Cellular immunity to beta 2-glycoprotein-1 in patients with the antiphospholipid syndrome.

Patients with antiphospholipid syndrome (APS) suffer recurrent thromboses, thrombocytopenia, and/or fetal loss in association with Abs that can be detected in phospholipid-dependent assays. Despite the name, the Igs associated with APS are predominantly directed against epitopes on phospholipid-binding plasma proteins, such as beta 2-glycoprotein-1 (beta 2GP1) and prothrombin. The aim of this study was to examine the cellular immune response to beta 2GP1 in patients with APS. Using a serum-free stimulation assay, PBMCs from 8 of 18 patients with APS proliferated to purified beta 2GP1 or to the beta 2GP1 present in serum, whereas no stimulation was observed by PBMCs from healthy individuals, patients with other autoimmune diseases, or anticardiolipin Ab-positive patients without histories of thromboses or fetal loss. The immune response was Ag-specific, requiring class II molecules, CD4+ T cells, and APCs, and was associated with a selective expansion of CD4+ but not CD8+ T cells. The proliferating T cells produced IFN-gamma but not IL-4, indicating a bias toward a type 1 immune response. Chronic low grade stimulation of autoreactive beta 2GP1-specific, IFN-gamma-producing Th1 CD4+ T cells may contribute to the high risk of thromboses and pregnancy failure in patients with APS.     

Masters of manipulation: Viral modulation of the immunological synapse

In order to thrive, viruses have evolved to manipulate host cell machinery for their own benefit. One major obstacle faced by pathogens is the immunological synapse. To enable efficient replication and latency in immune cells, viruses have developed a range of strategies to manipulate cellular processes involved in immunological synapse formation to evade immune detection and control T‐cell activation. In vitro, viruses such as human immunodeficiency virus 1 and human T‐lymphotropic virus type 1 utilise structures known as virological synapses to aid transmission of viral particles from cell to cell in a process termed trans‐infection. The formation of the virological synapse provides a gateway for virus to be transferred between cells avoiding the extracellular space, preventing antibody neutralisation or recognition by complement. This review looks at how viruses are able to subvert intracellular signalling to modulate immune function to their advantage and explores the role synapse formation has in viral persistence and cell‐to‐cell transmission.     

CNS recruitment of CD8+ T lymphocytes specific for a peripheral virus infection triggers neuropathogenesis during polymicrobial challenge

Although viruses have been implicated in central nervous system (CNS) diseases of unknown etiology, including multiple sclerosis and amyotrophic lateral sclerosis, the reproducible identification of viral triggers in such diseases has been largely unsuccessful. Here, we explore the hypothesis that viruses need not replicate in the tissue in which they cause disease; specifically, that a peripheral infection might trigger CNS pathology. To test this idea, we utilized a transgenic mouse model in which we found that immune cells responding to a peripheral infection are recruited to the CNS, where they trigger neurological damage. In this model, mice are infected with both CNS-restricted measles virus (MV) and peripherally restricted lymphocytic choriomeningitis virus (LCMV). While infection with either virus alone resulted in no illness, infection with both viruses caused disease in all mice, with ～50% dying following seizures. Co-infection resulted in a 12-fold increase in the number of CD8+ T cells in the brain as compared to MV infection alone. Tetramer analysis revealed that a substantial proportion (>35%) of these infiltrating CD8+ lymphocytes were LCMV-specific, despite no detectable LCMV in CNS tissues. Mechanistically, CNS disease was due to edema, induced in a CD8-dependent but perforin-independent manner, and brain herniation, similar to that observed in mice challenged intracerebrally with LCMV. These results indicate that T cell trafficking can be influenced by other ongoing immune challenges, and that CD8+ T cell recruitment to the brain can trigger CNS disease in the apparent absence of cognate antigen. By extrapolation, human CNS diseases of unknown etiology need not be associated with infection with any particular agent; rather, a condition that compromises and activates the blood-brain barrier and adjacent brain parenchyma can render the CNS susceptible to pathogen-independent immune attack.     

Expansion of effector memory TCR Vbeta4+ CD8+ T cells is associated with latent infection-mediated resistance to transplantation tolerance

Therapies that control largely T cell-dependent allograft rejection in humans also possess the undesirable effect of impairing T cell function, leaving transplant recipients susceptible to opportunistic viruses. Prime among these opportunists are the ubiquitous herpesviruses. To date, studies are lacking that address the effect of viruses that establish a true latent state on allograft tolerance or the effect of tolerance protocols on the immune control of latent viruses. By using a mixed chimerism-based tolerance-induction protocol, we found that mice undergoing latent infection with gammaHV68, a murine gamma-herpesvirus closely related to human gamma-herpesviruses such as EBV and Kaposi's sarcoma-associated herpesvirus, significantly resist tolerance to allografts. Limiting the degree of virus reactivation or innate immune response did not reconstitute chimerism in latently infected mice. However, gammaHV68-infected mice showed increased frequency of CD8+ T cell alloreactivity and, interestingly, expansion of virus-induced, alloreactive, "effector/effector memory" TCR Vbeta4+CD8+ T cells driven by the gammaHV68-M1 gene was associated with resistance to tolerance induction in studies using gammaHV68-M1 mutant virus. These results define the viral gene and immune cell types involved in latent infection-mediated resistance to allograft tolerance and underscore the influence of latent herpesviruses on allograft survival.     

Fighting a virus with a virus: a dynamic model for HIV-1 therapy

A mathematical model examined a potential therapy for controlling viral infections using genetically modified viruses. The control of the infection is an indirect effect of the selective elimination by an engineered virus of infected cells that are the source of the pathogens. Therefore, this engineered virus could greatly compensate for a dysfunctional immune system compromised by AIDS. In vitro studies using engineered viruses have been shown to decrease the HIV-1 load about 1000-fold. However, the efficacy of this potential treatment for reducing the viral load in AIDS patients is unknown. The present model studied the interactions among the HIV-1 virus, its main host cell (activated CD4+ T cells), and a therapeutic engineered virus in an in vivo context; and it examined the conditions for controlling the pathogen. This model predicted a significant drop in the HIV-1 load, but the treatment does not eradicate HIV. A basic estimation using a currently engineered virus indicated an HIV-1 load reduction of 92% and a recovery of host cells to 17% of their normal level. Greater success (98% HIV reduction, 44% host cells recovery) is expected as more competent engineered viruses are designed. These results suggest that therapy using viruses could be an alternative to extend the survival of AIDS patients.     

Recognition of rabies and rabies-related viruses by T cells derived from human vaccine recipients.

Human peripheral blood mononuclear cells and T-cell lines and clones from individuals immunized with rabies PM vaccine were tested for the ability to recognize antigenic determinants in rabies and rabies-related viruses in an antigen-induced proliferation assay. Some, but not all, of the T cells from these individuals cross-reacted with various laboratory strains of rabies virus with rabies-related viruses such as Duvenhage and Mokola. In addition, these T cells were shown to react with epitopes of either the ribonucleoprotein or the viral glycoprotein. Rabies-specific cytotoxic T-cell responses by a CD4+ T-cell line were evident against antigenic determinants of the ribonucleoprotein and glycoprotein.     

Impaired effector function of hepatitis C virus-specific CD8+ T cells in chronic hepatitis C virus infection

The cellular immune response contributes to clearance of hepatitis C virus (HCV) and persists for decades after recovery from infection. The immunological basis for the inefficiency of the cellular immune response in chronically infected persons is not known. Here, we used four HLA-A2 tetramers, specific for two HCV core and two HCV NS3 epitopes, to investigate at the single-cell level effector function and phenotype of HCV-specific CD8+ T cells in 20 chronically infected and 12 long-term recovered patients. Overall, HCV-specific, tetramer+ T cells were more frequently found in PBMCs of chronically infected patients than in those of recovered patients. However, when compared with HCV-tetramer+ T cells of recovered patients, they displayed an impaired proliferative capacity. As a result of the impaired proliferative capacity, HCV-specific T cell lines derived from chronically infected patients displayed less peptide-specific cytotoxicity than those from recovered patients. In addition, proliferation and ex vivo IFN-gamma production of HCV-tetramer+ cells, but not influenza-virus-specific T cells, were defective in chronically infected patients and could not be restored by in vitro stimulation with peptide and IL-2. At least three distinct phenotypes of HCV-specific CD8+ T cells were identified and associated with certain functional characteristics. In addition, impairment of proliferative, cytokine, and cytotoxic effector functions of tetramer+ T cells in viremic patients was associated with weak ex vivo HCV-specific CD4+ T cell responses. Thus, the defective functions of HCV-specific CD8+ T cells might contribute to viral persistence in chronically infected patients, and knowledge on their reversibility may facilitate the development of immunotherapeutic vaccines.     

Does apoptosis contribute to CD4 T cell depletion in human immunodeficiency virus infection ?

Despite an extensive knowledge of the molecular characteristics of the human immunodeficiency virus (HIV) identified more than ten years ago as the cause of AIDS (acquired immune deficiency syndrome) (Barre-Sinoussi et al. 1983) some critical questions have not been answered yet: Is the progressive disappearance of CD4+ helper T lymphocytes, the hallmark of AIDS, directly related to the killing of infected cells by the virus? If not, how do CD4+T cells die? Is HIV using its viral factory to kill uninfected bystander cells? What causes the immune system collapse in HIV infection? In the past three years some important studies have provided stimulating clues suggesting that AIDS is not only related to the killing of host cells by HIV but is also a consequence of mechanisms of misactivation of the immune system, leading to anergy or apoptosis of non-infected effector cells. We discuss some of the in vivo and in vitro models providing evidence that HIV is able to kill and cripple the immune system either by acting directly on its targets or indirectly in bystander T cells keeping in mind that HIV disease must be considered as a multifactorial process.     

A soluble form of lymphocyte activation gene-3 (IMP321) induces activation of a large range of human effector cytotoxic cells

The principal antitumor immune response is mediated through the activation of type 1 cytotoxic (Tc1) CD8 T cells, NK cells, and monocytes/macrophages. In this study, we investigated the potency of a clinical-grade soluble form of lymphocyte activation gene-3 protein (IMP321), a physiological high-affinity MHC class II binder, at inducing in PBMCs an appropriate cytotoxic-type response in short-term ex vivo assays. We found that IMP321 binds to a minority (<10%) of MHC class II + cells in PBMCs, including all myeloid dendritic cells, and a small fraction of monocytes. Four hours after addition of IMP321 to PBMCs, these myeloid cells produce TNF-alpha and CCL4 as determined by intracellular staining. At 18 h, 1% of CD8+ T cells and 3.7% NK cells produce Tc1 cytokines such as IFN-gamma and/or TNF-alpha (mean values from 60 blood donors). Similar induction was observed in metastatic cancer patient PBMCs, but the values were lower for the NK cell subset. Early APC activation by IMP321 is needed for this Tc1-type activation because pure sorted CD8+ T cells could not be activated by IMP321. Only Ag-experienced, fully differentiated granzyme+ CD8 T cells (effector and effector memory but not naive or central memory T cells) are induced by IMP321 to full Tc1 activation. In contrast to IMP321, TLR1-9 agonists induce IL-10 and are therefore unable to induce this Tc1 IFN-gamma+ response. Thus, IMP321 has many properties that confirm its potential to be a new class of immunopotentiator in cancer patients.     

CD4+ T cell priming accelerates the clearance of Sendai virus in mice, but has a negative effect on CD8+ T cell memory

Current vaccines designed to promote humoral immunity to respiratory virus infections also induce potent CD4+ T cell memory. However, little is known about the impact of primed CD4+ T cells on the immune response to heterologous viruses that are serologically distinct, but that share CD4+ T cell epitopes. In addition, the protective capacity of primed CD4+ T cells has not been fully evaluated. In the present study, we addressed these two issues using a murine Sendai virus model. Mice were primed with an HN421-436 peptide that represents the dominant CD4+ T cell epitope on the hemagglutinin-neuraminidase (HN) of Sendai virus. This vaccination strategy induced strong CD4+ T cell memory to the peptide, but did not induce Abs specific for the Sendai virus virion. Subsequent Sendai virus infection of primed mice resulted in 1) a substantially accelerated virus-specific CD4+ T cell response in the pneumonic lung; 2) enhanced primary antiviral Ab-forming cell response in the mediastinal lymph nodes; and 3) accelerated viral clearance. Interestingly, the virus-specific CD8+ T cell response in the lung and the development of long-term memory CD8+ T cells in the spleen were significantly reduced. Taken together, our data demonstrate that primed CD4+ T cells, in the absence of pre-existing Ab, can have a significant effect on the subsequent immune responses to a respiratory virus infection.     

Activation of extracellular signaling regulated kinase in natural killer cells and monocytes following IL-2 stimulation in vitro and in patients undergoing IL-2 immunotherapy: analysis via dual parameter flow-cytometric assay

Interleukin-2 (IL-2) activates extracellular signal-regulated protein kinase (ERK) within immune cells. To examine the profile of phosphorylated ERK (p-ERK) in IL-2 stimulated immune cells of normal donors and patients receiving IL-2 therapy, we developed a dual parameter flow-cytometric assay. An analysis of PBMCs stimulated with IL-2 indicated that IL-2 exposure induced p-ERK in CD56bright NK cells and CD14+ monocytes, but not in CD3+ T cells or CD21+ B cells. CD3+ T cells that were induced to express functional high-affinity IL-2R did not exhibit enhanced p-ERK following IL-2 treatment. Measurement of p-ERK within PBMCs from cancer patients 1 h following their first dose of IL-2 revealed a complete absence of circulating NK cells, consistent with earlier observations. However, the total number of circulating CD14+ monocytes increased in these samples and 97% of these cells exhibited ERK activation. p-ERK was not observed in T cells post-IL-2 therapy. Analysis of PBMCs obtained 3 weeks post-IL-2 therapy revealed high-p-ERK levels in CD56bright NK cells in a subset of patients, while levels of p-ERK returned to baseline in monocytes. These studies reveal an effective method to detect ERK activation in immune cells and demonstrate that IL-2 activates ERK in a subset of NK cells and monocytes but not T cells.     

International Network for Comparison of HIV Neutralization Assays: The NeutNet Report

Background

Neutralizing antibody assessments play a central role in human immunodeficiency virus type-1 (HIV-1) vaccine development but it is unclear which assay, or combination of assays, will provide reliable measures of correlates of protection. To address this, an international collaboration (NeutNet) involving 18 independent participants was organized to compare different assays.

Methods

Each laboratory evaluated four neutralizing reagents (TriMab, 447-52D, 4E10, sCD4) at a given range of concentrations against a panel of 11 viruses representing a wide range of genetic subtypes and phenotypes. A total of 16 different assays were compared. The assays utilized either uncloned virus produced in peripheral blood mononuclear cells (PBMCs) (virus infectivity assays, VI assays), or their Env-pseudotyped (gp160) derivatives produced in 293T cells (PSV assays) from molecular clones or uncloned virus. Target cells included PBMC and genetically-engineered cell lines in either a single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs that included extracellular or intracellular p24 antigen detection, RNA quantification and luciferase and beta-galactosidase reporter gene expression.

Findings

PSV assays were generally more sensitive than VI assays, but there were important differences according to the virus and inhibitor used. For example, for TriMab, the mean IC50 was always lower in PSV than in VI assays. However, with 4E10 or sCD4 some viruses were neutralized with a lower IC50 in VI assays than in the PSV assays. Inter-laboratory concordance was slightly better for PSV than for VI assays with some viruses, but for other viruses agreement between laboratories was limited and depended on both the virus and the neutralizing reagent.

Conclusions

The NeutNet project demonstrated clear differences in assay sensitivity that were dependent on both the neutralizing reagent and the virus. No single assay was capable of detecting the entire spectrum of neutralizing activities. Since it is not known which in vitro assay correlates with in vivo protection, a range of neutralization assays is recommended for vaccine evaluation.     

Impaired cell mediated immunity in haemophilia in the absence of infection with human immunodeficiency virus.

The cell mediated immune response was evaluated in vivo in 29 patients with clinically severe haemophilia by means of the dinitrochlorobenzene skin test. All patients had a response below the median normal value, and in 19 the response was on or below the lower limit of the normal range. There was no difference in skin response between patients positive and negative for the human immunodeficiency virus (HIV; formerly known as human T cell lymphotropic virus III or lymphadenopathy associated virus). In the whole group, and in seronegative patients (n = 17), there was an inverse relation between exposure to clotting factor and skin response. In seropositive patients (n = 12) no such association was apparent. This study shows that clotting factor concentrate impairs the cell mediated immune response to a new antigen in the absence of infection with HIV.     

Cutting edge: impairment of dendritic cells and adaptive immunity by Ebola and Lassa viruses

Acute infection of humans with Ebola and Lassa viruses, two principal etiologic agents of hemorrhagic fevers, often results in a paradoxical pattern of immune responses: early infection, characterized by an outpouring of inflammatory mediators such as TNF-alpha, IL-1 beta, and IL-6, vs late stage infections, which are associated with poor immune responses. The mechanisms underlying these diverse outcomes are poorly understood. In particular, the role played by cells of the innate immune system, such as dendritic cells (DC), is not known. In this study, we show that Ebola and Lassa viruses infect human monocyte-derived DC and impair their function. Monocyte-derived DC exposed to either virus fail to secrete proinflammatory cytokines, do not up-regulate costimulatory molecules, and are poor stimulators of T cells. These data represent the first evidence for a mechanism by which Ebola and Lassa viruses target DC to impair adaptive immunity.     

Biology and significance of T-cell apoptosis in the liver

The liver has emerged as an organ with distinct immunological properties. In this review, we summarize evidence that shows that the liver can remove apoptotic, or non-apoptotic but activated, CD8+ T cells from the circulation and induce apoptosis in these activated T cells by either active or passive mechanisms. Hepatitis viruses, particularly hepatitis C virus, often establish persistent infection. We review evidence that suggests that these viruses exploit intrahepatic tolerance mechanisms to protect themselves from immune attack.     

Acyclovir Therapy Reduces the CD4+ T Cell Response against the Immunodominant pp65 Protein from Cytomegalovirus in Immune Competent Individuals

Cytomegalovirus (CMV) infects the majority of the global population and leads to the development of a strong virus-specific immune response. The CMV-specific CD4+ and CD8+ T cell immune response can comprise between 10 and 50% of the T cell pool within peripheral blood and there is concern that this may impair immunity to other pathogens. Elderly individuals with the highest magnitude of CMV-specific immune response have been demonstrated to be at increased risk of mortality and there is increasing interest in interventions that may serve to moderate this. Acyclovir is an anti-viral drug with activity against a range of herpes viruses and is used as long term treatment to suppress reactivation of herpes simplex virus. We studied the immune response to CMV in patients who were taking acyclovir to assess if therapy could be used to suppress the CMV-specific immune response. The T cell reactivity against the immunodominant late viral protein pp65 was reduced by 53% in people who were taking acyclovir. This effect was seen within one year of therapy and was observed primarily within the CD4+ response. Acyclovir treatment only modestly influenced the immune response to the IE-1 target protein. These data show that low dose acyclovir treatment has the potential to modulate components of the T cell response to CMV antigen proteins and indicate that anti-viral drugs should be further investigated as a means to reduce the magnitude of CMV-specific immune response and potentially improve overall immune function.     

Antigen-driven expansion and contraction of CD8+-activated T cells in primary EBV infection.

The origin of the increased numbers of CD8+ atypical lymphocytes, expressing activated markers such as HLA-DR or CD45RO, in the peripheral blood of patients with infectious mononucleosis (IM) has been debated. Using a recently developed assay to detect intracellular accumulation of IFN-gamma in EBV-reactive T cells by FACS, we have demonstrated that 34-54% of HLA-DR+/CD8+ and 34-60% of CD45RO+/CD8+ T cells in the PBMCs of febrile patients suffering from IM are EBV-specific. The EBV-specific CD8+ T cell counts in the PBMCs of four febrile patients suffering from IM ranged between 2,260 and 8,200/microl, decreasing to 5.1% and 7.9% of the counts in the first samples over 10 days in two donors. The decline of CD8+ T cell subpopulations, namely HLA-DR+, CD45RO+, and EBV-specific T cells, was in parallel with the drop in the EBV genome load. These data indicate that the Ag-driven expansion of CD8+ T cells and subsequent contraction with the Ag decline in vivo in humans is effective for clearing virus-infected cells with minimal disturbance of the homeostasis of the immune system.