Jonathan E. Fogle

CD4+CD25+ REGULATORY T CELLS ARE INFECTED AND ACTIVATED DURING ACUTE FIV INFECTION

HIV-induced AIDS may be mediated by the activation of immunosuppressive CD4+CD25+ T regulatory cells (Treg cells). Treg cells have been shown to regulate CD4+ and CD8+ immune responses to HIV and FIV antigens in vitro. We tested the hypothesis that Treg cells become infected and activated during the acute infection with FIV leading to the suppression of CD4+ T helper cell responses. Cats were experimentally infected with FIV-NCSU1 and blood and lymph node cells were collected at weekly intervals following inoculation. Real-time RT-PCR was used to determine plasma viremia and the relative expression of FIV, FoxP3, TGF-beta, and GAPDH mRNA copies in CD4+CD25+ and CD4+CD25- T cell subsets. Flow cytometry was used to assess the absolute numbers of each cell type and the expression of surface TGF-beta and intracellular FoxP3 in CD4+CD25+ and CD4+CD25- T cells at each time-point. Treg suppression of IL-2 production in CD4+ T helper cells was assessed by ELISPOT assays. Our results showed that peak viremia occurred at 2 weeks post infection and correlated with maximal infectivity in CD4+CD25+ T cell populations. FIV-gag-mRNA levels were higher in CD4+CD25+ T cells than CD4+CD25- T cells throughout the acute phase of infection. Induction of FoxP3 and TGF-beta indicated activation of Treg cells during the acute stage infection, which was confirmed by Treg cell suppression of IL-2 production by CD4+ Th cells in an ELISPOT assay. Our findings support the hypothesis that early activation of Treg immunosuppressor function may limit an effective anti-FIV response, contributing to the establishment of chronic infection and the immunodeficiency caused by this virus.

Lymphoma immunophenotype of dogs determined by immunohistochemistry, flow cytometry, and polymerase chain reaction for antigen receptor rearrangements.

BACKGROUND Immunohistochemistry (IHC), flow cytometry (FC), and PCR for antigen receptor rearrangements (PARR) are 3 widely utilized tests to determine immunophenotype in dogs with lymphoma (LSA). OBJECTIVES This study evaluated the ability of FC and PARR to correctly predict immunophenotype as defined by IHC and to determine the level of agreement among the 3 tests. ANIMALS Sixty-two dogs with lymphoma. METHODS Retrospective study. Medical records were searched to identify dogs with LSA that had concurrent IHC, FC, and PARR performed. Immunophenotype results were categorized as B-cell, T-cell, dual immunophenotype (B- and T-cell), or indeterminate. The results of FC and PARR were evaluated for correctly classifying B- and T-cell LSA as compared with IHC. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were evaluated in addition to concordance between each test. RESULTS The sensitivity of FC was significantly higher than PARR for both B-cell (91% versus 67%; P < 0.0072) and T-cell (100% versus 75%; P < 0.0312) LSA. The percent agreement between FC and IHC was 94%, between PARR and IHC was 69%, between FC and PARR was 63%, and among all 3 tests was 63%. CONCLUSIONS AND CLINICAL IMPORTANCE Flow cytometry is superior to PARR in correctly predicting immunophenotype when evaluating lymph nodes from dogs already diagnosed with B- or T-cell LSA. If fresh samples are not available for FC, PARR is an acceptable assay for determination of immunophenotype given its high specificity.

CD4+CD25+ T Regulatory Cells Inhibit CD8+ IFN-γ Production During Acute and Chronic FIV Infection Utilizing a Membrane TGF-β-Dependent Mechanism

CD8(+) lymphocytes are critical to the control and elimination of viral pathogens. Impaired CD8(+) responses are well recognized in lentiviral infections; however, the mechanisms underlying CD8(+) impairment remain elusive. Using the feline immunodeficiency virus (FIV) model for human AIDS, we reported previously that CD4(+)CD25(+) Treg cells in both the acute and long-term, asymptomatic phase of infection are constitutively activated and suppress CD4(+)CD25(-) T cell responses. In the current study, we have demonstrated that CD4(+)CD25(+) Treg cells suppress CD8(+) responses to immune stimulation during both the acute and chronic, asymptomatic phase of FIV infection and that the mechanism of suppression may be mediated by membrane-associated TGF-beta (mTGF-beta) on CD4(+)CD25(+) lymphocytes. Depletion of CD4(+)CD25(+) lymphocytes from lymph node suspensions significantly enhanced production of IFN-gamma during the acute phase of infection and coculture of CD8(+) lymphocytes with CD4(+)CD25(+) lymphocytes resulted in suppression of CD8(+) IFN-gamma during both the acute and chronic stages of infection. FACS analysis indicated that there was TGF-betaRII upregulation on CD8(+) cells from FIV(+) cats during the acute and chronic stage of infection. In addition, there was upregulation of mTGF-beta on the CD4(+)CD25(+) subset in chronically infected cats. In support of activation of the TGF-beta signaling pathway, Western blotting showed Smad 2 phosphorylation in CD8(+) targets following CD4(+)CD25(+)/CD8(+) coculture. These results demonstrate the suppressive effect CD4(+)CD25(+) Treg cells have on the CD8(+) immune response during the acute and chronic stages of FIV infection and suggest that the mechanism of suppression may be mediated by mTGF-beta.

CD4 + CD25 + T regulatory cells activated during feline immunodeficiency virus infection convert T helper cells into functional suppressors through a membrane-bound TGFβ / GARP-mediated mechanism

BackgroundWe and others have previously reported that cell membrane-bound TGFβ (mTGFβ) on activated T regulatory (Treg) cells mediates suppressor function. Current findings suggest that a novel protein known as Glycoprotein A Repetitions Predominant (GARP) anchors mTGFβ to the Treg cell surface and facilitates suppressor activity. Recently, we have described that GARP+TGFβ+ Treg cells expand during the course of FIV infection. Because Treg cells are anergic and generally exhibit poor proliferative ability, we asked how Treg homeostasis is maintained during the course of feline immunodeficiency virus (FIV) infection.ResultsHere, we report that Treg cells from FIV+ cats express GARP and mTGFβ and convert T helper (Th) cells into phenotypic and functional Treg cells. Th to Treg conversion was abrogated by anti-TGFβ or anti-GARP treatment of Treg cells or by anti-TGFβRII treatment of Th cells, suggesting that Treg cell recruitment from the Th pool is mediated by TGFβ/TGFβRII signaling and that cell-surface GARP plays a major role in this process.ConclusionsThese findings suggest Th to Treg conversion may initiate a cascade of events that contributes to the maintenance of virus reservoirs, progressive Th cell immunosuppression, and the development of immunodeficiency, all of which are central to the pathogenesis of AIDS lentivirus infections.

CD4+CD25+ T regulatory cells from FIV+ cats induce a unique anergic profile in CD8+ lymphocyte targets

BackgroundUsing the FIV model, we reported previously that CD4+CD25+ T regulatory (Treg) cells from FIV+ cats are constitutively activated and suppress CD4+CD25- and CD8+ T cell immune responses. In an effort to further explore Treg-mediated suppression, we asked whether Treg cells induce anergy through the alteration of production of cyclins, cyclin-dependent kinases and their inhibitors.ResultsLymphocytes were obtained from control or FIV+ cats and sorted by FACS into CD4+CD25+ and CD8+ populations. Following co-culture with CD4+CD25+ cells, CD8+ targets were examined by Western blot for changes in cyclins D3, E and A, retinoblastoma (Rb) protein, as well as the cyclin dependent kinase inhibitor p21cip1. Following co-culture with CD4+CD25+cells, we observed up-regulation of p21cip1 and cyclin E, with down-regulation of cyclin D3, in CD8+ cells from FIV+ cats. As expected, CD8+ targets from control cats were quiescent with little up-regulation of p21cip1 and cyclin E. There was also a lack of Rb phosphorylation in CD8+ targets consistent with late G1 cell cycle arrest. Further, IL-2 mRNA was down regulated in CD8+ cells after co-culture with CD4+CD25+ Treg cells. Following CD4+CD25+ co-culture, CD8+ targets from FIV+ cats also had increased Foxp3 mRNA expression; however, these CD8+Foxp3+ cells did not exhibit suppressor function.ConclusionsCollectively, these data suggest that CD4+CD25+ Treg cells from FIV+ cats induce CD8+ anergy by disruption of normal G1 to S cell cycle progression.

Infection with Feline Immunodeficiency Virus Directly Activates CD4+ CD25+ T Regulatory Cells

ABSTRACT Lentivirus infection activates CD4+ CD25+ T regulatory (Treg) cells. Activation of Treg cells may be due to direct virus infection or chronic antigenic stimulation. Herein we demonstrate that in vitro feline immunodeficiency virus (FIV) infection, but not UV-inactivated virus, activates Treg cells as measured by immunosuppressive function and upregulation of GARP, FoxP3, and membrane-bound transforming growth factor β (TGF-β). These data demonstrate for the first time that AIDS lentiviruses infect and activate Treg cells, potentially contributing to immune dysfunction.

Feline Glycoprotein A Repetitions Predominant Anchors Transforming Growth Factor Beta on the Surface of Activated CD4+CD25+ Regulatory T Cells and Mediates AIDS Lentivirus-Induced T Cell Immunodeficiency

Using the feline immunodeficiency virus (FIV) model for AIDS-lentivirus infection, our laboratory has previously demonstrated that T regulatory (Treg) cell-mediated immune T and B cell dysfunction contributes to lentivirus persistence and chronic disease through membrane bound transforming growth factor beta (mTGFb). Studying Treg cells in the context of infection has been problematic as no inducible marker for activated Treg cells had been identified. However, recent reports in human Treg studies have described a novel protein, glycoprotein A repetitions predominant (GARP), as a unique marker of activated human Treg cells that anchors mTGFb. Herein we extend these studies to the feline Treg system, identifying feline GARP and demonstrating that human and feline GARP proteins are homologous in structure, expression pattern, and ability to form a complex with TGFb. We further demonstrate that GARP and TGFb form a complex on the surface of activated Treg cells and that these GARP(+)TGFb(+) Treg cells are highly efficient suppressor cells. Analysis of expression of this Treg activation marker in the FIV-AIDS model reveals an up-regulation of GARP expressing Treg cells during chronic FIV infection. We demonstrate that the GARP(+) Treg cells from FIV-infected cats suppress T helper cells in vivo and that blocking GARP or TGFb eliminates this suppression. These data suggest that GARP is expressed in complex with TGFb on the surface of activated Treg cells and plays an important role in TGFb(+) Treg-mediated T cell immune suppression during lentivirus infection.

Fozivudine tidoxil as single-agent therapy decreases plasma and cell-associated viremia during acute feline immunodeficiency virus infection.

BACKGROUND Feline immunodeficiency virus (FIV) is a lentivirus that infects domestic and wild felidae and the course of disease is similar to that of human immunodeficiency virus infection. The thymidine nucleoside analog fozivudine (FZD) tidoxil is a lipid-zidovudine (ZDV) conjugate and member of the family of nucleoside reverse transcriptase (RT) inhibitors (NRTIs). HYPOTHESIS FZD administration to cats during acute FIV infection produces antiviral activity with fewer adverse effects than its parent compound ZDV (AZT). ANIMALS Male, neutered cats approximately 7 months of age (n = 12). METHODS FZD (45 mg/kg q12h, n = 6) or placebo (n = 6) was administered PO in a nonblinded trial for 6 weeks to cats infected with the NCSU(1) isolate of FIV. Peripheral blood was collected preinfection and at 2, 4, and 6 weeks postinfection for CBC, evaluation of CD4(+) and CD8(+) cell counts by flow cytometry, and quantification of plasma and cell-associated viremia by real time RT-PCR. RESULTS Treatment of cats with FZD during the acute stage of FIV infection decreased plasma and cell-associated viremia during the first 2 weeks of infection, but was not protective against FIV, as all cats were infected by 6 weeks. CONCLUSIONS At the dosage used in this study, treatment with FZD results in a short-term decrease in viral load with no adverse effects. Further investigation of FZD is warranted to assess pharmacokinetics, optimal dosage, and to directly compare the antiviral activity of FZD to ZDV in naturally infected cats.

Modulating DNA Methylation in Activated CD8+ T Cells Inhibits Regulatory T Cell–Induced Binding of Foxp3 to the CD8+ T Cell IL-2 Promoter

We have previously demonstrated that CD4+CD25+ regulatory T cells (Tregs) activated during the course of feline immunodeficiency virus (FIV) infection suppress CD8+ CTL function in a TGF-β–dependent fashion, inhibiting IFN-γ and IL-2 production and inducing G1 cell-cycle arrest. In this article, we describe the molecular events occurring at the IL-2 promoter leading to suppression of IL-2 production. These experiments demonstrate that Foxp3 induced by lentivirus-activated Tregs in the CD8+ target cells binds to the IL-2 promoter, actively repressing IL-2 transcription. We further demonstrate that the chronic activation of CD8+ T cells during FIV infection results in chromatin remodeling at the IL-2 promoter, specifically, demethylation of CpG residues. These DNA modifications occur during active transcription and translation of IL-2; however, these changes render the IL-2 promoter permissive to Foxp3-induced transcriptional repression. These data help explain, in part, the seemingly paradoxical observations that CD8+ T cells displaying an activation phenotype exhibit altered antiviral function. Further, we demonstrate that blocking demethylation of CpG residues at the IL-2 promoter inhibits Foxp3 binding, suggesting a potential mechanism for rescue and/or reactivation of CD8+ T cells. Using the FIV model for lentiviral persistence, these studies provide a framework for understanding how immune activation combined with Treg-mediated suppression may affect CD8+ T cell IL-2 transcription, maturation, and antiviral function.

CD45+ and CD45- lymphocyte populations identified by flow cytometry from dogs with lymphoma exhibit similar morphology and the same clonal (B cell or T cell) lineage.

Flow cytometric analysis of canine lymphoma sometimes demonstrates a mixed population of CD45+ and CD45- lymphocytes. Recently, indolent forms of canine lymphoma have been described which are associated with the loss of CD45 expression, warranting further investigation of the role of CD45 in canine lymphoma. The purpose of this study was to compare morphology and assess clonal origin between CD45+ and CD45- lymphocyte populations identified by flow cytometry in confirmed cases of canine B- and T-cell lymphoma. Our hypothesis was that the CD45- population of lymphocytes represented a phenotypic variant of the CD45+ population. Fifteen client-owned dogs with lymphoma and distinct CD45+ and CD45- lymphocyte populations identified by flow cytometry were identified for a blinded, prospective assessment of morphology and clonal origin (B cell or T cell) between populations of sorted CD45+ and CD45- cells. Lymphocytes were isolated from 11 dogs for paired cytologic evaluation. In 10/11 dogs, the CD45+ and CD45- samples were similar (95% C.I., 0.301-1.00). DNA was harvested from sorted populations of CD45+ and CD45- cells from 12/15 dogs and PARR analysis produced amplicons of identical size from both populations, indicating that 100% (12/12) were of the same lineage, B cell or T cell (95% C.I., 0.757-1.00). Collectively, our data suggests that the CD45- population identified in dogs with lymphoma represents a phenotypic variant of the CD45+ population.