The role of CD4+CD25+ regulatory T cells in viral infections.

Many virus infections result in the suppression of one or more functions of the immune system. Multiple mechanisms have been proposed to explain viral-induced immunosuppression, including an imbalance in the cellular Th1/Th2 or cytokine profile, induction of anergy, depletion of effector cells and most recently the activation of CD4+CD25+ regulatory T (T reg) cells. CD4+CD25+ T reg cells are a subset of circulating CD4+ T cells with suppressive properties. CD4+CD25+ T reg cells were first identified in mice as cells capable of maintaining self-tolerance by suppressing autoreactive T cells. This review focuses on interactions between CD4+CD25+ T reg cells and viral pathogens. Most cases in which CD4+CD25+ T reg cells participate in response to infection reported so far involve chronic or persistent viral infections. Examples have been growing recently and include members of different viral families including retroviridae, herpesviridae and picornaviridae. It is currently not known how microbes are recognized by CD4+CD25+ T reg cells and whether exoantigen-specific T reg cells are of the same lineage as self-reacting natural T reg cells or represent peripherally induced counterparts derived from CD4+CD25- T cells. The findings that T reg cells influence the functional immunity during viral infections, however, might indicate that, in some cases, virus-specific T reg cells not only influence immune pathology or prevent pathogen elimination but also can promote a generalized state of immunosuppression in vivo such that the host is more susceptible to secondary infections with other pathogens or has reduced resistance to tumors. Conceivably, the activities of T reg cells might be one of the contributing reasons why it has been difficult so far to produce effective vaccines against some persisting viral infections.

Spontaneous T cell apoptosis in feline immunodeficiency virus (FIV)-infected cats is inhibited by IL2 and anti-B7.1 antibodies.

Lymph node (LN) T cells from feline immunodeficiency virus (FIV)-infected cats have an increased expression of B7 co-stimulatory molecules as well as their ligand CTLA4, resembling an activation phenotype shown to induce anergy and apoptosis in activated T cells. In addition, LN T cells from FIV-infected cats also show increased spontaneous apoptosis compared to uninfected animals. The apoptosis observed in these animals occurs primarily in T cells expressing B7 and CTLA4, suggesting a role for B7 and CTLA4 interactions in the induction of anergy/apoptosis. In order to investigate the role of B7 and CTLA4 interactions on T cell apoptosis in LN T cells from FIV-infected cats, we performed blocking experiments by measuring T cell apoptosis in LN T cell cultures treated with anti-feline B7.1, B7.2, and CTLA4 specific antibodies, as well as interleukin (IL)-2. The addition of IL2, the primary cytokine produced by B7/CD28 interactions, resulted in a significant decrease of T cell apoptosis in cultured LN cells as assessed by two-color flow cytometry and TUNEL assay. The addition of anti-B7.1 antibodies significantly inhibited T cell apoptosis in FIV-infected cats with low-level plasma viremia, while addition of anti-B7.2 and anti-CTLA4 antibodies had no affect. These results suggest a role of B7 signaling in the increased spontaneous apoptosis observed in LN T cells from FIV-infected animals.

Feline immunodeficiency virus infection phenotypically and functionally activates immunosuppressive CD4+CD25+ T regulatory cells.

Disease progression of feline immunodeficiency virus (FIV) infection is characterized by up-regulation of B7.1 and B7.2 costimulatory molecules and their ligand CTLA4 on CD4(+) and CD8(+) T cells. The CD4(+)CTLA4(+)B7(+) phenotype described in FIV(+) cats is reminiscent of CD4(+)CD25(+)CTLA4(+) cells, a phenotype described for immunosuppressive T regulatory (Treg) cells. In the present study, we describe the phenotypic and functional characteristics of CD4(+)CD25(+) T cells in PBMC and lymph nodes (LN) of FIV(+) and control cats. Similar to Treg cells, feline CD4(+)CD25(+) but not CD4(+)CD25(-) T cells directly isolated from LN of FIV(+) cats do not produce IL-2 and fail to proliferate in response to mitogen stimulation. Unstimulated CD4(+)CD25(+) T cells from FIV(+) cats significantly suppress the proliferative response and the IL-2 production of Con A-stimulated autologous CD4(+)CD25(-) T cells compared with unstimulated CD4(+)CD25(+) T cells from FIV(-) cats. Flow-cytometric analysis confirmed the apparent activation phenotype of the CD4(+)CD25(+) cells in LN of chronically FIV(+) cats, because these cells showed significant up-regulation of expression of costimulatory molecules B7.1, B7.2, and CTLA4. These FIV-activated, anergic, immunosuppressive CD25(+)CTLA4(+)B7(+)CD4(+) Treg-like cells may contribute to the progressive loss of T cell immune function that is characteristic of FIV infection.

Mechanism of feline immunodeficiency virus envelope glycoprotein-mediated fusion.

Feline immunodeficiency virus (FIV) shares remarkable homology to primate lentiviruses, human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). The process of lentiviral env glycoprotein-mediated fusion of membranes is essential for viral entry and syncytia formation. A detailed understanding of this phenomenon has helped identify new targets for antiviral drug development. Using a model based on syncytia formation between FIV env-expressing cells and a feline CD4+ T cell line we have studied the mechanism of FIV env-mediated fusion. Using this model we show that FIV env-mediated fusion mechanism and kinetics are similar to HIV env. Syncytia formation could be blocked by CXCR4 antagonist AMD3100, establishing the importance of this receptor in FIV gp120 binding. Interestingly, CXCR4 alone was not sufficient to allow fusion by a primary isolate of FIV, as env glycoprotein from FIV-NCSU(1) failed to induce syncytia in several feline cell lines expressing CXCR4. Syncytia formation could be inhibited at a post-CXCR4 binding step by synthetic peptide T1971, which inhibits interaction of heptad repeat regions of gp41 and formation of the hairpin structure. Finally, using site-directed mutagenesis, we also show that a conserved tryptophan-rich region in the membrane proximal ectodomain of gp41 is critical for fusion, possibly at steps post hairpin structure formation.

B7+CTLA4+ T cells engage in T-T cell interactions that mediate apoptosis: a model for lentivirus-induced T cell depletion.

Apoptosis in lymph node (LN) T cells of feline immunodeficiency virus (FIV)-infected cats is associated with cells co-expressing B7.1 and B7.2 costimulatory molecules, and their ligand CTLA4. To study the possibility of B7.1/B7.2-CTLA4 mediated T-T interactions and the predicted induction of T cell apoptosis in vitro, costimulatory molecules were up-regulated on CD4+ and CD8+ T cells by mitogen stimulation. B7.1 expression on in vitro stimulated CD4+ and CD8+ cells increased within 24h; B7.2 and CTLA4 expression increased after 48-72 h. Apoptosis, as analyzed by terminal deoxynucleotidyl transferase (transferase nick end labeling, TUNEL)-based staining followed by three color flow cytometric analysis, correlated to the cells expressing B7 and/or CTLA4. Blocking experiments revealed that CD4+ and CD8+ T cell apoptosis could be significantly inhibited with anti-B7 antibodies. As FIV infection results in immune activation with a T cell phenotype similar to that of the in vitro activated T cells, the data support the hypothesis that the chronic expansion of B7+CTLA4+ LN T cells in infected cats allows for T-T cell interactions resulting in T cell depletion and eventually the development of AIDS.

Preferential replication of FIV in activated CD4(+)CD25(+)T cells independent of cellular proliferation.

Studies attempting to identify reservoirs of HIV-1 latency have documented that the virus persists as both a latent and productive infection in subsets of CD4(+) cells. Reports regarding establishment of a stable HIV-1 infection in quiescent T cells in vitro, however, are controversial. In the present study, we investigated the susceptibility of naive and activated CD4(+) cell subsets (distinguished by differential expression of CD25) to feline immunodeficiency virus (FIV) infection, their ability to replicate the virus, and potentially act as a reservoir for virus persistence in infected animals. While both CD4(+)CD25(+) and CD4(+)CD25(-) cells are susceptible to FIV infection in vitro and in vivo, only CD4(+)CD25(+) cells produce infectious virions when cultured with interleukin-2 (IL-2). Latently infected CD4(+)CD25(-) cells produce infectious virions following ConcanvalinA (ConA) stimulation, which correlates with upregulated surface expression of CD25. In contrast to CD4(+)CD25(-) cells, CD4(+)CD25(+) cells remain unresponsive to mitogen stimulation and are relatively resistant to apoptosis whether or not infected with FIV. The ability of CD4(+)CD25(+) cells to replicate FIV efficiently in the presence of IL-2 but remain anergic and unresponsive to apoptotic signaling suggests that these cells may provide a reservoir of productive FIV infection. On the contrary, CD4(+)CD25(-) cells seem to establish as latent viral reservoirs capable of being reactivated after stimulation.

Evaluation of T lymphocytes in captive african lions (Panthera leo) infected with feline immunodeficiency virus.

OBJECTIVE: To determine whether FIV infection in captive African lions is associated with changes in immune cell variables similar to those detected in domestic cats infected with FIV. ANIMALS: 5 captive African lions naturally infected with FIV (FIV+) and 5 lions not infected with FIV (FIV-). PROCEDURE: Peripheral blood samples were collected from FIV+ lions during annual examinations conducted during a 7-year period and at a single time point from the FIV- lions. From results of CBC and flow cytometry, lymphocyte subsets were characterized and compared. RESULTS: Flow cytometric analysis revealed that the percentage and absolute number of CD4+ and CD8+ T cells were significantly lower in FIV+ lions, compared with these values in FIV- lions. In FIV+ lions, severe depletion in the absolute number of CD4+ and CD8+ T cells was detected, although this did not correlate with clinical signs. Muscle wasting was the most consistent clinical sign of infection. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that FIV+ African lions develop lymphocyte deficiencies, including significant decreases in the absolute number of CD4+ and CD8+ T cells; these findings of immune dysfunction are similar to those defined for FIV+ domestic cats. It is important to monitor the number of CD4+ T cells in infected animals as a measure of disease progression.

Evaluation of FIV protein-expressing VEE-replicon vaccine vectors in cats.

Venezuelan equine encephalitis (VEE) virus-replicon particles (VRP) were used to generate feline immunodeficiency virus (FIV) Gag- and ENV-expressing vaccine vectors. Serum and mucosal FIV-specific antibody was detected in cats immunized subcutaneously, once monthly for 5 months, with FIV-expressing VRP. Expansion of the CD8+ L-selectin negative phenotype and transient CD8+ noncytolytic suppressor activity were seen in cats immunized with FIV-expressing or control VRP. Despite induction of FIV-specific immune responses and nonspecific suppressor responses, all cats became infected following vaginal challenge with high dose, pathogenic cell-associated FIV-NCSU(1) although relative early maintenance of CD4+ cells was seen in FIV-immunized cats.

Polymorphic expression in the CD8alpha chain surface receptor of African lions (Panthera leo).

Free-ranging African lion (Panthera leo) peripheral blood mononuclear cells (PBMC) were examined using flow cytometry and antibodies developed for use in the domestic cat to determine if phenotypic changes occurred in lion lymphocytes as a result of feline immunodeficiency virus (FIV) infection. The percentage of CD8 cells from lion peripheral blood was considerably lower than in the domestic cat. Lions with elevated levels of CD8+ cells were typically infected with FIV, similar to observations in the domestic cat. Antibodies against the alpha chain of the CD8 receptor (monoclonal antibody (mAb) 3.357) did not react consistently in all lions examined. Flow cytometric analysis determined that approximately 82 and 80% of the animals from Kruger and Hluhluwe-Umfolozi National Parks in South Africa reacted with the monoclonal antibody against the alpha chain of CD8 receptor, while only 17% of the lions in Etosha National Park in Namibia cross-reacted with the CD8alpha chain. There was no apparent correlation between FIV status and CD8alpha chain reactivity. The relative isolation of Etosha from the other two parks could explain the marked difference in CD8alpha chain expression and suggests that lions similar to other mammalian species demonstrate polymorphic expression of the CD8alpha chain (197).