The discovery of CD4⁺CD25⁺ regulatory T cells (T_regs) in 1995 was accompanied by compelling evidence of their critical role in the maintenance of self-tolerance and prevention of autoimmunity [1]. These cells, characterized by the expression of the transcription factor FoxP3, comprise approximately 5–10 % of CD4⁺ T cells in normal naive mice and are of thymic origin (natural T_regs; nT_regs) [1, 2]. Studies have shown that it is possible to induce T_regs from naïve peripheral CD4⁺CD25⁻ T cells, depending on the stimulus and cytokine milieu [3], and that these adaptive T_regs (iT_regs) also possess the ability to regulate and suppress the immune response, even in the absence of nT_regs [4]. T_reg therapy has emerged as a promising tolerogenic approach for a variety of immune-mediated conditions, including autoimmune disease and Graft-Versus-Host-Disease (GVHD); in transplantation models, T_regs have been shown to actively regulate allogeneic T effector (T_eff) cell responses and promote transplantation tolerance [5, 6]. Although our knowledge of the wide spectrum of their suppressive properties is constantly expanding [7, 8], the homeostatic mechanisms involved in maintaining the population and functions of T_regs have been largely unexplored [9]. Given the increasing number of clinical trials testing the safety and efficacy of T_reg-based immunotherapy [10], the need to establish the mechanisms that determine the survival and longevity of T_regs, and, consequently, the means of monitoring these parameters, is paramount.

Secreted reporters in the blood or serum have been shown to be important tools for the detection, quantitation and noninvasive monitoring of different biological processes, including cell tracking, in experimental models [11–18]; indeed, they have several advantages over conventional in vivo imaging methods for monitoring gene expression [19, 20]. In culture, secreted reporters can be quantified in cell-free conditioned medium without the need for cell lysis, thereby leaving the cells intact and available for confirmation analysis. In vivo, secreted reporters can be easily monitored in animals by withdrawing a few microliters of blood or urine and assaying for their activity, allowing real-time monitoring of in vivo processes; furthermore, bioluminescence imaging can be performed to localize cell activity in vivo, although this method requires expensive instrumentation (cooled CCD camera), is time-consuming, involves frequent anesthesia and repeated systemic substrate injections, and is limited by photon absorption by tissues as a function of depth [20].

The most commonly used serum reporter is the secreted alkaline phosphatase or SEAP [12, 13, 15–17, 21–25]. Normally, alkaline phosphatase is not secreted; however, it has been shown that placental alkaline phosphatase is expressed and released efficiently into the conditioned medium of mammalian cells that have been engineered to express this reporter [21]. The level of SEAP in the conditioned medium is linearly related to the changes in its intracellular mRNA level as well as cell number [21, 22]. It is also possible to quantitatively detect SEAP in the serum of animals. In 1997, Abruzzese et al. used the SEAP serum level to monitor the induction of gene expression followed by gene transfer to the muscles of adult mice [26]. Since then, the SEAP reporter assay has been used as a surrogate serum marker to monitor different in vivo processes including tumor growth and drug efficacy [13], DNA vaccination [27]; promoter and transcription factors activation and inhibition [12, 16, 25]; viral gene transfer [28]; inflammatory events [17]; viral infection and replication [29]; and endoplasmic reticulum stress [23].

We have recently established and reported a novel secreted luciferase reporter from the marine copepod Gaussia princeps known as Gaussia luciferase (Gluc) [11, 14, 18, 30, 31]. We have shown that this reporter is naturally secreted from mammalian cells in an active form and that the levels of Gluc in the conditioned medium correlate linearly with respect to cell number, growth and proliferation [11, 31]. The Gluc reporter system has several advantages over the SEAP assay including a much reduced assay time (1–10 min vs. 1.5–2 h), greatly increased sensitivity (20,000-fold in vitro and 1,000-fold in vivo), and a linear range covering over 5 orders of magnitude of cell number. In vivo, the Gluc reporter assay has been used to localize tumors and to monitor tumor formation and proliferation using bioluminescence imaging [31, 32]. Since Gluc is naturally secreted, we have shown that its level in the blood or urine can be used as a marker to monitor different in vivo biological events such as tumor growth and response to therapy, viral infection and replication as well as the viability of circulating cells [18]. This is a crucial advantage when investigating the behavior of widely circulating cells where a lack of focal accumulation/cell density precludes the use of typical imaging techniques to localize the cells; in these instances, blood or serum levels can be used to reflect cell activity.

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⁺ T_regs and CD4⁺CD25⁻ T_con cells) in vivo by transducing the cells with different lentiviruses bearing distinct color signatures. One further advantage offered by this method of cell identification is that even if the cell loses its normal identifier due to cell plasticity, it is still possible to identify the original lineage of the cell, as it will retain its transduced color signature. This has become particularly important as we become more aware of cell plasticity, e.g., induced T_regs arising from conventional T cell precursors can lose FoxP3 expression and regulatory function over time, becoming effector T cells [33].