DNA-binding of the Tet-transactivator curtails antigen-induced lymphocyte activation in mice

Abstract

The Tet-On/Off system for conditional transgene expression constitutes state-of-the-art technology to study gene function by facilitating inducible expression in a timed and reversible manner. Several studies documented the suitability and versatility of this system to trace lymphocyte fate and to conditionally express oncogenes or silence tumour suppressor genes in vivo. Here, we show that expression of the tetracycline/doxycycline-controlled Tet-transactivator, while tolerated well during development and in immunologically unchallenged animals, impairs the expansion of antigen-stimulated T and B cells and thereby curtails adaptive immune responses in vivo. Transactivator-mediated cytotoxicity depends on DNA binding, but can be overcome by BCL2 overexpression, suggesting that apoptosis induction upon lymphocyte activation limits cellular and humoral immune responses. Our findings suggest a possible system-intrinsic biological bias of the Tet-On/Off system in vivo that will favour the outgrowth of apoptosis resistant clones, thus possibly confounding data published using such systems.

The Tetracycline-Controlled Transactivator (Tet-On/Off) System in β-Cells Reduces Insulin Expression and Secretion in Mice

“…There is increasing evidence that transgenes believed to be inert (e.g., Cre recombinase, GFP, bacterial transactivators) can have significant effects on cell function and viability ( 20 , 21 ). Based on these concerns, we investigated the impact of Tet-Off system components on glucose metabolism.…”

Expression of a tetracycline-controlled transactivator (Tet-On/Off system) in beta cells reduces insulin expression and secretion in mice

“…It was recently shown that common drugs used to control transgene expression can have off-target effects on mitochondrial and metabolic function 28 . Moreover, there is increasing evidence illustrating that overexpression of transgenes previously believed to be inert (e.g Crerecombinase, GFP, transactivator) can have significant effects on cell function and viability 34,35,[41][42][43] . Based on these concerns, we performed a series of in vivo experiments to investigate impact of the Tet-Off system components on glucose metabolism in males and females over time.…”

“…The improved tTA transactivator (itTA) 32 has been successfully used in combination with the MIP promoter to drive islet transgene expression 33 . However, in light of recent challenges in other models 14,15,31,34,35 , additional studies are required to evaluate the efficiency, specificity, and potential off-target effects of this system for beta cell targeting.…”

Immune Relevant and Immune Deficient Mice: Options and Opportunities in Translational Research

“…458,459 Tetracycline/doxycycline-inducible Tet-Off/Tet-On system Apoptotic response in activated lymphocytes resulting from DNA binding by tTA/rtTA. 464 Classical reporter molecules Increase in the CTL response against transplanted eGFP-expressing leukemia cells in BALB/c mice; IFNγ response to the dominant CTL epitope of Luc, with consequent restricted growth and metastatic activity of the reporter-labelled tumor cells in a mouse model of mammary adenocarcinoma; antigen specific activation of T cells to the reporter gene β-galactosidase, with loss of transgene expression. 465 The "mild" cold stress caused by standard sub-thermoneutral housing temperatures affects immune responses, tumor growth, and other experimental outcomes.…”

“…These findings indicate that a major experimental bias exists in the use of the Tet-On/Off system for lymphocyte targeting as the approach may (1) limit the extent of the adaptive immune reaction and (2) favor the outgrowth of apoptosis-resistant subpopulations of lymphoid cells. 464 Expression of fluorescent or enzymatic reporters driven by gene-specific regulatory elements is used to study in vivo or ex vivo activity and distribution of specific molecular targets or mutant alleles in GEM models. However, an increasing number of studies show that reporters can be highly immunogenic.…”