripheral Treg pool.

Chloroquine enhances human CD8+ T cell responses against soluble antigens in vivo.

The presentation of exogenous protein antigens in a major histocompatibility complex class I-restricted fashion to CD8+ T cells is called cross-presentation. We demonstrate that cross-presentation of soluble viral antigens (derived from hepatitis C virus [HCV], hepatitis B virus [HBV], or human immunodeficiency virus) to specific CD8+ T cell clones is dramatically improved when antigen-presenting dendritic cells (DCs) are pulsed with the antigen in the presence of chloroquine or ammonium chloride, which reduce acidification of the endocytic system. The export of soluble antigen into the cytosol is considerably higher in chloroquine-treated than in untreated DCs, as detected by confocal microscopy of cultured cells and Western blot analysis comparing endocytic and cytosolic fractions. To pursue our findings in an in vivo setting, we boosted groups of HBV vaccine responder individuals with a further dose of hepatitis B envelope protein vaccine with or without a single dose of chloroquine. Although all individuals showed a boost in antibody titers to HBV, six of nine individuals who were administered chloroquine showed a substantial CD8+ T cell response to HBV antigen, whereas zero of eight without chloroquine lacked a CD8 response. Our results suggest that chloroquine treatment improves CD8 immunity during vaccination.

Altered trafficking of CD8+ memory T cells after implantation of rapamycin-eluting stents in patients with coronary artery disease.

Aim of this study was to investigate the effects of implantation of different coronary drug-eluting stents on trafficking of central (T(CM)) or effector (T(EM)) memory T cells in the coronary sinus of patients with coronary artery disease (CAD) undergoing percutaneous coronary revascularization. Thirty-two patients presenting with stable coronary disease and angiographically proven stenosis of left descending coronary artery were randomly assigned to treatment with rapamycin-eluting, paclitaxel-eluting or bare metal stents. Heparinized blood samples were obtained from the coronary sinus either before or 20 min after stent implantation. Mononuclear cells were stained with mAbs specific for CD3, CD4, CD8, CD45R0, and CD27 molecules. Analysis of surface phenotype was performed by four-color flow cytometry and data on both CD4+ and CD8+ T(CM) and T(EM) cells were expressed either as absolute cell numbers/microL of blood or as percentages relative to the corresponding total memory T cell populations in the individual patients. We found that the number of CD8+ T(EM), as defined by CD3+CD45R0+CD8+CD27- phenotype, was significantly reduced in patients receiving a rapamycin-eluting stent as compared with basal values. Conversely, the number of CD8+ T(CM) (CD3+CD45R0+CD8+CD27+) was increased in the same treatment group after the revascularization procedure. No changes in the absolute number of CD4+ and CD8+ total (T(CM) plus T(EM)) memory T cells before and after the procedure were observed. These findings suggest that rapamycin eluted from medicated coronary stents rapidly induce a redistribution of memory CD8+ T lymphocyte subsets, with a significant decrease of T(EM) and a corresponding increase of T(CM) increase circulating within the coronary sinus. This anti-inflammatory effect could partially explain the reduction of coronary in-stent restenosis rate associated with the clinical use of this type of device.

Responses of peptide-specific T cells to stimulation with polystyrene beads carrying HLA class I molecules loaded with single peptides.

Cell-sized microbeads carrying single peptide-loaded HLA class I molecules were prepared for HLA-A2 and HLA-B7 by a simple procedure which transfers single peptide-loaded HLA class I molecules from cultured cells to polystyrene beads using anti-peptide antibodies directed to an intracellular segment of HLA-A alpha chains. The surface density of peptide-loaded HLA class I molecules on beads was comparable to that on the peptide-loaded cells. HLA-A2 beads loaded with an HCV peptide HCV1073 were tested for stimulation activity on an HCV1073-specific CD8+ T cell clone NS3-1. A substantial level of gamma-IFN production was induced. The stimulation was peptide-specific. The efficiency was dependent on the bead concentration and the surface HLA class I density on beads and enhanced significantly by co-coupling of anti-CD28 to peptide-loaded beads. The peptide-loading efficiency on HLA class I molecules and the transfer efficiency of HLA class I molecules to polystyrene beads were reasonably high for HLA-A2 and HLA-B7. Thus, polystyrene beads carrying these single peptide-loaded HLA class I molecules are potentially useful in further analysis of the co-stimulatory or inhibitory factors involved in CD8+ T cell responses and eventually in detection of cytotoxic T cells in PBLs.

Hepatic expansion of a virus-specific regulatory CD8(+) T cell population in chronic hepatitis C virus infection.

Regulatory T (T(R)) cells consist of phenotypically and functionally distinct CD4(+) and CD8(+) T cell subsets engaged both in maintaining self-tolerance and in preventing anti-non-self effector responses (microbial, tumor, transplant, and so on) that may be harmful to the host. Here we propose that the proinflammatory function of virus-specific memory effector CCR7(-)CD8(+) T cells, which are massively recruited in the liver, are inefficient (in terms of IFN-gamma production) in patients with chronic hepatitis C virus (HCV) infection because of the concomitant presence of virus-specific CCR7(-)CD8(+) T(R) cells producing considerable amounts of IL-10. These CD8(+) T(R) cells are antigen specific, as they can be stimulated by HCV epitopes and suppress T cell responses that are in turn restored by the addition of neutralizing anti-IL-10. This study provides for the first time to our knowledge direct evidence of the existence of virus-specific CD8(+) T(R) cells that infiltrate the livers of patients with chronic HCV infection, identifies IL-10 as a soluble inhibitory factor mediating suppression, and suggests that these cells play a pivotal role in controlling hepatic effector CD8(+) T cell responses.

Subversion of effector CD8+ T cell differentiation in acute hepatitis C virus infection: exploring the immunological mechanisms.

Hallmark of acute hepatitis C virus (HCV) infection is a severe virus-specific effector CD8(+) T cell dysfunction that seems to be a critical factor in preventing the resolution of infection and in favoring the onset of chronic liver immunopathology. We suggest that this dysfunction is critical in the establishment of HCV persistence, unless it is compensated by multispecific responses, as found in individuals resolving infection. Analyses on purified populations indicate that central memory HCV-specific CCR7(+)/CD8(+) T cells efficiently proliferate and differentiate in vitro, although the large population of memory effector CCR7(-) cells found in the peripheral blood of acutely infected patients display poor effector functions ex vivo (semi-effectors). However, we report strong evidence in support of IL-2 being capable of pushing semi-effector CTL to complete their effector cell program. Therefore, IL-2 deficiency during T cell activation may be responsible for the dichotomy between memory CTL expansion and incomplete effector differentiation shown in patients with acute HCV infection. These data are consistent with the possible therapeutic treatment with IL-2 to rebuild the effector T cell pool in these patients.

Subversion of effector CD8+ T cell differentiation in acute hepatitis C virus infection: the role of the virus.

In a companion study, we showed a dichotomy between the expansion of central memory (CCR7(+)) hepatitis C virus (HCV)-specific CTL and the incomplete memory effector differentiation in patients with acute HCV infection. Indeed, effector cells were unable to perform immediate functions, despite expressing the tissue-homing phenotype of effector memory cells (CCR7(-); semi-effectors). However, since they promptly differentiated into full-effectors upon IL-2 contact, we suggested that the inhibitory effect by environmental (possibly viral) factors on IL-2 production may have a pivotal role in generating the large population of semi-effector CCR7(-)/IFN-gamma(-) CTL. In accord with this view, we report here strong evidence in support of circulating HCVcore protein (HCVcore) playing a central role in inhibiting effector CTL differentiation, but not memory CTL expansion. The regulatory HCVcore effect is related to inhibition of the signal transduction pathway instrumental for IL-2 production, supporting the evidence that IL-2 was capable both of pushing semi-effector CTL to complete their effector cell program and of restoring the HCVcore-dependent inhibitory effect. Therefore, the strength of CTL activation is dependent on the balance between the threshold of stimulatory signals and the viral interference capacities provided during priming.