We have reported that CD4+CD25+ T-regulatory (Treg) cells are fully activated for suppressor function in feline immunodeficiency virus (FIV)-infected cats. Studies have suggested that surface transforming growth factor-beta (TGFbeta; membrane TGFbeta [mTGFbeta]) is a feature of activated CD4+CD25+ Treg cells and may play a role in Treg homeostasis and suppressor function. Herein, we explore the role of TGFbeta in feline Treg homeostasis and suppressor function and what effect FIV infection of cats might have on these processes. Stimulation of CD4+CD25- T helper (Th) cells with Concanavalin A (ConA) plus TGFbeta converts them to Treg-like cells capable of suppressor function. Reverse-transcription polymerase chain reaction and flow cytometry revealed that these ConA/TGFbeta-converted Treg cells upregulate Foxp3 and mTGFbeta. ConA stimulation of CD4+CD25- T cells upregulates TGFbeta receptor II (RII), and pretreatment of these cells with anti-TGFbeta-RII antibodies blocks the TGFbeta-induced conversion to Treg cells. Pretreatment of ConA/TGFbeta-converted Treg cells with anti-TGFbeta antibodies also abrogates their suppressor function, suggesting that Treg homeostasis and suppressor function may be mediated by mTGFbeta. Finally, we show that treatment of CD4+CD25+ mTGFbeta-positive Treg cells from FIV-infected cats with anti-TGFbeta antibodies or treatment of ConA-stimulated CD4+CD25- Th target cells with anti-TGFbeta-RII antibodies diminishes suppressor function. These data suggest that the recruitment of Treg cells from the Th pool and suppressor function of Treg cells are dependent on the TGFbeta/TGFbeta-RII signaling pathway and that this pathway is constitutively upregulated in asymptomatic chronically FIV-infected cats.

Regulatory T cells (Treg) are increased and directly infected by feline immunodeficiency virus (FIV) and likely play a role in other feline autoimmune, neoplastic, and infectious diseases. Phenotypically, Treg are best characterized by surface expression of CD4 and CD25 and intranuclear expression of the forkhead transcription factor Foxp3. Our objective was to clone and sequence feline FOXP3 for the purpose of developing assays to enhance studies of feline Treg. We determined the feline FOXP3 is 1293 nucleotides in length and codes for a protein that shares high homology to other species. A splice variant devoid of exon 2 was also identified. A real-time PCR assay was developed and used to show Foxp3 mRNA expression occurs primarily in CD4+CD25+ T cells. Two crossreacting antibodies were identified by immunocytochemical staining of HEK293 cells transfected with feline FOXP3. The antibody labeling confirmed the nuclear localization of the protein. A flow cytometric assay was also validated and used to correlate the phenotypic and functional characteristics of feline Treg induced by treatment of lymph node lymphocytes with flagellin or LPS in combination with mitogen or IL2. Together, these studies provide useful tools to further investigate Foxp3 and Tregs in cats. KeywordsFoxp3; feline; Treg; regulatory T cells; Toll-like receptors ReportRegulatory T cells (Treg) can functionally suppress CD4 and CD8 T cells, B cells, NK cells, NKT cells, monocytes/macrophages, dendritic cells and neutrophils, thereby playing a key role in limiting both innate and adaptive immune responses (Azuma et al., 2003;Fallarino et al., 2003;Lewkowicz et al., 2006;Lim et al., 2005;Piccirillo and Shevach, 2004;Ralainirina et al., 2007;Taams et al., 2005;Wing et al., 2005 Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. The forkhead transcription factor Foxp3 has been identified as essential and sufficient to impart Treg function in mice (Ziegler, 2006). This understanding has come from two lines of study. The first involved determination of the genetic basis of autoimmune diseases observed in the scurfy mouse ) and human X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy (IPEX) (Bennett et al., 2001). In both cases, mutations in the FOXP3 gene were shown to be the sole cause of these diseases that result from a lack of Treg. Second, transfection of FOXP3 into CD4+CD25− cells imparts Treg suppressive function (Fontenot et al., 2003;Hori et al., 2003;Khattri et al., 2003). NIH Public AccessRegulatory T cells have been phenotypically and functionally characterized in the cat ...

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.

The immune response to recombinant adenoviruses is the most significant impediment to their clinical use for immunization. We test the hypothesis that specific virus-antibody combinations dictate the type of immune response generated against the adenovirus and its transgene cassette under certain physiological conditions while minimizing vector-induced toxicity. In vitro and in vivo assays were used to characterize the transduction efficiency, the T and B cell responses to the encoded transgene, and the toxicity of 1 ؋ 10