The genetic targeting of gene products to T cells, but not their thymic precursors, is a lingering challenge in animal transgenesis and stem cell therapies. We therefore sought to modulate expression from a constitutive promoter by subjecting it to endogenous miR-181a regulation. We show here that lentiviral vector–encoded antigen receptor expression that is regulated by miR-181a enabled robust segregation between developing thymocytes and mature T cells. miR-181a–targeted transcripts were maximally repressed in late DN and DP cells (Figure , D and E), as expected from our miR-181a expression studies (Figure C) and the previous studies of Neilson et al. (33
) and Li et al. (32
). Our functional analyses show that antigen receptor expression was fully repressed in DP cells (Figure , D and E), a developmental stage in which miR-181a, which downregulates phosphatase expression, renders DP thymocytes exceptionally sensitive to TCR signaling (32
). The profound downregulation of the CAR 19z1 demonstrated that miR-181–mediated silencing of a relatively highly expressed transcript driven by the hEF1a promoter was sufficient to functionally prevent expression at the stages of positive and negative selection. Our studies with the D10 TCR further compound that antigen receptor is profoundly suppressed in all stages of thymocyte development in which negative selection occurs (including DN, DP, and the DP-to-SP transition; ref. 38
), averting the deletion of D10 TCR+
T cells in B10.D2 recipient mice (Figure and ref. 35
Transgene expression was readily detectable in peripheral T cells, as found with the 19z1 CAR (Figures and ) and the D10 TCR (Figure ). Expression was reinstated in peripheral T cells, consistent with the major decrease of miR-181a levels in post-thymic T cells (Figure B). Expression of both antigen receptors was functional, enabling mice harboring the 19z1 receptor to kill CD19+ tumors in vitro (Figure D) and resist challenge with CD19+ tumor cells (Figure C). Similarly, B10.D2 mice engrafted with the GD102 vector acquired the ability to respond to the cognate peptide, which was precluded in mice harboring the GD100 vector (Figure D). These results demonstrate the ability of the miR-181a MRE to developmentally regulate receptor expression within the T cell lineage, preserving receptor functionality.
Our findings also underscore the importance of MRE copy number to achieving desirable levels of transgene expression. Whereas vector-encoded transcripts bearing either 2 or 4 miR-181a MRE copies under the transcriptional control of either a human phosphoglycerate kinase (hPGK; vectors 19z12
) or a hEF1a (vector G19z14×181a
) promoter were similarly downregulated in thymocytes, the presence of either 2 or 4 copies yielded different outcomes in peripheral T cells (Figure ). Expression of 19z1 was lower in T cells expressing transcripts encoding 4 MRE copies, resulting in diminished cytolytic activity. This functional difference is consistent with the known importance of antigen receptor levels for T cell responsiveness. Higher expression of TCR chains may also be important for the transgenic TCR to successfully compete with endogenous TCR chains for rate-limiting amounts of CD3 to achieve cell surface expression (39
). miR-181a expression did not increase upon T cell activation (Figure B), whereas cell surface expression of the 19z1 (Figure B) and D10 (Figure C) receptors increased within 24 hours of mitogen activation, consistent with promoter activation (41
MRE-mediated repression may also vary in developing thymocytes. Whereas 19z1 expression was profoundly repressed in SP cells (Figure , D and E), the receptor was detected at very low levels in 2 of 10 mice, which also expressed low 19z1 levels in DP-stage cells (data not shown). The latter expressed reduced levels of both CD4 and CD8 (DPdull
), a phenotype corresponding to the immediate progeny of positive selection (42
), which possess reduced sensitivity to TCR signaling, potentially owing to reduced levels of expression of miR-181a (43
). Our in vitro and in vivo findings support the notion of MRE dose dependence of knockdown efficiency, corroborating data from transient reporter assays (44
) and stably integrated miRNA target sites in cell lines (29
MRE-regulated transgene expression is poised to find applications in basic biology as well as in therapeutic stem cell engineering. Posttranscriptional control superimposed onto classic transcriptional regulation is highly valuable to correct undesirable expression patterns or fine-tune developmentally regulated or inducible gene expression. Preventing transgene expression in professional APCs may attenuate the immunogenicity of foreign antigens (28
). As we showed here, miR-181–regulated transgene expression was effective in allowing temporal transgene regulation throughout lymphopoiesis, which to our knowledge was previously unattainable through pol II–mediated transcriptional regulation.
A major challenge of cancer immunotherapy is the generation of large numbers of T cells with the desired antitumor specificities that can persist long-term in vivo. Delivery of tumor-specific TCR genes into hematopoietic stem cells constitutes a recent promising strategy in this direction (7
). Antigen-specific T cells may also be provided by infusion of lymphoid progenitors that are educated and tolerized according to the recipient’s genetic background (45
). A predictable problem of this approach is negative selection of T lymphocytes recognizing self tumor antigens (46
) as a consequence of clonal deletion of developing thymocytes expressing autoreactive receptors. miR-181a regulation of transgenic TCRs targeted to either the hematopoietic stem cell or the lymphoid progenitor compartment may thus be very useful to circumvent negative selection of tumor-reactive T lymphocytes and afford durable immune surveillance.