title: Adenoviral transduction of naive CD4 T cells to study Treg differentiation.
abstract: Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGFβ and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown. Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44(low), CD62L(high)) and resting (CD25(-), CD69(-)) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.
authors: Institute for Molecular Immunology, Helmholtz Zentrum München.
authors: Sebastian C
authors: SC
authors: Warth
authors: Vigo
authors: V
authors: Heissmeyer
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000502:physiology* / Q000821:virology; D002454:Cell Differentiation / Q000235:genetics; D018014:Gene Transfer Techniques; D057809:HEK293 Cells; D006801:Humans; D050378:T-Lymphocytes, Regulatory / Q000166:cytology / Q000502:physiology* / Q000821:virology; D014161:Transduction, Genetic
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