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Supplementary Figures 1-8

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Cancer-associated fibroblasts (CAFs) are generally associated with poor clinical outcome. CAFs support tumor growth in a variety of ways and can suppress antitumor immunity and response to immunotherapy. However, a precise understanding of CAF contributions to tumor growth and therapeutic response is lacking. Discrepancies in this field of study may stem from heterogeneity in the composition and function of fibroblasts in the tumor microenvironment. Furthermore, it remains unclear whether CAFs directly interact with and suppress T cells. Here, mouse and human breast tumors were used to examine stromal cells expressing fibroblast activation protein (FAP), a surface marker for CAFs. Two discrete populations of FAP+ mesenchymal cells were identified on the basis of podoplanin (PDPN) expression: a FAP+PDPN+ population of CAFs and a FAP+PDPN− population of cancer-associated pericytes (CAPs). Although both subsets expressed extracellular matrix molecules, the CAF transcriptome was enriched in genes associated with TGFβ signaling and fibrosis compared with CAPs. In addition, CAFs were enriched at the outer edge of the tumor, in close contact with T cells, whereas CAPs were localized around vessels. Finally, FAP+PDPN+ CAFs suppressed the proliferation of T cells in a nitric oxide–dependent manner, whereas FAP+PDPN− pericytes were not immunosuppressive. Collectively, these findings demonstrate that breast tumors contain multiple populations of FAP-expressing stromal cells of dichotomous function, phenotype, and location.

The SARS-CoV-2 coronavirus is the causal agent of the current global pandemic. SARS-CoV-2 belongs to an order, Nidovirales, with very large RNA genomes. It is proposed that the fidelity of coronavirus (CoV) genome replication is aided by an RNA nuclease complex, comprising the non-structural proteins 14 and 10 (nsp14–nsp10), an attractive target for antiviral inhibition. Our results validate reports that the SARS-CoV-2 nsp14–nsp10 complex has RNase activity. Detailed functional characterization reveals nsp14–nsp10 is a versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3′-terminus. Consistent with a role in maintaining viral genome integrity during replication, we find that nsp14–nsp10 activity is enhanced by the viral RNA-dependent RNA polymerase complex (RdRp) consisting of nsp12–nsp7–nsp8 (nsp12–7–8) and demonstrate that this stimulation is mediated by nsp8. We propose that the role of nsp14–nsp10 in maintaining replication fidelity goes beyond classical proofreading by purging the nascent replicating RNA strand of a range of potentially replication-terminating aberrations. Using our developed assays, we identify drug and drug-like molecules that inhibit nsp14–nsp10, including the known SARS-CoV-2 major protease (Mpro) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for multifunctional inhibitors in COVID-19 treatment.

Cancer-associated fibroblasts (CAFs) are generally associated with poor clinical outcome. CAFs support tumor growth in a variety of ways and can suppress antitumor immunity and response to immunotherapy. However, a precise understanding of CAF contributions to tumor growth and therapeutic response is lacking. Discrepancies in this field of study may stem from heterogeneity in the composition and function of fibroblasts in the tumor microenvironment. Furthermore, it remains unclear whether CAFs directly interact with and suppress T cells. Here, mouse and human breast tumors were used to examine stromal cells expressing fibroblast activation protein (FAP), a surface marker for CAFs. Two discrete populations of FAP+ mesenchymal cells were identified on the basis of podoplanin (PDPN) expression: a FAP+PDPN+ population of CAFs and a FAP+PDPN− population of cancer-associated pericytes (CAPs). Although both subsets expressed extracellular matrix molecules, the CAF transcriptome was enriched in genes associated with TGFβ signaling and fibrosis compared with CAPs. In addition, CAFs were enriched at the outer edge of the tumor, in close contact with T cells, whereas CAPs were localized around vessels. Finally, FAP+PDPN+ CAFs suppressed the proliferation of T cells in a nitric oxide–dependent manner, whereas FAP+PDPN− pericytes were not immunosuppressive. Collectively, these findings demonstrate that breast tumors contain multiple populations of FAP-expressing stromal cells of dichotomous function, phenotype, and location.

Rana et al. demonstrate that white adipose tissue-resident multipotent stromal cells (WAT-MSC) support IL-33– and LFA-1/ICAM-1–mediated proliferation and type-2 cytokine expression by ILC2. Inversely, ILC2-derived IL-4 and IL-13 promote WAT-MSC eotaxin production and eosinophil recruitment, further maintaining type-2 immune homeostasis., Group-2 innate lymphoid cells (ILC2), type-2 cytokines, and eosinophils have all been implicated in sustaining adipose tissue homeostasis. However, the interplay between the stroma and adipose-resident immune cells is less well understood. We identify that white adipose tissue–resident multipotent stromal cells (WAT-MSCs) can act as a reservoir for IL-33, especially after cell stress, but also provide additional signals for sustaining ILC2. Indeed, we demonstrate that WAT-MSCs also support ICAM-1–mediated proliferation and activation of LFA-1–expressing ILC2s. Consequently, ILC2-derived IL-4 and IL-13 feed back to induce eotaxin secretion from WAT-MSCs, supporting eosinophil recruitment. Thus, MSCs provide a niche for multifaceted dialogue with ILC2 to sustain a type-2 immune environment in WAT., Graphical Abstract

The SARS-CoV-2 coronavirus (CoV) causes COVID-19, a current global pandemic. SARS-CoV-2 belongs to an order of Nidovirales with very large RNA genomes. It is proposed that the fidelity of CoV genome replication is aided by an RNA nuclease complex, formed of non-structural proteins 14 and 10 (nsp14-nsp10), an attractive target for antiviral inhibition. Here, we confirm that the SARS-CoV-2 nsp14-nsp10 complex is an RNase. Detailed functional characterisation reveals nsp14-nsp10 is a highly versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3’-terminus. We propose that the role of nsp14-nsp10 in maintaining replication fidelity goes beyond classical proofreading and purges the nascent replicating RNA strand of a range of potentially replication terminating aberrations. Using the developed assays, we identify a series of drug and drug-like molecules that potently inhibit nsp14-nsp10, including the known Sars-Cov-2 major protease (Mpro) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for bifunctional inhibitors in the treatment of COVID-19.

SummaryTissue homeostasis requires a tight control of stem cells to maintain quiescence in normal conditions, and ensure a balance between progenitor cell production and the need to preserve a stem cell pool in repair conditions. Using ex-vivo and in-vivo genetic approaches, we provide evidence that primary cilium-mediated repressive Hedgehog (Hh) signalling is required to maintain skeletal muscle stem cells (MuSCs) in a quiescent state. De-repression and further activation of Hh signalling initiates MuSC entry and progression through the cell cycle, and controls self-renewal to ensure efficient repair of injured muscles. We propose a model whereby disassembly of primary cilia upon MuSC activation induces a switch in Hh signalling from a repressive to active state that controls exit from quiescence. Positive Hh response in bi-potential muscle progenitor cells regulates also cell cycle progression and drives MuSC self-renewal. These findings identify Hh signalling as a major regulator of MuSC activity.HighlightsCilia-containing quiescent MuSCs are Hh signalling suppressedMuSC activation coincides with a switch to active Hh signallingSmomutation delays cell cycle entry and progression, and causes impaired self-renewalPtch1mutation promotes exit from quiescence, rapid cell cycle and increased self-renewalGraphical abstract

Summary Regeneration of vertebrate skeletal muscles requires satellite cells, a population of stem cells that are quiescent in normal conditions and divide, differentiate, and self-renew upon activation triggered by exercise, injury, and degenerative diseases. Satellite cell self-renewal is essential for long-term tissue homeostasis, and previous work has identified a number of external cues that control this process. However, little is known of the possible intrinsic control mechanisms of satellite cell self-renewal. Here, we show that quiescent satellite cells harbor a primary cilium, which is rapidly disassembled upon entry into the cell cycle. Contrasting with a commonly accepted belief, cilia reassembly does not occur uniformly in cells exiting the cell cycle. We found that primary cilia reassemble preferentially in cells committed to self-renew, and disruption of cilia reassembly causes a specific deficit in self-renewing satellite cells. These observations indicate that primary cilia provide an intrinsic cue essential for satellite cell self-renewal., Highlights • In skeletal muscles, primary cilia are associated with quiescent satellite cells • Primary cilia disassemble when satellite cells enter the cell cycle • Primary cilia reassemble preferentially in self-renewing satellite cells • Disruption of primary cilia reassembly impairs satellite cell self-renewal, In this article, Borycki and colleagues investigate the role of primary cilia in skeletal muscle satellite stem cells. They report on the specific association of primary cilia with quiescent and self-renewing satellite cells during adult myogenesis. Blocking cilia reassembly following cell division impairs satellite cell self-renewal, demonstrating that primary cilia are intrinsic cues required for stem cell self-renewal.

The microenvironment of lymphoid organs can aid healthy immune function through provision of both structural and molecular support. In mice, fibroblastic reticular cells (FRCs) create an essential T-cell support structure within lymph nodes, while human FRCs are largely unstudied. Here, we show that FRCs create a regulatory checkpoint in human peripheral T-cell activation through 4 mechanisms simultaneously utilised. Human tonsil and lymph node-derived FRCs constrained the proliferation of both naïve and pre-activated T cells, skewing their differentiation away from a central memory T-cell phenotype. FRCs acted unilaterally without requiring T-cell feedback, imposing suppression via indoleamine-2,3-dioxygenase, adenosine 2A Receptor, prostaglandin E2, and transforming growth factor beta receptor (TGFβR). Each mechanistic pathway was druggable, and a cocktail of inhibitors, targeting all 4 mechanisms, entirely reversed the suppressive effect of FRCs. T cells were not permanently anergised by FRCs, and studies using chimeric antigen receptor (CAR) T cells showed that immunotherapeutic T cells retained effector functions in the presence of FRCs. Since mice were not suitable as a proof-of-concept model, we instead developed a novel human tissue-based in situ assay. Human T cells stimulated using standard methods within fresh tonsil slices did not proliferate except in the presence of inhibitors described above. Collectively, we define a 4-part molecular mechanism by which FRCs regulate the T-cell response to strongly activating events in secondary lymphoid organs while permitting activated and CAR T cells to utilise effector functions. Our results define 4 feasible strategies, used alone or in combinations, to boost primary T-cell responses to infection or cancer by pharmacologically targeting FRCs.

The HIV integrase-binding domain (IBD) of lens epithelium-derived growth factor has been cloned, purified and crystallized, and its structure has been solved. IBD forms an unusual domain-swapped dimer that assembles into octamers in the crystal., Lens epithelium-derived growth factor (LEDGF)/p75 is the dominant binding partner of HIV-1 integrase in human cells. The crystal structure of the HIV integrase-binding domain (IBD) of LEDGF has been determined in the absence of ligand. IBD was overexpressed in Escherichia coli, purified and crystallized by sitting-drop vapour diffusion. X-ray diffraction data were collected at Diamond Light Source to a resolution of 2.05 Å. The crystals belonged to space group P21, with eight polypeptide chains in the asymmetric unit arranged as an unusual octamer composed of four domain-swapped IBD dimers. IBD exists as a mixture of monomers and dimers in concentrated solutions, but the dimers are unlikely to be biologically relevant.

Preventing the protein‐protein interaction of the cellular chromatin binding protein LEDGF and HIV integrase is an important strategy for alternative anti‐viral treatment for AIDS. While potent small molecules capable of performing this function have been difficult to develop, intracellular antibody fragments can readily be designed to perform with high affinity binding in vivo. We have isolated a single VH domain that binds to LEDGF and blocks the binding of HIV integrase to LEDGF. The crystal structure of the LEDGF‐VH complex reveals a remarkably similar region of the binding for the single domain antibody fragment and HIV integrase on LEDGF as a mimic of integrase. In cell assays, the intracellular domain antibody inhibits virus capsid p24 protein production after HIV virus infection into CD4‐expressing target T cells. Therefore, blocking the HIV interaction surface of cellular LEDGF with the potent antibody fragment macromolecule is a way to prevent HIV spread in infected patients. This antibody fragment is available for intracellular vaccination approaches in combating HIV‐based disease by inhibiting retroviral incorporation into the genome of infected cells in HIV‐positive patients.

Fat-associated lymphoid clusters (FALCs) are atypical lymphoid tissues that were originally identified in mouse and human mesenteries due to that they contain a high number of type 2 innate lymphoid cells/nuocytes/natural helper cells. FALCs are located on adipose tissues in mucosal surfaces such as the mediastinum, pericardium, and gonadal fat. Importantly, these clusters contain B1, B2 and T lymphocytes as well as myeloid and other innate immune cell populations. The developmental cues of FALC formation have started to emerge, showing that these clusters depend on a different set of molecules and cells than secondary lymphoid tissues for their formation. Here, we review the current knowledge on FALC formation, and we compare FALCs and omental milky spots and their responses to inflammation.