Cloning of feline FOXP3 and detection of expression in CD4+CD25+ regulatory T cells

Abstract

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 ...

Interplay of Regulatory T Cell and Th17 Cells during Infectious Diseases in Humans and Animals

“…In cats, an alternative splice variant of Foxp3 lacking exon 2 also exists, an observation similarly recorded for human Foxp3 (99). Surprisingly, when wild type and the variant lacking in exon 2 were expressed in a cell, the suppressive activity was enhanced, as compared a single version expressing cells suggesting a critical role of exon 2 in activity of Treg (111). Cytokines shown to promote Th17 responses in cats are IL-1β, IL-6, TGF-β, and IL-21 (113).…”

Flagellin suppresses experimental asthma by generating regulatory dendritic cells and T cells

“…The treatment of lymphocytes with flagellin has been reported to induce Treg cells in cats. 46 Human Treg cells have been reported to express TLR5, with flagellin treatment enhancing the function of Treg cells. 47 However, in our system the direct action of flagellin on T cells did not play a major role because DC adoptive transfer conferred the inhibitory effect almost completely.…”

Feline immunodeficiency virus latency

“…FIV has been shown to preferentially infect CD4 + CD25+ activated/regulatory T-cells (Tregs) [80], which correlates with both surface CXCR4 expression and binding of cellular transcription factors to the FIV promoter [81]. Feline Tregs have since been characterized using FoxP3 [82,83], so these studies are merely suggestive of infection in that subset. Importantly, the FIV receptor CD134 (OX40) is constitutively expressed on Tregs [84], lending support to this hypothesis.…”