R-spondin (RSpo) proteins amplify Wnt signaling and stimulate regeneration in a variety of tissues. to repair tissue in a tissue-specific manner, tissue-targeted RSPO mimetic molecules are desired. Here, we mutated RSPO (RSPO2 F105R/F109A) to eliminate LGR binding while preserving ZNRF3/RNF43 binding and targeted the mutated RSPO to a liver specific receptor, ASGR1. The resulting bi-specific molecule (αASGR1-RSPO2-RA) enhanced Wnt signaling effectively in vitro, and its activity was limited to ASGR1 expressing cells. Systemic administration of αASGR1-RSPO2-RA in mice specifically upregulated Wnt target genes and stimulated cell proliferation in liver but not intestine (which is more responsive to non-targeted RSPO2) in healthy mice, and improved liver function in diseased mice. These results not only suggest that a tissue-specific RSPO mimetic protein can stimulate regeneration in a cell-specific manner, but also provide a blueprint of how a tissue-specific molecule might be constructed for applications in a broader context. The Wnt ("Wingless-related integration site" or "Wingless and Int-1" or "Wingless-Int") signaling pathway plays a key role in the development, homeostasis and regeneration of many essential organs and tissues 1. Timely activation, modulation, or enhancement of Wnt signaling holds potential for the treatment of various degenerative diseases and pathologies in which tissue regeneration could confer a therapeutic benefit. R-spondins 1-4 (RSPO1-4) are a family of ligands that amplify Wnt signals through a receptor complex containing the zinc and ring finger 3 (ZNRF3) and ring finger protein 43 (RNF43) proteins and the coreceptor leucine-rich repeat-containing G-protein coupled receptors 4-6 (LGR4-6) 2-4. RSPOs contain two furin (Fu) repeats, Fu1 and Fu2, which in combination are sufficient to recapitulate Wnt signaling enhancing activity 5. Fu1 primarily interacts with ZNRF3/RNF43 and Fu2 interacts with LGR4-6 5-7. ZNRF3 and RNF43 are membranebound E3 ligases that specifically target Wnt receptors (FZD1-10 and LRP5 or LRP6) for degradation 8,9. Binding of RSPOs to ZNRF3/RNF43 and LGR4-6 causes clearance or sequestration of the ternary complex, which stabilizes Wnt receptors and amplifies Wnt signaling. In addition, R-spondins might work through mechanisms that are independent of LGRs 10,11. RSPOs may be beneficial in adult tissue repair 12,13 , particularly in situations where the expression of endogenous Wnt ligands is upregulated but signaling (presumably limited by receptor stability) is insufficient to overcome tissue damage. In liver, RSPO function is important for metabolic zonation and for hepatocyte proliferation and regeneration 12,14. Therefore, RSPO may provide therapeutic benefit for various acute and chronic liver injury and diseases. One major challenge to exploring RSPO for tissue repair and regeneration is limiting RSPO effects to specific tissue of interest such as liver, as LGR4-6 and ZNRF3/RNF43 are widely expressed in various tissues. The mucosa of the gastrointestinal tract h...

Regulatory T cells (Treg) are conventionally viewed to suppress endogenous and therapy-induced anti-tumor immunity; however, their role in modulating responses to immune checkpoint blockade (ICB) is unclear. In this study, we integrated single-cell RNAseq/TCRseq of >73,000 tumor-infiltrating Treg (TIL-Treg) from anti-PD-1-treated and treatment naive non-small cell lung cancers (NSCLC) with single cell analysis of tumor-associated antigen (TAA)-specific Treg derived from a murine tumor model. We identified 10 subsets of human TIL-Treg, most of which have high concordance with murine TIL-Treg subsets. Notably, one subset selectively expresses high levels of OX40 and GITR, whose engangement by cognate ligand mediated proliferative programs and NF-kB activation, as well as multiple genes involved in Treg suppression, in particular LAG3. Functionally, the OX40hiGITRhi subset in the most highly suppressive ex vivo and Treg expression of OX40, GITR and LAG3, correlated with resistance to PD-1 blockade. Surprisingly, in the murine tumor model, we found that virtually all TIL-Treg expressing T cell receptors that are specific for TAA fully develop a distinct Th1-like signature over a two-week period after entry into the tumor, down-regulating FoxP3 and up-regulating expression of TBX21 (Tbet), IFNy and certain pro-inflammatory granzymes. Application of a gene score from the murine TAA-specific Th1-like Treg subset to the human single-cell dataset revealed a highly analogous subcluster that was enriched in anti-PD-1 responding tumors. These findings demonstrate that TIL-Treg partition into multiple distinct transcriptionally-defined subsets with potentially opposing effects on ICB-induced anti-tumor immunity and suggest that TAA-specific TIL-Treg may positively contribute to anti-tumor responses.

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