Combined PD-L1/TGFß blockade allows expansion and differentiation of stem cell-like CD8 T cells in immune excluded tumors.

TGFß signaling is associated with non-response to immune checkpoint blockade in patients with advanced cancers, particularly in the immune-excluded phenotype. While previous work demonstrates that converting tumors from excluded to inflamed phenotypes requires attenuation of PD-L1 and TGFß signaling, the underlying cellular mechanisms remain unclear. Here, we show that TGFß and PD-L1 restrain intratumoral stem cell-like CD8 T cell (TSCL) expansion and replacement of progenitor-exhausted and dysfunctional CD8 T cells with non-exhausted T effector cells in the EMT6 tumor model in female mice. Upon combined TGFß/PD-L1 blockade IFNγhi CD8 T effector cells show enhanced motility and accumulate in the tumor. Ensuing IFNγ signaling transforms myeloid, stromal, and tumor niches to yield an immune-supportive ecosystem. Blocking IFNγ abolishes the anti-PD-L1/anti-TGFß therapy efficacy. Our data suggest that TGFß works with PD-L1 to prevent TSCL expansion and replacement of exhausted CD8 T cells, thereby maintaining the T cell compartment in a dysfunctional state.

Gremlin 1+ fibroblastic niche maintains dendritic cell homeostasis in lymphoid tissues.

Fibroblastic reticular cells (FRCs) are specialized stromal cells that define tissue architecture and regulate lymphocyte compartmentalization, homeostasis, and innate and adaptive immunity in secondary lymphoid organs (SLOs). In the present study, we used single-cell RNA sequencing (scRNA-seq) of human and mouse lymph nodes (LNs) to identify a subset of T cell-zone FRCs defined by the expression of Gremlin1 (Grem1) in both species. Grem1-CreERT2 knock-in mice enabled localization, multi-omics characterization and genetic depletion of Grem1+ FRCs. Grem1+ FRCs primarily localize at T-B cell junctions of SLOs, neighboring pre-dendritic cells and conventional dendritic cells (cDCs). As such, their depletion resulted in preferential loss and decreased homeostatic proliferation and survival of resident cDCs and compromised T cell immunity. Trajectory analysis of human LN scRNA-seq data revealed expression similarities to murine FRCs, with GREM1+ cells marking the endpoint of both trajectories. These findings illuminate a new Grem1+ fibroblastic niche in LNs that functions to maintain the homeostasis of lymphoid tissue-resident cDCs.

Pro-myogenic small molecules revealed by a chemical screen on primary muscle stem cells.

Satellite cells are the canonical muscle stem cells that regenerate damaged skeletal muscle. Loss of function of these cells has been linked to reduced muscle repair capacity and compromised muscle health in acute muscle injury and congenital neuromuscular diseases. To identify new pathways that can prevent loss of skeletal muscle function or enhance regenerative potential, we established an imaging-based screen capable of identifying small molecules that promote the expansion of freshly isolated satellite cells. We found several classes of receptor tyrosine kinase (RTK) inhibitors that increased freshly isolated satellite cell numbers in vitro. Further exploration of one of these compounds, the RTK inhibitor CEP-701 (also known as lestaurtinib), revealed potent activity on mouse satellite cells both in vitro and in vivo. This expansion potential was not seen upon exposure of proliferating committed myoblasts or non-myogenic fibroblasts to CEP-701. When delivered subcutaneously to acutely injured animals, CEP-701 increased both the total number of satellite cells and the rate of muscle repair, as revealed by an increased cross-sectional area of regenerating fibers. Moreover, freshly isolated satellite cells expanded ex vivo in the presence of CEP-701 displayed enhanced muscle engraftment potential upon in vivo transplantation. We provide compelling evidence that certain RTKs, and in particular RET, regulate satellite cell expansion during muscle regeneration. This study demonstrates the power of small molecule screens of even rare adult stem cell populations for identifying stem cell-targeting compounds with therapeutic potential.

Single-Cell RNA Sequencing Reveals Stromal Evolution into LRRC15+ Myofibroblasts as a Determinant of Patient Response to Cancer Immunotherapy.

With only a fraction of patients responding to cancer immunotherapy, a better understanding of the entire tumor microenvironment is needed. Using single-cell transcriptomics, we chart the fibroblastic landscape during pancreatic ductal adenocarcinoma (PDAC) progression in animal models. We identify a population of carcinoma-associated fibroblasts (CAF) that are programmed by TGFß and express the leucine-rich repeat containing 15 (LRRC15) protein. These LRRC15+ CAFs surround tumor islets and are absent from normal pancreatic tissue. The presence of LRRC15+ CAFs in human patients was confirmed in >80,000 single cells from 22 patients with PDAC as well as by using IHC on samples from 70 patients. Furthermore, immunotherapy clinical trials comprising more than 600 patients across six cancer types revealed elevated levels of the LRRC15+ CAF signature correlated with poor response to anti-PD-L1 therapy. This work has important implications for targeting nonimmune elements of the tumor microenvironment to boost responses of patients with cancer to immune checkpoint blockade therapy. SIGNIFICANCE: This study describes the single-cell landscape of CAFs in pancreatic cancer during in vivo tumor evolution. A TGFß-driven, LRRC15+ CAF lineage is associated with poor outcome in immunotherapy trial data comprising multiple solid-tumor entities and represents a target for combinatorial therapy.This article is highlighted in the In This Issue feature, p. 161.

Adaptive adipose tissue stromal plasticity in response to cold stress and antibody-based metabolic therapy.

In response to environmental and nutrient stress, adipose tissues must establish a new homeostatic state. Here we show that cold exposure of obese mice triggers an adaptive tissue remodeling in visceral adipose tissue (VAT) that involves extracellular matrix deposition, angiogenesis, sympathetic innervation, and adipose tissue browning. Obese VAT is predominated by pro-inflammatory M1 macrophages; cold exposure induces an M1-to-M2 shift in macrophage composition and dramatic changes in macrophage gene expression in both M1 and M2 macrophages. Antibody-mediated CSF1R blocking prevented the cold-induced recruitment of adipose tissue M2 macrophages, suggesting the role of CSF1R signaling in the process. These cold-induced effects in obese VAT are phenocopied by an administration of the FGF21-mimetic antibody, consistent with its action to stimulate sympathetic nerves. Collectively, these studies illuminate adaptive visceral adipose tissue plasticity in obese mice in response to cold stress and antibody-based metabolic therapy.

Tumor Elastography and Its Association with Collagen and the Tumor Microenvironment.

Purpose: The tumor microenvironment presents with altered extracellular matrix (ECM) and stroma composition, which may affect treatment efficacy and contribute to tissue stiffness. Ultrasound (US) elastography can visualize and quantify tissue stiffness noninvasively. However, the contributions of ECM and stromal components to stiffness are poorly understood. We therefore set out to quantify ECM and stroma density and their relation to tumor stiffness.Experimental Design: A modified clinical ultrasound system was used to measure tumor stiffness and perfusion during tumor growth in preclinical tumor models. In vivo measurements were compared with collagen mass spectroscopy and automatic analysis of matrix and stromal markers derived from immunofluorescence images.Results: US elastography estimates of tumor stiffness were positively correlated with tumor volume in collagen and myofibroblast-rich tumors, while no correlations were found for tumors with low collagen and myofibroblast content. US elastography measurements were strongly correlated with ex vivo mechanical testing and mass spectroscopy-based measurements of total collagen and immature collagen crosslinks. Registration of ultrasound and confocal microscopy data showed strong correlations between blood vessel density and T-cell density in syngeneic tumors, while no correlations were found for genetic tumor models. In contrast to collagen density, which was positively correlated with stiffness, no significant correlations were observed for hyaluronic acid density. Finally, localized delivery of collagenase led to a significant reduction in tumor stiffness without changes in perfusion 24 hours after treatment.Conclusions: US elastography can be used as a potential biomarker to assess changes in the tumor microenvironment, particularly changes affecting the ECM. Clin Cancer Res; 24(18); 4455-67. ©2018 AACR.

TGFß attenuates tumour response to PD-L1 blockade by contributing to exclusion of T cells.

Therapeutic antibodies that block the programmed death-1 (PD-1)-programmed death-ligand 1 (PD-L1) pathway can induce robust and durable responses in patients with various cancers, including metastatic urothelial cancer. However, these responses only occur in a subset of patients. Elucidating the determinants of response and resistance is key to improving outcomes and developing new treatment strategies. Here we examined tumours from a large cohort of patients with metastatic urothelial cancer who were treated with an anti-PD-L1 agent (atezolizumab) and identified major determinants of clinical outcome. Response to treatment was associated with CD8+ T-effector cell phenotype and, to an even greater extent, high neoantigen or tumour mutation burden. Lack of response was associated with a signature of transforming growth factor ß (TGFß) signalling in fibroblasts. This occurred particularly in patients with tumours, which showed exclusion of CD8+ T cells from the tumour parenchyma that were instead found in the fibroblast- and collagen-rich peritumoural stroma; a common phenotype among patients with metastatic urothelial cancer. Using a mouse model that recapitulates this immune-excluded phenotype, we found that therapeutic co-administration of TGFß-blocking and anti-PD-L1 antibodies reduced TGFß signalling in stromal cells, facilitated T-cell penetration into the centre of tumours, and provoked vigorous anti-tumour immunity and tumour regression. Integration of these three independent biological features provides the best basis for understanding patient outcome in this setting and suggests that TGFß shapes the tumour microenvironment to restrain anti-tumour immunity by restricting T-cell infiltration.

The clearance of cell remnants and the regeneration of the injured muscle depend on soluble pattern recognition receptor PTX3.

OBJECTIVE: The signals causing the resolution of muscle inflammation are only partially characterized. The long pentraxin PTX3, which modulates leukocyte recruitment and activation, could contribute. METHODS: We analysed the expression of ptx3 after muscle injury and verified whether hematopoietic precursors are a source of the protein. The kinetics of regeneration and leukocytes infiltration, the accumulation of cell remnants and anti-histidyl-t-RNA synthetase autoantibodies were compared in wild-type and ptx3-deficient mice. RESULTS: Ptx3 expression was up-regulated three-five days after injury and restricted to the extracellular matrix. Cellular debris and leukocytes persisted in the muscle of ptx3-deficient mice for a long time after wild-type animals had healed. ptx3-deficient macrophages expressed receptors involved in apoptotic cell clearance and engulfed dead cells in vitro. Accumulation of cell debris in a pro-inflammatory microenvironment was not sufficient to elicit autoantibodies. CONCLUSION: PTX3 generated in response to muscle injury prompts the clearance of debris and the termination of the inflammatory response.

Hedgehog-driven myogenic tumors recapitulate skeletal muscle cellular heterogeneity.

Hedgehog (Hh) pathway activation in R26-SmoM2;CAGGS-CreER mice, which carry a tamoxifen-inducible activated Smoothened allele (SmoM2), results in numerous microscopic tumor foci in mouse skeletal muscle. These tumors exhibit a highly differentiated myogenic phenotype and resemble human fetal rhabdomyomas. This study sought to apply previously established strategies to isolate lineally distinct populations of normal mouse myofiber-associated cells in order to examine cellular heterogeneity in SmoM2 tumors. We demonstrate that established SmoM2 tumors are composed of cells expressing myogenic, adipocytic and hematopoietic lineage markers and differentiation capacity. SmoM2 tumors thus recapitulate the phenotypic and functional hetereogeneity observed in normal mouse skeletal muscle. SmoM2 tumors also contain an expanded population of PAX7+ and MyoD+ satellite-like cells with extremely low clonogenic activity. Selective activation of Hh signaling in freshly isolated muscle satellite cells enhanced terminal myogenic differentiation without stimulating proliferation. Our findings support the conclusion that SmoM2 tumors represent an aberrant skeletal muscle state and demonstrate that, similar to normal muscle, myogenic tumors contain functionally distinct cell subsets, including cells lacking myogenic differentiation potential.

FOXP3+ T Cells Recruited to Sites of Sterile Skeletal Muscle Injury Regulate the Fate of Satellite Cells and Guide Effective Tissue Regeneration.

Muscle injury induces a classical inflammatory response in which cells of the innate immune system rapidly invade the tissue. Macrophages are prominently involved in this response and required for proper healing, as they are known to be important for clearing cellular debris and supporting satellite cell differentiation. Here, we sought to assess the role of the adaptive immune system in muscle regeneration after acute damage. We show that T lymphocytes are transiently recruited into the muscle after damage and appear to exert a pro-myogenic effect on muscle repair. We observed a decrease in the cross-sectional area of regenerating myofibers after injury in Rag2-/- γ-chain-/- mice, as compared to WT controls, suggesting that T cell recruitment promotes muscle regeneration. Skeletal muscle infiltrating T lymphocytes were enriched in CD4+CD25+FOXP3+ cells. Direct exposure of muscle satellite cells to in vitro induced Treg cells effectively enhanced their expansion, and concurrently inhibited their myogenic differentiation. In vivo, the recruitment of Tregs to acutely injured muscle was limited to the time period of satellite expansion, with possibly important implications for situations in which inflammatory conditions persist, such as muscular dystrophies and inflammatory myopathies. We conclude that the adaptive immune system, in particular T regulatory cells, is critically involved in effective skeletal muscle regeneration. Thus, in addition to their well-established role as regulators of the immune/inflammatory response, T regulatory cells also regulate the activity of skeletal muscle precursor cells, and are instrumental for the proper regeneration of this tissue.

Isolation of progenitors that exhibit myogenic/osteogenic bipotency in vitro by fluorescence-activated cell sorting from human fetal muscle.

Fluorescence-activated cell sorting (FACS) strategies to purify distinct cell types from the pool of fetal human myofiber-associated (hMFA) cells were developed. We demonstrate that cells expressing the satellite cell marker PAX7 are highly enriched within the subset of CD45(-)CD11b(-)GlyA(-)CD31(-)CD34(-)CD56(int)ITGA7(hi) hMFA cells. These CD45(-)CD11b(-)GlyA(-)CD31(-)CD34(-)CD56(int)ITGA7(hi) cells lack adipogenic capacity but exhibit robust, bipotent myogenic and osteogenic activity in vitro and engraft myofibers when transplanted into mouse muscle. In contrast, CD45(-)CD11b(-)GlyA(-)CD31(-)CD34(+) fetal hMFA cells represent stromal constituents of muscle that do not express PAX7, lack myogenic function, and exhibit adipogenic and osteogenic capacity in vitro. Adult muscle likewise contains PAX7(+) CD45(-)CD11b(-)GlyA(-)CD31(-)CD34(-)CD56(int)ITGA7(hi) hMFA cells with in vitro myogenic and osteogenic activity, although these cells are present at lower frequency in comparison to their fetal counterparts. The ability to directly isolate functionally distinct progenitor cells from human muscle will enable novel insights into muscle lineage specification and homeostasis.

A zebrafish embryo culture system defines factors that promote vertebrate myogenesis across species.

Ex vivo expansion of satellite cells and directed differentiation of pluripotent cells to mature skeletal muscle have proved difficult challenges for regenerative biology. Using a zebrafish embryo culture system with reporters of early and late skeletal muscle differentiation, we examined the influence of 2,400 chemicals on myogenesis and identified six that expanded muscle progenitors, including three GSK3ß inhibitors, two calpain inhibitors, and one adenylyl cyclase activator, forskolin. Forskolin also enhanced proliferation of mouse satellite cells in culture and maintained their ability to engraft muscle in vivo. A combination of bFGF, forskolin, and the GSK3ß inhibitor BIO induced skeletal muscle differentiation in human induced pluripotent stem cells (iPSCs) and produced engraftable myogenic progenitors that contributed to muscle repair in vivo. In summary, these studies reveal functionally conserved pathways regulating myogenesis across species and identify chemical compounds that expand mouse satellite cells and differentiate human iPSCs into engraftable muscle.

Selective up-regulation of the soluble pattern-recognition receptor pentraxin 3 and of vascular endothelial growth factor in giant cell arteritis: relevance for recent optic nerve ischemia.

OBJECTIVE: To assess local expression and plasma levels of pentraxin 3 (PTX3) in patients with giant cell arteritis (GCA). METHODS: Plasma and serum samples were obtained from 75 patients with GCA (20 of whom had experienced optic nerve ischemia in the previous 3 weeks and 24 of whom had experienced symptom onset in the previous 6 months and had no history of optic nerve ischemia) and 63 controls (35 age-matched healthy subjects, 15 patients with rheumatoid arthritis, and 13 patients with chronic stable angina). In 9 patients in whom GCA was recently diagnosed, circulating levels of interleukin-1ß (IL-1ß), IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12p70, CCL2/monocyte chemotactic protein 1, CCL3/macrophage inflammatory protein 1α (MIP-1α), CCL4/MIP-1ß, CCL11/eotaxin, CXCL9/monokine induced by interferon-γ, CXCL10/interferon-γ-inducible 10-kd protein, tumor necrosis factor α (TNFα), interferon-γ, vascular endothelial growth factor (VEGF), granulocyte-macrophage colony-stimulating factor, and FasL were measured via a multiplexed cytometric assay. PTX3 and VEGF concentrations were assessed by enzyme-linked immunosorbent assay. PTX3 and CD68 expression were determined by immunohistochemistry and immunofluorescence on temporal artery samples. RESULTS: GCA patients with very recent optic nerve ischemia had significantly higher PTX3 and VEGF levels compared to other GCA patients and controls. GCA patients with a disease duration of <6 months had significantly higher PTX3 levels compared to other GCA patients and controls. Immunohistochemistry revealed selective PTX3 expression in the wall of inflamed arteries. CONCLUSION: Our findings indicate that local expression of PTX3 is a feature of vascular inflammation in GCA; elevated circulating levels of PTX3 identify patients with very recent optic nerve ischemia or a recent diagnosis. Optic nerve ischemia is also associated with increased circulating VEGF levels.

High-mobility group box 1 release and redox regulation accompany regeneration and remodeling of skeletal muscle.

High-mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) molecules, favors tissue regeneration via recruitment and activation of leukocytes and stem cells. Here we demonstrate, in a model of acute sterile muscle injury, that regeneration is accompanied by active reactive oxygen species (ROS) production counterbalanced and overcome by the generation of antioxidant moieties. Mitochondria are initially responsible for ROS formation. However, they undergo rapid disruption with almost complete disappearance. Twenty-four hours after injury, we observed a strong induction of MURF1 and atrogin-1 ubiquitin ligases, key signals in activation of the proteasome system and induction of muscle atrophy. At later time points, ROS generation is maintained by nonmitochondrial sources. The antioxidant response occurs in both regenerating fibers and leukocytes that express high levels of free thiols and antioxidant enzymes, such as superoxide dismutase 1 (SOD1) and thioredoxin. HMGB1, a protein thiol, weakly expressed in healthy muscles, increases during regeneration in parallel with the antioxidant response in both fibers and leukocytes. A reduced environment may be important to maintain HMGB1 bioactivity. Indeed, oxidation abrogates both muscle stem cell migration in response to HMGB1 and their ability to differentiate into myofibers in vitro. We propose that the early antioxidant response in regenerating muscle limits HMGB1 oxidation, thus allowing successful muscle regeneration.

High-mobility group box 1 (HMGB1) as a master regulator of innate immunity.

Damage-associated molecular patterns (DAMPs) comprise intracellular molecules characterized by the ability to reach the extracellular environment, where they prompt inflammation and tissue repair. The high-mobility box group 1 (HMGB1) protein is a prototypic DAMP and is highly conserved in evolution. HMGB1 is released upon cell and tissue necrosis and is actively produced by immune cells. Evidence suggests that HMGB1 acts as a key molecule of innate immunity, downstream of persistent tissue injury, orchestrating inflammation, stem cell recruitment/activation, and eventual tissue remodeling.

Polarization dictates iron handling by inflammatory and alternatively activated macrophages.

BACKGROUND: Macrophages play a key role in iron homeostasis. In peripheral tissues, they are known to polarize into classically activated (or M1) macrophages and alternatively activated (or M2) macrophages. Little is known on whether the polarization program influences the ability of macrophages to store or recycle iron and the molecular machinery involved in the processes. DESIGN AND METHODS: Inflammatory/M1 and alternatively activated/M2 macrophages were propagated in vitro from mouse bone-marrow precursors and polarized in the presence of recombinant interferon-γ or interleukin-4. We characterized and compared their ability to handle radioactive iron, the characteristics of the intracellular iron pools and the expression of molecules involved in internalization, storage and export of the metal. Moreover we verified the influence of iron on the relative ability of polarized macrophages to activate antigen-specific T cells. RESULTS: M1 macrophages have low iron regulatory protein 1 and 2 binding activity, express high levels of ferritin H, low levels of transferrin receptor 1 and internalize--albeit with low efficiency -iron only when its extracellular concentration is high. In contrast, M2 macrophages have high iron regulatory protein binding activity, express low levels of ferritin H and high levels of transferrin receptor 1. M2 macrophages have a larger intracellular labile iron pool, effectively take up and spontaneously release iron at low concentrations and have limited storage ability. Iron export correlates with the expression of ferroportin, which is higher in M2 macrophages. M1 and M2 cells activate antigen-specific, MHC class II-restricted T cells. In the absence of the metal, only M1 macrophages are effective. CONCLUSIONS: Cytokines that drive macrophage polarization ultimately control iron handling, leading to the differentiation of macrophages into a subset which has a relatively sealed intracellular iron content (M1) or into a subset endowed with the ability to recycle the metal (M2).

Adjuvant role for cell death during chemo- and radiotherapy of cancer?

Evaluation of: Apetoh L, Ghiringhelli F, Tesniere A et al. Toll-like receptor 4-dependent contribution of the immune system to anti-cancer chemotherapy and radiotherapy. Nat. Med. 13, 1050-1059 (2007). Chemotherapy and radiotherapy are supposed to mediate their anticancer activity via the direct elimination of tumor cells. Dying cells, however, also release molecules that promote the activation and the functional maturation of the most potent antigen-presenting cells, the dendritic cells. Mature dendritic cells are endowed with the ability to cross-prime T cells against the antigens contained in the dead cells. This paper evaluates the results from a recent article in which the success of some protocols for anticancer therapy depends, besides their direct cytotoxic effect, on the induction of a specific immune response: the effect is mediated both in mice and humans through the release by dying tumor cells of the nuclear protein high-mobility group 1 protein and by its interaction with Toll-like receptor 4. Larger studies on various therapeutic regimens are needed to evaluate the general relevance of this observation.