Regulatory CD4 CD8 Double Negative T Cells

Peripheral αβTCR⁺ CD3⁺ CD4^– CD8^– NK1.1/CD56^– double-negative (DN) Treg cells are a relatively rare subset of regulatory cells found in both humans and mice, typically comprising less than 5% of the total peripheral T-cell pool. Numerous studies have shown that DN Tregs can inhibit CD4⁺ and CD8⁺ T-cell responses in vitro and in vivo using a variety of model systems [Zhang et al., Nature Medicine 6:782, 2000; Young et al., Blood 100:3408, 2002; Ford et al., Experimental Medicine 196:261, 2002; Young et al., Journal of Immunology 171:134, 2003; Ford et al., European Journal of Immunology 37:2234, 2007; Zhang et al., Blood 109:4071, 2007; Fischer et al., Blood 105:2828, 2005]. This chapter describes published methods for the phenotypic identification of DN Tregs, their isolation from secondary lymphoid organs of mice or human peripheral blood, activation and expansion, and assays for their ability to suppress T-cell proliferation, induce apoptosis, and promote tolerance to allografts in vivo.