Effects of in vivo injection of anti-chicken CD25 monoclonal antibody on regulatory T cell depletion and CD4 + CD25 À T cell properties in chickens Revathi Shanmugasundaram, Ramesh K. Selvaraj ⇑ Department of Animal Sciences, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH 44691, United States article info Article history: Received 16 August 2011 Revised 14 September 2011 Accepted 29 September 2011 Available online 6 October 2011 Keywords: Tregs T cells IL-2 IFNc abstract Regulatory T cells (Tregs) are defined as CD4 + CD25 + cells in chickens. This study examined the effects of an anti-chicken CD25 monoclonal antibody injection (0.5 mg/bird) on in vivo depletion of Tregs and the properties of CD4 + CD25 À cells in Treg-depleted birds. The CD4 + CD25 + cell percentage in the blood was lower at 8 d post injection than at 0 d. Anti-CD25-mediated CD4 + CD25 + cell depletion in blood was max- imum at 12 d post injection. The anti-CD25 antibody injection depleted CD4 + CD25 + cells in the spleen and cecal tonsils, but not in the thymus, at 12 d post antibody injection. CD4 + CD25 À cells from the spleen and cecal tonsils of birds injected with the anti-chicken CD25 antibody had higher proliferation and higher IL-2 and IFNc mRNA amounts than the controls at 12 d post injection. At 20 d post injection, CD4 + CD25 + cell percentages in the blood, spleen and thymus were comparable to that of the 0 d post injection. It could be concluded that anti-chicken CD25 injection temporarily depleted Treg population and increased and IL-2 and IFNc mRNA amounts in CD4 + CD25 À cells at 12 d post injection. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction CD4 + CD25 + FoxP3 + cells are described as natural regulatory T cells (Tregs) in several species (Adeegbe et al., 2006; Ouabed et al., 2008; Porter et al., 2007; Tiemessen et al., 2007). Tregs are a subset of T cells that specialize in immune suppression. Though CD25 was initially considered a Treg marker with no role in Treg function, later reports showed that CD25 is essential for the gener- ation, peripheral expansion, and maintenance of Tregs (Setiady et al., 2010). We previously described CD4 + CD25 + cells as Tregs in chickens (Shanmugasundaram and Selvaraj, 2011). Chicken Tregs produce high mRNA amounts of suppressive cytokines like IL-10 and TGF-b and do not produce IL-2 mRNA. In vitro suppres- sive properties of chicken Tregs can be abrogated by supplemental IL-2 (Shanmugasundaram and Selvaraj, 2011). An anti-CD25 antibody injection depletes Tregs in vivo in mice (Kottke et al., 2008; Larocque et al., 2010; Tenorio et al., 2010) and cats (Smithberg et al., 2008). In mice, an anti-CD25 injection depletes approximately 70% of Tregs with the remaining Tregs expressing low or no CD25. The anti-CD25 monoclonal antibody depletes Tregs through Fcc receptor-III-mediated phagocytosis. Natural killer cells and macrophages are major immune cells that express Fcc receptor III and they deplete Tregs through the anti- body-dependent-cellular cytotoxicity and antibody-mediated-cel- lular-phagocytosis pathway (Setiady et al., 2010). An anti-CD25 antibody injection has also been reported to induce functional inactivation of Tregs instead of their depletion (Kohm et al., 2006). Tregs suppress host T cell immune response, and depletion of Tregs using anti-CD25 neutralizing antibodies relieves the in vivo suppression of antiviral immune responses and contributes to fas- ter viral clearance. Depletion of Tregs enhances activity of natural killer cells and lymphokine-activated killer cells and enhances pro- duction of IFNc (Kottke et al., 2008). Anti-CD25-mediated Treg depletion increases the IFNc-producing cell percentage (Marin et al., 2010) and increases proliferation of T cells in mice (Liu et al., 2005). In vivo depletion of Tregs has been applied to study the role of Tregs in autoimmune diseases (Larocque et al., 2010), allergies (Schouten et al., 2011), tumor incidences (Kottke et al., 2008), Toxoplasma gondii infections (Tenorio et al., 2010), and feline immunodeficiency virus infections (Smithberg et al., 2008). We previously reported the production of an anti-chicken CD25 monoclonal antibody (Shanmugasundaram and Selvaraj, 2011). Two experiments were conducted to identify the effect of anti- chicken CD25 antibody injection in chickens. The first experiment studied the effect of the anti-chicken CD25 monoclonal injection on CD4 + CD25 + cell percentage in the blood at 0, 4, 8, 12, 16 and 20 d post-antibody injection. The second experiment studied the effects of the anti-chicken CD25 monoclonal injection on CD4 + CD25 + percentages in internal organs and properties of CD4 + CD25 À cells from internal organs at 12 and 20 d post-antibody injection. 0145-305X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2011.09.015 Abbreviation: Tregs, regulatory T cells. ⇑ Corresponding author. Tel.: +1 330 2633793; fax: +1 330 2633949. E-mail addresses: shanmugasundaram.2@osu.edu (R. Shanmugasundaram), selvaraj.7@osu.edu (R.K. Selvaraj). Developmental and Comparative Immunology 36 (2012) 578–583 Contents lists available at SciVerse ScienceDirect Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/dci