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

Abstract

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.

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

“…Studies have reported that TGF-␤ is expressed on the surface of activated Treg cells and plays a role in contact-dependent Tregmediated suppression (3,15,17,18). Furthermore, we have demonstrated that GARP forms a complex with membrane-bound TGF-␤ (mTGF-␤) on activated Treg cells (18).…”

“…Furthermore, we have demonstrated that GARP forms a complex with membrane-bound TGF-␤ (mTGF-␤) on activated Treg cells (18). As FIV infection induced GARP expression on CD4…”

“…More recently, we have demonstrated that activated feline Treg cells also display a surface marker, GARP (glycoprotein A repetitions predominant), that anchors mTGF-␤ on the surface of Treg suppressor cells (18). As Treg cells from FIV-infected cats display increased expression of mTGF-␤, GARP, and FoxP3, and express suppressor function (18), we asked if these characteristics could be replicated in Treg cells infected in vitro and thus establish a role for direct Treg cell activation by FIV infection.…”

“…To determine if virus infection alone was capable of inducing a Treg activation phenotype, FACS-purified CD4 ϩ CD25 ϩ cells from SPF cats were untreated, infected with FIV, or treated with UV-FIV and analyzed at 3 and 6 days postinfection for surface expression of GARP, TGF-␤, and FoxP3 protein by flow cytometry or for mRNA by real-time reverse transcription (RT)-PCR as described previously (18). As a positive control for activation, purified CD4…”

“…Purified CD4 ϩ CD25 ϩ cells were infected with FIV for 3 days and FACS sorted into GARP ϩ and GARP Ϫ populations. RNA was collected and analyzed for FIV gag mRNA load by real-time RT-PCR (18), or DNA was collected and analyzed for FIV provirus load using the same gag primers and protocol (18), except that the provirus load was normalized to the housekeeping gene coding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). While the FIV provirus loads were similar in GARP ϩ and GARP Ϫ cells (Fig.…”