Although microRNAs (miRNAs) function in the control of embryonic stem cell (ESC) pluripotency, a systems-level understanding is still being developed. Through the analysis of progressive Argonaute (Ago)-miRNA depletion and rescue, including stable Ago knockout mouse ESCs, we uncover transforming growth factor beta (TGF-β) pathway activation as a direct and early response to ESC miRNA reduction. Mechanistically, we link the derepression of weaker miRNA targets, including TGF-β receptor 1 (Tgfbr1), to the sensitive TGF-β pathway activation. In contrast, stronger miRNA targets impart a more robust repression, which dampens concurrent transcriptional activation. We verify such dampened induction for TGF-β antagonist Lefty. We find that TGF-β pathway activation contributes to the G1 cell-cycle accumulation of miRNA-deficient ESCs. We propose that miRNA target affinity is a determinant of the temporal response to miRNA changes, which enables the coordination of gene network responses.

Synthetic receptor signalling has the potential to endow adoptively transferred T cells with new functions that overcome major barriers in the treatment of solid tumours, including the need for conditioning chemotherapy^1,2. Here we designed chimeric receptors that have an orthogonal IL-2 receptor extracellular domain (ECD) fused with the intracellular domain (ICD) of receptors for common γ-chain (γ_c) cytokines IL-4, IL-7, IL-9 and IL-21 such that the orthogonal IL-2 cytokine elicits the corresponding γ_c cytokine signal. Of these, T cells that signal through the chimeric orthogonal IL-2Rβ-ECD-IL-9R-ICD (o9R) are distinguished by the concomitant activation of STAT1, STAT3 and STAT5 and assume characteristics of stem cell memory and effector T cells. Compared to o2R T cells, o9R T cells have superior anti-tumour efficacy in two recalcitrant syngeneic mouse solid tumour models of melanoma and pancreatic cancer and are effective even in the absence of conditioning lymphodepletion. Therefore, by repurposing IL-9R signalling using a chimeric orthogonal cytokine receptor, T cells gain new functions, and this results in improved anti-tumour activity for hard-to-treat solid tumours.

Defects in tumor-intrinsic interferon (IFN) signaling result in failure of immune checkpoint blockade (ICB) against cancer, but these tumors may still maintain sensitivity to T cell-based adoptive cell therapy (ACT). We generated models of IFN signaling defects in B16 murine melanoma observed in patients with acquired resistance to ICB. Tumors lacking Jak1 or Jak2 did not respond to ICB, whereas ACT was effective against Jak2 ^KO tumors, but not Jak1 ^KO tumors, where both type I and II tumor IFN signaling were defective. This was a direct result of low baseline class I major histocompatibility complex (MHC I) expression in B16 and the dependency of MHC I expression on either type I or type II IFN signaling. We used genetic and pharmacologic approaches to uncouple this dependency and restore MHC I expression. Through independent mechanisms, overexpression of NLRC5 (nucleotide-binding oligomerization domain-like receptor family caspase recruitment domain containing 5) and intratumoral delivery of BO-112, a potent nanoplexed version of polyinosinic:polycytidylic acid (poly I:C), each restored the efficacy of ACT against B16-Jak1 ^KO tumors. BO-112 activated double-stranded RNA (dsRNA) sensing (via protein kinase R and Toll-like receptor 3) and induced MHC I expression via nuclear factor κB, independent of both IFN signaling and NLRC5. In summary, we demonstrated that in the absence of tumor IFN signaling, MHC I expression is essential and sufficient for the efficacy of ACT. For tumors lacking MHC I expression due to deficient IFN signaling, activation of dsRNA sensors by BO-112 affords an alternative approach to restore the efficacy of ACT.