Simultaneous in vivo monitoring of regulatory and effector T lymphocytes using secreted Gaussia luciferase, Firefly luciferase, and secreted alkaline phosphatase.

Regulatory T cells (Tregs) are amongst the most widely studied cells in a variety of immune-mediated conditions, including transplantation and Graft Versus Host Disease (GVHD), cancer and autoimmunity; indeed, there is great interest in the tolerogenic potential of Treg-based therapy. Consequently, the need to establish the mechanisms that determine Treg survival and longevity, in addition to developing new tools to monitor these parameters, is paramount. Using both a mouse model of GVHD and a mouse model of Type 1 Diabetes (T1D), we describe herein a dual reporter system based on Gluc and multiplexed with SEAP and non-secreted Firefly luciferase (Fluc), which permits simultaneous imaging and noninvasive tracking of two different T-cell populations (CD4(+)CD25(+) Tregs and CD4(+)CD25(-) Tcon cells) in vivo by transducing the cells with different lentiviruses bearing distinct color signatures. This new technology promises to overcome the limitations of the conventional methods currently available to study lymphocyte survival in vivo. Furthermore, this novel technique has applications not only in autoimmunity and alloimmunity, but also in the wider field of immunology.