Type I interferons trigger systemic, partial lymphocyte activation in response to viral infection

The vast majority of both T and B cells in mice were found to up-regulate cell surface expression of the early activation markers CD69 and CD86, but not CD25, within 24 h of infection with Semliki Forest virus. Kinetics and magnitude of activation marker expression was dependent on live virus, dose, and correlated with strain virulence. Activation marker expression declined to baseline levels over the next 96 h. This very early "activation" of such a high percentage of lymphocytes required the presence of type I IFN receptor genes, was inducible with poly(I:C), and correlated with IFN-I levels in serum. We conclude that virus-induced IFN-I release systemically affects most of the hosts T and B cells by triggering them rapidly and independently of Ag-reactivity into a semiactivated state.     

Roles of macrophages in measles virus infection of genetically modified mice

Knowledge of the mechanisms of virus dissemination in acute measles is cursory, but cells of the monocyte/macrophage (MM) lineage appear to be early targets. We characterized the dissemination of the Edmonston B vaccine strain of measles virus (MV-Ed) in peripheral blood mononuclear cells (PBMC) of two mouse strains expressing the human MV-Ed receptor CD46 with human-like tissue specificity and efficiency. In one strain the alpha/beta interferon receptor is defective, allowing for efficient MV-Ed systemic spread. In both mouse strains the PBMC most efficiently infected were F4/80-positive MMs, regardless of the inoculation route used. Circulating B lymphocytes and CD4-positive T lymphocytes were infected at lower levels, but no infected CD8-positive T lymphocytes were detected. To elucidate the roles of MMs in infection, we depleted these cells by clodronate liposome treatment in vivo. MV-Ed infection of splenic MM-depleted mice caused strong activation and infection of splenic dendritic cells (DC), followed by enhanced virus replication in the spleen. Similarly, depletion of lung macrophages resulted in strong activation and infection of lung DC. Thus, in MV infections of genetically modified mice, blood monocytes and tissue macrophages provide functions beneficial for both the virus and the host: they support virus replication early after infection, but they also contribute to protecting other immune cells from infection. Human MM may have similar roles in acute measles.     

Cutting edge: Antigen is not required for the activation and maintenance of virus-specific memory CD8+ T cells in the lung airways

Respiratory virus infections establish a population of memory CD8(+) T cells in the lung airways that persist for months after infection. However, the relationship between Ag-specific memory T cells in the lung airways and the systemic memory T cell pool is not well understood. The majority of lung airway memory T cells express a highly activated phenotype (CD69(+)/CD127(-)), suggesting that recent Ag stimulation is required to drive T cell activation and recruitment to the lung airways. In this study, we demonstrate that the lung airway environment itself in the absence of cognate Ag alters the expression of acute activation markers such as CD69 and CD127 on memory CD8(+) T cells. Furthermore, the steady-state recruitment of virus-specific memory CD8(+) T cells to the lung airways from the circulation can occur without recent Ag stimulation. These findings alter the current perceptions concerning the contribution of Ag to the maintenance of peripheral T cell memory.     

Immature CD4+CD8+ Thymocytes Are Preferentially Infected by Measles Virus in Human Thymic Organ Cultures

Cells of the human immune system are important target cells for measles virus (MeV) infection and infection of these cells may contribute to the immunologic abnormalities and immune suppression that characterize measles. The thymus is the site for production of naïve T lymphocytes and is infected during measles. To determine which populations of thymocytes are susceptible to MeV infection and whether strains of MeV differ in their ability to infect thymocytes, we used ex vivo human thymus organ cultures to assess the relative susceptibility of different subpopulations of thymocytes to infection with wild type and vaccine strains of MeV. Thymocytes were susceptible to MeV infection with the most replication in immature CD4⁺CD8⁺ double positive cells. Susceptibility correlated with the level of expression of the MeV receptor CD150. Wild type strains of MeV infected thymocytes more efficiently than the Edmonston vaccine strain. Thymus cultures from children ≥3 years of age were less susceptible to MeV infection than cultures from children 5 to 15 months of age. Resistance in one 7 year-old child was associated with production of interferon-gamma suggesting that vaccination may result in MeV-specific memory T cells in the thymus. We conclude that immature thymocytes are susceptible to MeV infection and thymocyte infection may contribute to the immunologic abnormalities associated with measles.     

CD4 T cell depletion is linked directly to immune activation in the pathogenesis of HIV-1 and HIV-2 but only indirectly to the viral load

The causal relationships among CD4 cell depletion, HIV replication, and immune activation are not well understood. HIV-2 infection, "nature's experiment" with inherently attenuated HIV disease, provides additional insights into this issue. We report the finding that in HIV-2 and HIV-1 patients with a comparable degree of CD4 depletion the imbalance in the relative sizes of the naive and memory T cell populations and the up-regulation of CD4 and CD8 cell activation markers (HLA-DR, CD38, CD69, Fas molecules) are similar, even though the viral load in the plasma of HIV-2-infected patients is two orders of magnitude lower than in HIV-1 patients and HIV-2 patients are known to have slower rates of CD4 T cell decline and a better clinical prognosis. Moreover, we found a similar increase in the frequency of cycling CD4 T cells (Ki67+), which was in strong correlation with the expression of activation markers. Finally, the level of T cell anergy, as assessed by the proliferative responses to CD3 stimulation and to a panel of microbial Ags, proved to be comparable in HIV-1 and HIV-2 patients with a similar degree of CD4 depletion despite large differences in viral load. Our data are consistent with a direct causal relationship between immune activation and CD4 cell depletion in HIV disease and an only indirect relation of these parameters to the virus replication rate. Invoking the concept of proximal immune activation and virus transmission, which links efficient transmission of virus to local cell activation and proliferation in response to Ags and inflammation, we propose an integrative interpretation of the data and suggest that strongly elevated immune activation induces CD4 cell depletion and not vice versa, with potential implications for the choice of treatment strategies.     

Cytokine expression, natural killer cell activation, and phenotypic changes in lymphoid cells from rhesus macaques during acute infection with pathogenic simian immunodeficiency virus

We studied the innate and adaptive immune system of rhesus macaques infected with the virulent simian immunodeficiency virus isolate SIVmac251 by evaluating natural killer (NK) cell activity, cytokine levels in plasma, humoral and virological parameters, and changes in the activation markers CD25 (interleukin 2R [IL-2R] α chain), CD69 (early activation marker), and CD154 (CD40 ligand) in lymphoid cells. We found that infection with SIVmac251 induced the sequential production of interferon-α/β (IFN-α/β), IL-18, and IL-12. IFN-γ, IL-4, and granulocyte-macrophage colony-stimulating factor were undetected in plasma by the assays used. NK cell activity peaked at 1 to 2 weeks postinfection and paralleled changes in viral loads. Maximum expression of CD69 on CD3⁻CD16⁺ lymphocytes correlated with NK cytotoxicity during this period. CD25 expression, which is associated with proliferation, was static or slightly down-regulated in CD4⁺ T cells from both peripheral blood (PB) and lymph nodes (LN). CD69, which is normally present in LN CD4⁺ T cells and absent in peripheral blood leukocyte (PBL) CD4⁺ T cells, was down-regulated in LN CD4⁺ T cells and up-regulated in PBL CD4⁺ T cells immediately after infection. CD8⁺ T cells increased CD69 but not CD25 expression, indicating the activation of this cellular subset in PB and LN. Finally, CD154 was transiently up-regulated in PBL CD4⁺ T cells but not in LN CD4⁺ T cells. Levels of antibodies to SIV Gag and Env did not correlate with the level of activation of CD154, a critical costimulatory molecule for T-cell-dependent immunity. In summary, we present the first documented evidence that the innate immune system of rhesus macaques recognizes SIV infection by sequential production of proinflammatory cytokines and transient activation of NK cytotoxic activity. Additionally, pathogenic SIV induces drastic changes in the level of activation markers on T cells from different anatomic compartments. These changes involve activation in the absence of proliferation, indicating that activation-induced cell death may cause some of the reported increase in lymphocyte turnover during SIV infection.The immune system of higher vertebrates consists of innate and adaptive components. Innate immunity exhibits immediate recognition and response without prior sensitization. Cells of the innate immune system (i.e., monocytes/macrophages, natural killer [NK] cells, and polymorphonuclear leukocytes) recognize pathogen-associated molecular patterns and activate events such as phagocytosis, induction of the synthesis of antimicrobial peptides, expression of inflammatory and effector cytokines and chemokines, induction of nitric oxide synthase in macrophages, and expression of costimulatory molecules on antigen-presenting cells. The adaptive immune system uses somatically generated antigen receptors that are clonally distributed on T and B lymphocytes. Generally, adaptive immune recognition in the absence of innate immune recognition results in inactivation of lymphocytes that express receptors involved in the identification events (20). Thus, innate immune responses have critical consequences in adaptive immune responses.Little is known of the contribution of the innate immune system during infection with the human immunodeficiency virus (HIV). Based on similarities of biologic and genetic features, simian immunodeficiency virus (SIV) infection of rhesus macaques provides the best animal model of HIV infection and AIDS. Accordingly, this animal model is critical for the elucidation of mechanisms of pathogenesis and for the development of vaccines and antiviral therapies (12). As with almost all viral infections, the innate immune system is thought to be the first component of the immune system that recognizes SIV infection. However, few studies have methodically analyzed the changes induced in cell phenotype and cytokine levels by SIV infection. Recent studies have demonstrated that SIV infection results in a generalized increase in lymphocyte turnover (23) and that the primary site for viral replication is activated memory CD4⁺ T cells that are present in the intestinal lamina propia (46). Although cellular changes are not that dramatic at this early stage in peripheral lymphoid tissue, peripheral blood (PB) and lymph nodes (LN) still reflect the pathologic changes induced by the viral infection and are readily available for longitudinal studies.To analyze changes in the activation state of cells from the innate and adaptive immune system after SIV infection, we evaluated NK activity, cytokine levels in plasma, and changes in activation markers on lymphoid cells of rhesus macaques after infection with pathogenic SIVmac251. We found the sequential appearance in plasma of interferon-α/β (IFN-α/β) interleukin-18 (IL-18) and IL-12, whereas IL-4, IFN-γ and granulocyte-macrophage colony-stimulating factor (GM-CSF) remained undetectable. We also found transient activation of NK cells during the peak of viral replication, and this activation was not predictive of disease progression. Finally, we observed that after SIV infection, both CD4⁺ and CD8⁺ T cells became activated in the absence of markers for proliferation, suggesting that the increased turnover of these cells reflects activation-induced cell death rather than differential compartmentalization.     

Toll-like receptor ligands induce human T cell activation and death, a model for HIV pathogenesis

BACKGROUND: Recently, heightened systemic translocation of microbial products was found in persons with chronic HIV infection and this was linked to immune activation and CD4(+) T cell homeostasis. METHODOLOGY: We examined here the effects of microbial Toll-like receptor (TLR) ligands on T cell activation in vitro. CONCLUSIONS/FINDINGS: We show that exposure to TLR ligands results in activation of memory and effector CD4(+) and CD8(+) T cells. After exposure to each of 8 different ligands that activate TLRs 2, 3, 4, 5, 7, 8, and 9, CD8(+) T cells are activated and gain expression of the C type lectin CD69 that may promote their retention in lymphoid tissues. In contrast, CD4(+) T cells rarely increase CD69 expression but instead enter cell cycle. Despite activation and cell cycle entry, CD4(+) T cells divide poorly and instead, disproportionately undergo activation-induced cell death. Systemic exposure to TLR agonists may therefore increase immune activation, effector cell sequestration in lymphoid tissues and T cell turnover. These events may contribute to the pathogenesis of immune dysfunction and CD4+ T cell losses in chronic infection with the human immunodeficiency virus.     

The role of tumor cells in the modification of T lymphocytes activity--the expression of the early CD69+, CD71+ and the late CD25+, CD26+, HLA/DR+ activation markers on T CD4+ and CD8+ cells in squamous cell laryngeal carcinoma. Part I

The role of interactions between tumor cells and autologous immunocompetent cells, the impact on the modulation of the activity of T CD4(+) and CD8(+) lymphocytes, as well as the influence on the regulation and determination of antitumor cellular immune response in patients with head and neck squamous cell carcinomas (HNSCC) is not completely clear. The aim of this study was to analyze early and late activation antigens expression on T cells subpopulations modified under the influence of the presence of cancer cells to investigate the regulatory mechanisms of the local cellular immune response in carcinoma of the larynx. Cytofluorymetric analysis of the early (CD69(+), CD71(+)) and late activation markers (CD25(+) (high), CD26(+), HLA/DR(+)) expression on T CD3(+)CD4(+) and CD3(+)CD8(+) cells subpopulations in mixed cellular cultures of freshly isolated tumor cells (MLTMC) and non-cancerous normal epithelial cells (MLNCC) with immunocompetent cells was performed in 55 cases of squamous cell laryngeal carcinoma. The whole peripheral blood concentrations of IL-10 and IFN-γ in 21 h and 72 h of experiments were also measured by ELISA. The relationships between the activation markers expression depending on the type of cells used in co-cultures, as well as the level of secreted cytokines, were investigated. Our work has revealed a statistically significant dependence of cytofluorymetric results on the presence of TMC or NCC in mixed cellular cultures. Increased expression of CD69(+), CD71(+) and CD25(+) (high), CD26(+), HLA/DR(+) antigens on T CD3(+)CD4(+) and CD3(+)CD8(+) cells was higher in MLTMC cultures, in comparison with MLNCC. We demonstrated negative significant relationships of IFN-γ and IL-10 secretion with regard to CD4(+)CD69(+), CD8(+)CD69(+), CD4(+)CD71(+), CD8(+)CD71(+) antigens expression in 21 h of experiments without mitogenic stimulation. Furthermore, this study revealed negative significant relationships of IFN-g secretion with regard to CD4(+)HLA/DR(+) and CD8(+)HLA/DR(+) as well as between IL-10 concentration and CD4(+)HLA/DR(+) in trials without PHA stimulation. Our findings have confirmed a key role for tumor cells in determining the function of T cells involved in the immunological processes and impact of neoplastic cells on modulating the activity of T CD4(+) and CD8(+) lymphocytes in laryngeal carcinoma.     

Distinct expression patterns of CD69 in mucosal and systemic lymphoid tissues in primary SIV infection of rhesus macaques

Although the intestinal tract plays a major role in early human immunodeficiency virus (HIV) infection, the role of immune activation and viral replication in intestinal tissues is not completely understood. Further, increasing evidence suggests the early leukocyte activation antigen CD69 may be involved in the development or regulation of important T cell subsets, as well as a major regulatory molecule of immune responses. Using the simian immunodeficiency virus (SIV) rhesus macaque model, we compared expression of CD69 on T cells from the intestine, spleen, lymph nodes, and blood of normal and SIV-infected macaques throughout infection. In uninfected macaques, the majority of intestinal lamina propria CD4+ T cells had a memory (CD95+) phenotype and co-expressed CD69, and essentially all intestinal CCR5+ cells co-expressed CD69. In contrast, systemic lymphoid tissues had far fewer CD69+ T cells, and many had a naïve phenotype. Further, marked, selective depletion of intestinal CD4+CD69+ T cells occurred in early SIV infection, and this depletion persisted throughout infection. Markedly increased levels of CD8+CD69+ T cells were detected after SIV infection in virtually all tissues, including the intestine. Further, confocal microscopy demonstrated selective, productive infection of CD3+CD69+ T cells in the intestine in early infection. Combined, these results indicate CD69+CD4+ T cells are a major early target for viral infection, and their rapid loss by direct infection may have profound effects on intestinal immune regulation in HIV infected patients.     

Pivotal role of antibody and subsidiary contribution of CD8+ T cells to recovery from infection in a murine model of Japanese encephalitis

The immunological correlates for recovery from primary Japanese encephalitis virus (JEV) infection in humans and experimental animals remain poorly defined. To investigate the relative importance of the adaptive immune responses, we have established a mouse model for Japanese encephalitis in which a low-dose virus inoculum was administered into the footpads of adult C57BL/6 mice. In this model, ~60% of the mice developed a fatal encephalitis and a virus burden in the central nervous system (CNS). Using mice lacking B cells (μMT(-/-) mice) and immune B cell transfer to wild-type mice, we show a critically important role for humoral immunity in preventing virus spread to the CNS. T cell help played an essential part in the maintenance of an effective antibody response necessary to combat the infection, since mice lacking major histocompatibility complex class II showed truncated IgM and blunted IgG responses and uniformly high lethality. JEV infection resulted in extensive CD8(+) T cell activation, judged by upregulation of surface markers CD69 and CD25 and cytokine production after stimulation with a JEV NS4B protein-derived H-2D(b)-binding peptide and trafficking of virus-immune CD8(+) T cells into the CNS. However, no significant effect of CD8(+) T cells on the survival phenotype was found, which was corroborated in knockout mice lacking key effector molecules (Fas receptor, perforin, or granzymes) of cytolytic pathways triggered by T lymphocytes. Accordingly, CD8(+) T cells are mostly dispensable for recovery from infection with JEV. This finding highlights the conflicting role that CD8(+) T cells play in the pathogenesis of JEV and closely related encephalitic flaviviruses such as West Nile virus.     

HIV-induced type I interferon and tryptophan catabolism drive T cell dysfunction despite phenotypic activation

Infection by the human immunodeficiency virus (HIV) is characterized by functional impairment and chronic activation of T lymphocytes, the causes of which are largely unexplained. We cultured peripheral blood mononuclear cells (PBMC) from HIV-uninfected donors in the presence or absence of HIV. HIV exposure increased expression of the activation markers CD69 and CD38 on CD4 and CD8 T cells. IFN-alpha/beta, produced by HIV-activated plasmacytoid dendritic cells (pDC), was necessary and sufficient for CD69 and CD38 upregulation, as the HIV-induced effect was inhibited by blockade of IFN-alpha/beta receptor and mimicked by recombinant IFN-alpha/beta. T cells from HIV-exposed PBMC showed reduced proliferation after T cell receptor stimulation, partially prevented by 1-methyl tryptophan, a competitive inhibitor of the immunesuppressive enzyme indoleamine (2,3)-dioxygenase (IDO), expressed by HIV-activated pDC. HIV-induced IDO inhibited CD4 T cell proliferation by cell cycle arrest in G1/S, and prevented CD8 T cell from entering the cell cycle by downmodulating the costimulatory receptor CD28. Finally, the expression of CHOP, a marker of the stress response activated by IDO, was upregulated by HIV in T cells in vitro and is increased in T cells from HIV-infected patients. Our data provide an in vitro model for HIV-induced T cell dysregulation and support the hypothesis that activation of pDC concomitantly contribute to phenotypic T cell activation and inhibition of T cell proliferative capacity during HIV infection.     

Differential effect of Listeria monocytogenes infection on cytokine production and cytotoxicity of CD8 T cells

Bacterial infection induces a shift to type 1 CD4 T cell subset in an infected host and this shift is important for protection of the host from disease development. Many researchers think that the shift is antigen-dependent, but we previously demonstrated an initial induction step for CD4 T cell subsets during Listeria monocytogenes (Lm) infection is antigen-independent. Although Listeria is a TLR2 ligand, the immune system of the Lm-infected host responded to the pathogen to induce expression of CD69 but not CD25 on CD4 T cells, CD8 T cells and B cells even in the absence of TLR2 or MyD88. The antigen-independent activation of type 1 CD4 T cells accelerate the clearance of pathogens by activating innate immune cells with type 1 cytokines. Type 1 CD4 T cells and CD8 T cells also collaborate to protect the host from intracellular Lm infection. Since CD8 T cells function mainly as cytotoxic T cells and CD69-positive CD8 T cells increase during Lm-infection, cytotoxic activity of CD8 T cells was evaluated during Lm-infection. Although CD8 T cells were activated to produce IFN-gamma, the cytotoxic function of CD8 T cells in Lymphocytic choriomeningitis virus (LCMV) p14 TCR-transgenic mouse was not augmented by Lm-infection. Therefore, Lm-infection differentially influences on cytokine production and cytotoxicity of CD8 T cells.     

Invariant NKT Cell Response to Dengue Virus Infection in Human

Background

Dengue viral infection is a global health threat without vaccine or specific treatment. The clinical outcome varies from asymptomatic, mild dengue fever (DF) to severe dengue hemorrhagic fever (DHF). While adaptive immune responses were found to be detrimental in the dengue pathogenesis, the roles of earlier innate events remain largely uninvestigated. Invariant natural killer T (iNKT) cells represent innate-like T cells that could dictate subsequent adaptive response but their role in human dengue virus infection is not known. We hypothesized that iNKT cells play a role in human dengue infection.

Methods

Blood samples from a well-characterized cohort of children with DF, DHF, in comparison to non-dengue febrile illness (OFI) and healthy controls at various time points were studied. iNKT cells activation were analyzed by the expression of CD69 by flow cytometry. Their cytokine production was then analyzed after α-GalCer stimulation. Further, the CD1d expression on monocytes, and CD69 expression on conventional T cells were measured.

Results

iNKT cells were activated during acute dengue infection. The level of iNKT cell activation associates with the disease severity. Furthermore, these iNKT cells had altered functional response to subsequent ex vivo stimulation with α-GalCer. Moreover, during acute dengue infection, monocytic CD1d expression was also upregulated and conventional T cells also became activated.

Conclusion

iNKT cells might play an early and critical role in the pathogenesis of severe dengue viral infection in human. Targeting iNKT cells and CD1d serve as a potential therapeutic strategy for severe dengue infection in the future.     

Inverted immunodominance and impaired cytolytic function of CD8+ T cells during viral persistence in the central nervous system.

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (JHMV) clear infectious virus; nevertheless, virus persists in the CNS as noninfectious RNA, resulting in ongoing primary demyelination. Phenotypic and functional analysis of CNS infiltrating cells during acute infection revealed a potent regional CD8+ T cell response comprising up to 50% virus-specific T cells. The high prevalence of virus-specific T cells correlated with ex vivo cytolytic activity and efficient reduction in viral titers. Progressive viral clearance coincided with the loss of cytolytic activity, but retention of IFN-gamma secretion and increased expression of the early activation marker CD69, indicating differential regulation of effector function. Although the total number of infiltrating T cells declined following clearance of infectious virus, CD8+ T cells, both specific for the dominant viral epitopes and of unknown specificity, were retained within the CNS, suggesting an ongoing T cell response during persistent CNS infection involving a virus-independent component. Reversed immunodominance within the virus-specific CD8+ T cell population further indicated epitope-specific regulation, supporting ongoing T cell activation. Even in the absence of infectious virus, the CNS thus provides an environment that maintains both unspecific and Ag-specific CD8+ T cells with restricted effector function. Chronic T cell stimulation may thus play a role in preventing viral recrudescence, while increasing the risk of pathological conditions, such as demyelination.     

Preferential infection of mature dendritic cells by mouse hepatitis virus strain JHM

Mouse hepatitis virus strain JHM (MHV-JHM) causes acute encephalitis and acute and chronic demyelinating diseases in mice. Dendritic cells (DCs) are key cells in the initiation of innate and adaptive immune responses, and infection of these cells could potentially contribute to a dysregulated immune response; consistent with this, recent results suggest that DCs are readily infected by another strain of mouse hepatitis virus, the A59 strain (MHV-A59). Herein, we show that the JHM strain also productively infected DCs. Moreover, mature DCs were at least 10 times more susceptible than immature DCs to infection with MHV-JHM. DC function was impaired after MHV-JHM infection, resulting in decreased stimulation of CD8 T cells in vitro. Preferential infection of mature DCs was not due to differential expression of the MHV-JHM receptor CEACAM-1a on mature or immature cells or to differences in apoptosis. Although we could not detect infected DCs in vivo, both CD8(+) and CD11b(+) splenic DCs were susceptible to infection with MHV-JHM directly ex vivo. This preferential infection of mature DCs may inhibit the development of an efficient immune response to the virus.     

CD8+ T cells specific for EBV, cytomegalovirus, and influenza virus are activated during primary HIV infection

Primary viral infections, including primary HIV infection, trigger intense activation of the immune system, with marked expansion of CD38(+)CD8(+) T cells. Whether this expansion involves only viral-specific cells or includes a degree of bystander activation remains a matter of debate. We therefore examined the activation status of EBV-, CMV-, and influenza virus (FLU)-specific CD8(+) T cells during primary HIV infection, in comparison to HIV-specific CD8(+) T cells. The activation markers CD38 and HLA-DR were strongly expressed on HIV-specific CD8(+) T cells. Surprisingly, CD38 expression was also up-regulated on CD8(+) T cells specific for other viruses, albeit to a lesser extent. Activation marker expression returned to normal or near-normal values after 1 year of highly active antiretroviral therapy. HIV viral load correlated with CD38 expression on HIV-specific CD8(+) T cells but also on EBV-, CMV-, and FLU-specific CD8(+) T cells. In primary HIV infection, EBV-specific CD8(+) T cells also showed increased Ki67 expression and decreased Bcl-2 expression, compared with values observed in HIV-seronegative control subjects. These results show that bystander activation occurs during primary HIV infection, even though HIV-specific CD8(+) T cells express the highest level of activation. The role of this bystander activation in lymphocyte homeostasis and HIV pathogenesis remains to be determined.     

Carboxyfluorescein succinimidyl ester-based proliferative assays for assessment of T cell function in the diagnostic laboratory

Immune deficiency diseases are often accompanied by abnormalities in one or both arms of the specific immune system. Impairment can often be detected as a decrease in the number of T or B lymphocytes or their products in the circulation, but questions are often asked as to the functional capabilities of T lymphocytes in patients with recurrent infections. Function of T cells has traditionally been measured by their uptake of [3H]- thymidine following stimulation with antigen or mitogen in vitro. However, the ability of carboxyfluorescein succinimidyl ester (CFSE) to label lymphocytes intracellularly and track their mitotic activity by progressive two-fold reduction in fluorescence intensity prompted an alternative methodology based on flow cytometry, an approach which has the advantage of allowing specific gating on particular T cell subsets and simultaneous assessment of activation markers. This method was therefore evaluated for T cell responses to mitogen and antigen. Phytohaemagglutinin-induced blast transformation of CFSE-labelled T cells was reflected by an increase in forward and orthogonal light scatter and a progressive two-fold decrease in CFSE fluorescence intensity. These changes allowed the derivation of various measures of mitotic activity, which correlated well with [3H]-thymidine uptake. Patients with T cell functional deficiencies showed impairment in their responses by both assays, whereas the CFSE-based assay demonstrated that impaired blastogenesis was not simply due to depressed T cell numbers. Concomitant measurement of the activation markers CD69 and CD25 showed that CD69 was rapidly expressed on non-mitotic cells and that this expression was progressively diluted with subsequent rounds of cell division. In contrast, CD25 expression was unaffected by cell cycle, but was expressed in proportion to the PHA dose. Antigen-specific responsiveness to Candida was also assessed using a CFSE-based assay. Initial gating on the relatively minor population of T cells that underwent blast transformation demonstrated progressive twofold dilutions of CFSE intensity in responsive cells. These normal Candida responses, found in patients who had recovered from Candida infection, contrasted with those who had not been infected with Candida or who had chronic recurrent infection, in whom neither blast transformation nor significant mitosis could be detected. Again, there was good correlation with [3H]-thymidine uptake. The CFSE-based assays are equivalent to traditional measures of mitogen- and antigen-specific T cell responsiveness in the diagnostic laboratory and have significant advantages in terms of decreased labour intensiveness, avoidance of radioactivity, the ability to gate on a specific population of lymphocytes and the concomitant measurement of activation markers.     

Antigen-dependent and -independent mechanisms of T and B cell hyperactivation during chronic HIV-1 infection

Continuous loss of CD4(+) T lymphocytes and systemic immune activation are hallmarks of untreated chronic HIV-1 infection. Chronic immune activation during HIV-1 infection is characterized by increased expression of activation markers on T cells, elevated levels of proinflammatory cytokines, and B cell hyperactivation together with hypergammaglobulinemia. Importantly, hyperactivation of T cells is one of the best predictive markers for progression toward AIDS, and it is closely linked to CD4(+) T cell depletion and sustained viral replication. Aberrant activation of T cells is observed mainly for memory CD4(+) and CD8(+) T cells and is documented, in addition to increased expression of surface activation markers, by increased cell cycling and apoptosis. Notably, the majority of these activated T cells are neither HIV specific nor HIV infected, and the antigen specificities of hyperactivated T cells are largely unknown, as are the exact mechanisms driving their activation. B cells are also severely affected by HIV-1 infection, which is manifested by major changes in B cell subpopulations, B cell hyperactivation, and hypergammaglobulinemia. Similar to those of T cells, the mechanisms underlying this aberrant B cell activation remain largely unknown. In this review, we summarized current knowledge about proposed antigen-dependent and -independent mechanisms leading to lymphocyte hyperactivation in the context of HIV-1 infection.