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.
Key wordsDouble-negative T cells Regulatory cells Transplant tolerance Autoimmunity Isolation Activation/expansion Suppression Apoptosis
The authors would like to thank Ramesh Vanama and Betty Joe for technical assistance. E.Y.K and S.C.J. are recipients of Terry Fox Foundation Research Fellowships from the National Cancer Institute of Canada. S.C.J. was also supported by the Clinician-Scientist Training Program, Department of Medicine, University of Toronto. This work was supported by Canadian Institutes of Health Research grant # 14431 and CCS grant # 17157 to LZ.
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