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The concept of sustainability is becoming familiar and has already led to many initiatives of different types to put the concept into practice. There is a range of approaches, existing tools and methods that focus on the challenge of sustainability of European urban infrastructure. The actual problem is the transfer of knowledge to practice, the suggestion of ways of action and the integration of information. The Practical Evaluation Tool for Urban Sustainability (PETUS) research outcomes are presented here, on one side, as reflections upon these topics, and on the other side, as an operational framework developed with these objectives. Key Words Sustainability * Tools * Framework * Urban infrastructure * European research, Introduction To get to the nub of sustainable urban infrastructure this paper will first summarise the general concept of sustainable development that has emerged progressively at an international level for [...]

OBJECTIVE: Accumulating evidence indicates an important role of neutrophils in the development of rheumatoid arthritis (RA). Recruitment of neutrophils to the joint space and release of proteolytic enzymes can exacerbate tissue damage and the inflammatory response related to RA. Engagement of beta2 integrin and subsequent activation of downstream signaling have been shown to be fundamental for activation of neutrophil effector functions. The aim of this study was to test the hypothesis that Vav and phospholipase Cgamma2 (PLCgamma2), two molecules involved in integrin signaling, are required for arthritis generation and neutrophil activation in a mouse model of arthritis. METHODS: Arthritis was induced in wild-type (WT), Vav(null), and PLCgamma2(-/-) mice using the K/BxN serum-transfer model. Neutrophil function was assessed by analyses of adhesion, spreading, and degranulation on integrin-dependent substrates. Regulation of integrin signaling was determined by analyzing the phosphorylation of Pyk-2, Src, and ERK. RESULTS: Vav(null) and PLCgamma2(-/-) mice were protected from inflammation and bone erosion in the K/BxN serum-transfer model of arthritis. Mechanistically, Vav and PLCgamma2 control neutrophils mediated spreading and degranulation on integrin-dependent substrates. Consequently, the Vav/PLCgamma2 axis, acting downstream of the integrin receptor, modulated the activation of Pyk-2, Src, and ERK. CONCLUSION: Our findings show that Vav cooperates with PLCgamma2 in modulating neutrophil activation downstream of the integrin receptor. This study identifies a Vav/PLCgamma2-dependent signaling pathway as a possible therapeutic target for the treatment of inflammation and bone disruption in arthritis. [ABSTRACT FROM AUTHOR]

Background: NIS793 is a human IgG2 monoclonal antibody that binds to transforming growth factor beta (TGF-β). This first-in-human study investigated NIS793 plus spartalizumab treatment in patients with advanced solid tumors., Methods: Patients received NIS793 (0.3-1 mg/kg every 3 weeks (Q3W)) monotherapy; following evaluation of two dose levels, dose escalation continued with NIS793 plus spartalizumab (NIS793 0.3-30 mg/kg Q3W and spartalizumab 300 mg Q3W or NIS793 20-30 mg/kg every 2 weeks [Q2W] and spartalizumab 400 mg every 4 weeks (Q4W)). In dose expansion, patients with non-small cell lung cancer (NSCLC) resistant to prior anti-programmed death ligand 1 or patients with microsatellite stable colorectal cancer (MSS-CRC) were treated at the recommended dose for expansion (RDE)., Results: Sixty patients were treated in dose escalation, 11 with NIS793 monotherapy and 49 with NIS793 plus spartalizumab, and 60 patients were treated in dose expansion (MSS-CRC: n=40; NSCLC: n=20). No dose-limiting toxicities were observed. The RDE was established as NIS793 30 mg/kg (2100 mg) and spartalizumab 300 mg Q3W. Overall 54 (49.5%) patients experienced ≥1 treatment-related adverse event, most commonly rash (n=16; 13.3%), pruritus (n=10; 8.3%), and fatigue (n=9; 7.5%). Three partial responses were reported: one in renal cell carcinoma (NIS793 30 mg/kg Q2W plus spartalizumab 400 mg Q4W), and two in the MSS-CRC expansion cohort. Biomarker data showed evidence of target engagement through increased TGF-β/NIS793 complexes and depleted active TGF-β in peripheral blood. Gene expression analyses in tumor biopsies demonstrated decreased TGF-β target genes and signatures and increased immune signatures., Conclusions: In patients with advanced solid tumors, proof of mechanism of NIS793 is supported by evidence of target engagement and TGF-β pathway inhibition., Trial Registration Number: NCT02947165., Competing Interests: Competing interests: TMB has received support for the present manuscript from Novartis (to institution); consulting fees from AstraZeneca, Bayer, Blueprint, Lilly, and Pfizer; and has received honoraria from Bayer, Lilly and Pfizer. ASa has received consulting fees from Incyte and Sanofi; has received honoraria from AbbVie, Amgen, AstraZeneca, Bayer, Bristol Myers Squibb, Celgene, Eisai, Gilead, Lilly, Merck Sharp & Dohme, Novartis, Pfizer, Roche, Sandoz, Servier, and Takeda; and has participated in advisory boards for Bayer, Bristol Myers Squibb, Eisai, Gilead, Merck Sharp & Dohme, Pfizer, and Servier. C-CL has received consulting fees from Bayer, Blueprint Medicines, Boehringer Ingelheim, Bristol Myers Squibb, Daiichi Sankyo, Merck KGaA, Novartis, and PharmaEngine; has received honoraria from Boehringer Ingelheim, Novartis, and Roche; has received attendance/travel support from BeiGene, Daiichi Sankyo, and Eli Lilly; and has participated in an advisory board for Novartis. IG-L has received consulting fees from Jazz, Kanaph, OncXer and SOTIO; has received research support funding from Bayer, BridgeBio Pharma, Bristol Myers Squibb, GlaxoSmithKline, Incyte, Jacobio, Lilly, MedImmune, Novartis, Pfizer, Repare Therapeutics, Sumitomo Dainippon Pharma Oncology (to institution), and Tolero Pharmaceuticals. MJ has received research support from Basilea Pharmaceutica, Bristol Myers Squibb, Immunophotonics, Innomedica, Merck Sharp & Dohme, and Novartis (to institution); has received attendance/travel support from Bristol Myers Squibb and Roche; and has participated on an advisory board for Sanofi. RG has received consulting fees from AbbVie, AstraZeneca, Bristol Myers Squibb, Celgene, Daiichi Sankyo, Gilead, Janssen, Merck, Merck Sharp & Dohme, Novartis, Roche, Sanofi, and Takeda; has received honoraria from AbbVie, Amgen, AstraZeneca, Bristol Myers Squibb, Celgene, Daiichi Sankyo, Gilead, Merck, Merck Sharp & Dohme, Novartis, Roche, Sandoz, Sanofi, and Takeda; has received attendance/travel support from AbbVie, Amgen, AstraZeneca, Bristol Myers Squibb, Celgene, Daiichi Sankyo, Gilead, Janssen, Merck Sharp & Dohme, Novartis, and Roche; has participated in advisory boards for AbbVie, AstraZeneca, Bristol Myers Squibb, Celgene, Daiichi Sankyo, Gilead, Janssen, Merck, Merck Sharp & Dohme, Novartis, Roche, Sanofi, and Takeda. ASp received research support from Alkermes, Amgen, Array Biopharma/Pfizer, AstraZeneca/Medimmune, Bayer, Bristol Myers Squibb, GlaxoSmithKline, Janssen Oncology/Johnson & Johnson, Merck, Novartis, Northern Biologics, NuBiyota, Oncorus, Surface Oncology, Symphogen, Regeneron, Roche, and Treadwell; has participated in advisory boards for Bristol Myers Squibb, Janssen, Medison & Immunocore Merck, and Oncorus. TY has received support for the present manuscript from Novartis (institution); has received consulting fees from AstraZeneca, Bristol Myers Squibb, Eisai, Ipsen, and Merck Sharp & Dohme; has received support for attending meetings and/or travel from Roche and Bayer; holds stock with Moderna; has received receipt of medical writing from Ipsen and Taiho; has received payments (institution) for clinical trial investigatorship from AstraZeneca, Bristol Myers Squibb, Eli Lilly, Exelixis, Merck Sharp & Dohme, Roche, and Taiho. M-EG has received honoraria from AstraZeneca, Bristol Myers Squibb, Janssen, and Novartis; has participated in advisory boards for Bristol Myers Squibb and Janssen-Cilag. MLH-K has received support for attending meetings and/or travel from Jazz pharmaceuticals; has participated in advisory boards for Grifols. P-EJ and CF are former employees of Novartis. DL, MP, LB, and VC are employees of Novartis and hold stock with Novartis. HE is an employee of Novartis and holds Novartis shares. TD has received research support funding from AbbVie, Bristol Myers Squibb, Boehringer Ingelheim, Chugai Pharma, Daiichi Sankyo, Eisai, IQVIA, Janssen, Lilly Merck, Merck Sharp & Dohme, Novartis, Pfizer, SHIONOGI, Sumitomo Dainippon Pharma Oncology (to institution), and Taiho; consulting fees from A2 Health Care, AbbVie, Bayer, Chugai Pharma, KAKEN Pharma, KYOWA KIRIN, Noil Immune, Otsuka Pharma, PRA Health Science Rakuten Medical, SHIONOGI, Sumitomo Dainippon, Taiho, and Takeda; has received honoraria from AstraZeneca, Bristol Myers Squibbs, Daiichi Sankyo, Ono Pharma, and Rakuten Medical; has participated in advisory boards for AbbVie, Amgen, Astellas Pharma, Bayer Boehringer Ingelheim, Daiichi Sankyo, Janssen Pharma, Merck Sharp & Dohme, and Novartis. AP has no conflicts of interests to declare., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background & Aims: Transforming growth factor-b (TGFb) plays pleiotropic roles in pancreatic cancer, including promoting metastasis, attenuating CD8 T-cell activation, and enhancing myofibroblast differentiation and deposition of extracellular matrix. However, single-agent TGFb inhibition has shown limited efficacy against pancreatic cancer in mice or humans., Methods: We evaluated the TGFβ-blocking antibody NIS793 in combination with gemcitabine/nanoparticle (albumin-bound)-paclitaxel or FOLFIRINOX (folinic acid [FOL], 5-fluorouracil [F], irinotecan [IRI] and oxaliplatin [OX]) in orthotopic pancreatic cancer models. Single-cell RNA sequencing and immunofluorescence were used to evaluate changes in tumor cell state and the tumor microenvironment., Results: Blockade of TGFβ with chemotherapy reduced tumor burden in poorly immunogenic pancreatic cancer, without affecting the metastatic rate of cancer cells. Efficacy of combination therapy was not dependent on CD8 T cells, because response to TGFβ blockade was preserved in CD8-depleted or recombination activating gene 2 (RAG2 -/- ) mice. TGFβ blockade decreased total α-smooth muscle actin-positive fibroblasts but had minimal effect on fibroblast heterogeneity. Bulk RNA sequencing on tumor cells sorted ex vivo revealed that tumor cells treated with TGFβ blockade adopted a classical lineage consistent with enhanced chemosensitivity, and immunofluorescence for cleaved caspase 3 confirmed that TGFβ blockade increased chemotherapy-induced cell death in vivo., Conclusions: TGFβ regulates pancreatic cancer cell plasticity between classical and basal cell states. TGFβ blockade in orthotropic models of pancreatic cancer enhances sensitivity to chemotherapy by promoting a classical malignant cell state. This study provides scientific rationale for evaluation of NIS793 with FOLFIRINOX or gemcitabine/nanoparticle (albumin-bound) paclitaxel chemotherapy backbone in the clinical setting and supports the concept of manipulating cancer cell plasticity to increase the efficacy of combination therapy regimens., (Copyright © 2023 AGA Institute. Published by Elsevier Inc. All rights reserved.)

High levels of IL1β can result in chronic inflammation, which in turn can promote tumor growth and metastasis. Inhibition of IL1β could therefore be a promising therapeutic option in the treatment of cancer. Here, the effects of IL1β blockade induced by the mAbs canakinumab and gevokizumab were evaluated alone or in combination with docetaxel, anti-programmed cell death protein 1 (anti-PD-1), anti-VEGFα, and anti-TGFβ treatment in syngeneic and humanized mouse models of cancers of different origin. Canakinumab and gevokizumab did not show notable efficacy as single-agent therapies; however, IL1β blockade enhanced the effectiveness of docetaxel and anti-PD-1. Accompanying these effects, blockade of IL1β alone or in combination induced significant remodeling of the tumor microenvironment (TME), with decreased numbers of immune suppressive cells and increased tumor infiltration by dendritic cells (DC) and effector T cells. Further investigation revealed that cancer-associated fibroblasts (CAF) were the cell type most affected by treatment with canakinumab or gevokizumab in terms of change in gene expression. IL1β inhibition drove phenotypic changes in CAF populations, particularly those with the ability to influence immune cell recruitment. These results suggest that the observed remodeling of the TME following IL1β blockade may stem from changes in CAF populations. Overall, the results presented here support the potential use of IL1β inhibition in cancer treatment. Further exploration in ongoing clinical studies will help identify the best combination partners for different cancer types, cancer stages, and lines of treatment., (©2023 American Association for Cancer Research.)

Objectives: Sabatolimab is a humanized monoclonal antibody (hIgG4, S228P) directed against human T-cell immunoglobulin domain and mucin domain-3 (TIM-3). Herein, we describe the development and characterization of sabatolimab., Methods: Sabatolimab was tested for binding to its target TIM-3 and blocking properties. The functional effects of sabatolimab were tested in T-cell killing and myeloid cell cytokine assays. Antibody-mediated cell phagocytosis (ADCP) by sabatolimab was also assessed., Results: Sabatolimab was shown to (i) enhance T-cell killing and inflammatory cytokine production by dendritic cells (DCs); (ii) facilitate the phagocytic uptake of TIM-3-expressing target cells; and (iii) block the interaction between TIM-3 and its ligands PtdSer/galectin-9., Conclusion: Taken together, our results support both direct anti-leukemic effects and immune-mediated modulation by sabatolimab, reinforcing the notion that sabatolimab represents a novel immunotherapy with immuno-myeloid activity, holding promise for the treatment of myeloid cell neoplasms., (© The Author(s) 2022. Published by Oxford University Press on behalf of the British Society for Immunology.)

Chronic inflammation is often associated with the development of tissue fibrosis, but how mesenchymal cell responses dictate pathological fibrosis versus resolution and healing remains unclear. Defining stromal heterogeneity and identifying molecular circuits driving extracellular matrix deposition and remodeling stands to illuminate the relationship between inflammation, fibrosis, and healing. We performed single-cell RNA-sequencing of colon-derived stromal cells and identified distinct classes of fibroblasts with gene signatures that are differentially regulated by chronic inflammation, including IL-11-producing inflammatory fibroblasts. We further identify a transcriptional program associated with trans-differentiation of mucosa-associated fibroblasts and define a functional gene signature associated with matrix deposition and remodeling in the inflamed colon. Our analysis supports a critical role for the metalloprotease Adamdec1 at the interface between tissue remodeling and healing during colitis, demonstrating its requirement for colon epithelial integrity. These findings provide mechanistic insight into how inflammation perturbs stromal cell behaviors to drive fibroblastic responses controlling mucosal matrix remodeling and healing., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: R.J.X. is a cofounder of Celsius Therapeutics and Jnana Therapeutics.

High-grade serous ovarian cancers (HGSOC) represent the most common subtype of ovarian malignancies. Due to the frequency of late-stage diagnosis and high rates of recurrence following standard of care treatments, novel therapies are needed to promote durable responses. We investigated the anti-tumor activity of CD3 T cell engaging bispecific antibodies (TCBs) directed against the PAX8 lineage-driven HGSOC tumor antigen LYPD1 and demonstrated that anti-LYPD1 TCBs induce T cell activation and promote in vivo tumor growth inhibition in LYPD1-expressing HGSOC. To selectively target LYPD1-expressing tumor cells with high expression while sparing cells with low expression, we coupled bivalent low-affinity anti-LYPD1 antigen-binding fragments (Fabs) with the anti-CD3 scFv. In contrast to the monovalent anti-LYPD1 high-affinity TCB (VHP354), the bivalent low-affinity anti-LYPD1 TCB (QZC131) demonstrated antigen density-dependent selectivity and showed tolerability in cynomolgus monkeys at the maximum dose tested of 3 mg/kg. Collectively, these data demonstrate that bivalent TCBs directed against LYPD1 have compelling efficacy and safety profiles to support its use as a treatment for high-grade serous ovarian cancers., (© 2021. The Author(s).)

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-CreER T2 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.

SHP2 is a ubiquitous tyrosine phosphatase involved in regulating both tumor and immune cell signaling. In this study, we discovered a novel immune modulatory function of SHP2. Targeting this protein with allosteric SHP2 inhibitors promoted anti-tumor immunity, including enhancing T cell cytotoxic function and immune-mediated tumor regression. Knockout of SHP2 using CRISPR/Cas9 gene editing showed that targeting SHP2 in cancer cells contributes to this immune response. Inhibition of SHP2 activity augmented tumor intrinsic IFNγ signaling resulting in enhanced chemoattractant cytokine release and cytotoxic T cell recruitment, as well as increased expression of MHC Class I and PD-L1 on the cancer cell surface. Furthermore, SHP2 inhibition diminished the differentiation and inhibitory function of immune suppressive myeloid cells in the tumor microenvironment. SHP2 inhibition enhanced responses to anti-PD-1 blockade in syngeneic mouse models. Overall, our study reveals novel functions of SHP2 in tumor immunity and proposes that targeting SHP2 is a promising strategy for cancer immunotherapy.

Despite the increasing interest in targeting stromal elements of the tumor microenvironment, we still face tremendous challenges in developing adequate therapeutics to modify the tumor stromal landscape. A major obstacle to this is our poor understanding of the phenotypic and functional heterogeneity of stromal cells in tumors. Herein, we perform an unbiased interrogation of tumor mesenchymal cells, delineating the co-existence of distinct subsets of cancer-associated fibroblasts (CAFs) in the microenvironment of murine carcinomas, each endowed with unique phenotypic features and functions. Furthermore, our study shows that neutralization of TGFβ in vivo leads to remodeling of CAF dynamics, greatly reducing the frequency and activity of the myofibroblast subset, while promoting the formation of a fibroblast population characterized by strong response to interferon and heightened immunomodulatory properties. These changes correlate with the development of productive anti-tumor immunity and greater efficacy of PD1 immunotherapy. Along with providing the scientific rationale for the evaluation of TGFβ and PD1 co-blockade in the clinical setting, this study also supports the concept of plasticity of the stromal cell landscape in tumors, laying the foundation for future investigations aimed at defining pathways and molecules to program CAF composition for cancer therapy.

Lymph node fibroblastic reticular cells (FRCs) respond to signals from activated T cells by releasing nitric oxide, which inhibits T cell proliferation and restricts the size of the expanding T cell pool. Whether interactions with FRCs also support the function or differentiation of activated CD8 + T cells is not known. Here we report that encounters with FRCs enhanced cytokine production and remodeled chromatin accessibility in newly activated CD8 + T cells via interleukin-6. These epigenetic changes facilitated metabolic reprogramming and amplified the activity of pro-survival pathways through differential transcription factor activity. Accordingly, FRC conditioning significantly enhanced the persistence of virus-specific CD8 + T cells in vivo and augmented their differentiation into tissue-resident memory T cells. Our study demonstrates that FRCs play a role beyond restricting T cell expansion-they can also shape the fate and function of CD8 + T cells.

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., (©2018 American Association for Cancer Research.)

Over the past 25 years, research in cancer therapeutics has largely focused on two distinct lines of enquiry. In one approach, efforts to understand the underlying cell-autonomous, genetic drivers of tumorigenesis have led to the development of clinically important targeted agents that result in profound, but often not durable, tumour responses in genetically defined patient populations. In the second parallel approach, exploration of the mechanisms of protective tumour immunity has provided several therapeutic strategies - most notably the 'immune checkpoint' antibodies that reverse the negative regulators of T cell function - that accomplish durable clinical responses in subsets of patients with various tumour types. The integration of these potentially complementary research fields provides new opportunities to improve cancer treatments. Targeted and immune-based therapies have already transformed the standard-of-care for several malignancies. However, additional insights into the effects of targeted therapies, along with conventional chemotherapy and radiation therapy, on the induction of antitumour immunity will help to advance the design of combination strategies that increase the rate of complete and durable clinical response in patients.

Systemic sclerosis (SSc) is a potentially fatal autoimmune disorder with limited therapeutic options. Sclerodermatous graft versus host disease (sclGvHD), induced by transfer of B10.D2 splenocytes into BALB/c Rag2 -/- mice, models an inflammatory subset of SSc characterized by a prominent IL13-induced gene expression signature in the skin. Host mice deficient in IL4RA, a subunit of the type II IL4/IL13 receptor, are protected from sclGvHD. While IL4RA has a well-established role in Th2 differentiation and alternative macrophage activation, we report here a previously unappreciated function for IL4RA in lymphatic endothelial cells (LECs): regulation of activated T cell egress. Seven days after splenocyte transfer, Il4ra -/- hosts had increased numbers of activated graft CD4 + T cells in skin draining lymph nodes (dLNs) but fewer T cells in efferent lymph, blood, and skin. Sphingosine-1 phosphate (S1P), master regulator of lymphocyte egress from LNs, was lower in dLNs of Il4ra -/- hosts with a corresponding decrease of S1P kinase 1 ( Sphk1 ) expression in LECs. Bypassing the efferent lymphatics via i.v. injection of CD4 + T cells from dLNs of Il4ra -/- sclGvHD mice restored clinical GvHD in secondary Il4ra -/- recipients. These results identify a role for IL4RA and suggest that modulation of lymphocyte egress from LNs may be effective in SSc and GvHD.

Fibroblastic reticular cells (FRCs) form the cellular scaffold of lymph nodes (LNs) and establish distinct microenvironmental niches to provide key molecules that drive innate and adaptive immune responses and control immune regulatory processes. Here, we have used a graph theory-based systems biology approach to determine topological properties and robustness of the LN FRC network in mice. We found that the FRC network exhibits an imprinted small-world topology that is fully regenerated within 4 wk after complete FRC ablation. Moreover, in silico perturbation analysis and in vivo validation revealed that LNs can tolerate a loss of approximately 50% of their FRCs without substantial impairment of immune cell recruitment, intranodal T cell migration, and dendritic cell-mediated activation of antiviral CD8+ T cells. Overall, our study reveals the high topological robustness of the FRC network and the critical role of the network integrity for the activation of adaptive immune responses.

A dynamic and mutualistic interaction between tumour cells and the surrounding stroma promotes the initiation, progression, metastasis and chemoresistance of solid tumours. Far less understood is the relationship between the stroma and tumour-infiltrating leukocytes; however, emerging evidence suggests that the stromal compartment can shape antitumour immunity and responsiveness to immunotherapy. Thus, there is growing interest in elucidating the immunomodulatory roles of the stroma that evolve within the tumour microenvironment. In this Review, we discuss the evidence that stromal determinants interact with leukocytes and influence antitumour immunity, with emphasis on the immunological attributes of stromal cells that may foster their protumorigenic function.

Increased diacylglycerol (DAG) levels are observed in numerous pathologies, including conditions associated with bone loss. However, the effects of DAG accumulation on the skeleton have never been directly examined. Because DAG is strictly controlled by tissue-specific diacylglycerol kinases (DGKs), we sought to examine the biological consequences of DAG accumulation on bone homeostasis by genetic deletion of DGKζ, a highly expressed DGK isoform in osteoclasts (OCs). Strikingly, DGKζ(-/-) mice are osteoporotic because of a marked increase in OC numbers. In vitro, DGKζ(-/-) bone marrow macrophages (BMMs) form more numerous, larger, and highly resorptive OCs. Surprisingly, although increased DAG levels do not alter receptor activator of NF-κB (RANK)/RANK ligand (RANKL) osteoclastogenic pathway, DGKζ deficiency increases responsiveness to the proliferative and pro-survival cytokine macrophage colony-stimulating factor (M-CSF). We find that M-CSF is responsible for increased DGKζ(-/-) OC differentiation by promoting higher expression of the transcription factor c-Fos, and c-Fos knockdown in DGKζ(-/-) cultures dose-dependently reduces OC differentiation. Using a c-Fos luciferase reporter assay lacking the TRE responsive element, we also demonstrate that M-CSF induces optimal c-Fos expression through DAG production. Finally, to demonstrate the importance of the M-CSF/DGKζ/DAG axis on regulation of c-Fos during osteoclastogenesis, we turned to PLCγ2(+/-) BMMs, which have reduced DAG levels and form fewer OCs because of impaired expression of the master regulator of osteoclastogenesis NFATc1 and c-Fos. Strikingly, genetic deletion of DGKζ in PLCγ2(+/-) mice rescues OC formation and normalizes c-Fos levels without altering NFATc1 expression. To our knowledge, this is the first report implicating M-CSF/DGKζ/DAG axis as a critical regulator of bone homeostasis via its actions on OC differentiation and c-Fos expression., (© 2015 American Society for Bone and Mineral Research.)

Epidemiologic studies discovered an inverse association between immunoglobulin E (IgE)-mediated allergies and cancer, implying tumor-protective properties of IgE. However, the underlying immunologic mechanisms remain poorly understood. Antigen cross-presentation by dendritic cells (DCs) is of key importance for anti-tumor immunity because it induces the generation of cytotoxic CD8 + T lymphocytes (CTLs) with specificity for tumor antigens. We demonstrate that DCs use IgE and FcεRI, the high-affinity IgE receptor, for cross-presentation and priming of CTLs in response to free soluble antigen at low doses. Importantly, IgE/FcεRI-mediated cross-presentation is a distinct receptor-mediated pathway because it does not require MyD88 signals or IL-12 induction in DCs. Using passive immunization with tumor antigen-specific IgE and DC-based vaccination experiments, we demonstrate that IgE-mediated cross-presentation significantly improves anti-tumor immunity and induces memory responses in vivo. Our findings suggest a cellular mechanism for the tumor-protective features of IgE and expand the known physiological functions of this immunoglobulin., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

In lymph nodes, fibroblastic reticular cells (FRCs) form a collagen-based reticular network that supports migratory dendritic cells (DCs) and T cells and transports lymph. A hallmark of FRCs is their propensity to contract collagen, yet this function is poorly understood. Here we demonstrate that podoplanin (PDPN) regulates actomyosin contractility in FRCs. Under resting conditions, when FRCs are unlikely to encounter mature DCs expressing the PDPN receptor CLEC-2, PDPN endowed FRCs with contractile function and exerted tension within the reticulum. Upon inflammation, CLEC-2 on mature DCs potently attenuated PDPN-mediated contractility, which resulted in FRC relaxation and reduced tissue stiffness. Disrupting PDPN function altered the homeostasis and spacing of FRCs and T cells, which resulted in an expanded reticular network and enhanced immunity.

Fibroblastic reticular cells (FRCs) are known to inhabit T cell-rich areas of lymphoid organs, where they function to facilitate interactions between T cells and dendritic cells. However, in vivo manipulation of FRCs has been limited by a dearth of genetic tools that target this lineage. Here, using a mouse model to conditionally ablate FRCs, we demonstrated their indispensable role in antiviral T cell responses. Unexpectedly, loss of FRCs also attenuated humoral immunity due to impaired B cell viability and follicular organization. Follicle-resident FRCs established a favorable niche for B lymphocytes via production of the cytokine BAFF. Thus, our study indicates that adaptive immunity requires an intact FRC network and identifies a subset of FRCs that control B cell homeostasis and follicle identity.

Myeloid cells play important regulatory roles within the tumor environment by directly promoting tumor progression and modulating the function of tumor-infiltrating lymphocytes, and as such, they represent a potential therapeutic target for the treatment of cancer. Although distinct subsets of tumor-associated myeloid cells have been identified, a broader analysis of the complete myeloid cell landscape within individual tumors and also across different tumor types has been lacking. By establishing the developmental and transcriptomic signatures of infiltrating myeloid cells from multiple primary tumors, we found that tumor-associated macrophages (TAM) and tumor-associated neutrophils (TAN), while present within all tumors analyzed, exhibited strikingly different frequencies, gene expression profiles, and functions across cancer types. We also evaluated the impact of anatomic location and circulating factors on the myeloid cell composition of tumors. The makeup of the myeloid compartment was determined by the tumor microenvironment rather than the anatomic location of tumor development or tumor-derived circulating factors. Protumorigenic and hypoxia-associated genes were enriched in TAMs and TANs compared with splenic myeloid-derived suppressor cells. Although all TANs had an altered expression pattern of secretory effector molecules, in each tumor type they exhibited a unique cytokine, chemokine, and associated receptor expression profile. One such molecule, haptoglobin, was uniquely expressed by 4T1 TANs and identified as a possible diagnostic biomarker for tumors characterized by the accumulation of myeloid cells. Thus, we have identified considerable cancer-specific diversity in the lineage, gene expression, and function of tumor-infiltrating myeloid cells., (©2014 American Association for Cancer Research.)

Recent clinical trials showed that targeting of inhibitory receptors on T cells induces durable responses in a subset of cancer patients, despite advanced disease. However, the regulatory switches controlling T-cell function in immunosuppressive tumours are not well understood. Here we show that such inhibitory mechanisms can be systematically discovered in the tumour microenvironment. We devised an in vivo pooled short hairpin RNA (shRNA) screen in which shRNAs targeting negative regulators became highly enriched in murine tumours by releasing a block on T-cell proliferation upon tumour antigen recognition. Such shRNAs were identified by deep sequencing of the shRNA cassette from T cells infiltrating tumour or control tissues. One of the target genes was Ppp2r2d, a regulatory subunit of the PP2A phosphatase family. In tumours, Ppp2r2d knockdown inhibited T-cell apoptosis and enhanced T-cell proliferation as well as cytokine production. Key regulators of immune function can therefore be discovered in relevant tissue microenvironments.

Multiple sclerosis (MS) is a presumed autoimmune disease directed against central nervous system (CNS) myelin, in which diet and obesity are implicated as risk factors. Immune responses can be influenced by molecules produced by fat cells, called adipokines. Adiponectin is an adipokine with anti-inflammatory effects. We tested the hypothesis that adiponectin has a protective role in the EAE model for MS, that can be induced by immunization with myelin antigens or transfer of myelin-specific T lymphocytes. Adiponectin deficient (ADPKO) mice developed worse EAE with greater CNS inflammation, demyelination, and axon injury. Lymphocytes from myelin-immunized ADPKO mice proliferated more, produced higher amounts of IFN-γ, IL-17, TNF-α, IL-6, and transferred more severe EAE than wild type (WT) lymphocytes. At EAE peak, the spleen and CNS of ADPKO had fewer regulatory T (Treg) cells than WT mice and during EAE recovery, Foxp3, IL-10 and TGF-β expression levels in the CNS were reduced in ADPKO compared with WT mice. Treatment with globular adiponectin in vivo ameliorated EAE, and was associated with an increase in Treg cells. These data indicate that adiponectin is an important regulator of T-cell functions during EAE, suggesting a new avenue of investigation for MS treatment., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Bone homeostasis requires stringent regulation of osteoclasts, which secrete proteolytic enzymes to degrade the bone matrix. Despite recent progress in understanding how bone resorption occurs, the mechanisms regulating osteoclast secretion, and in particular the trafficking route of cathepsin K vesicles, remain elusive. Using a genetic approach, we describe the requirement for protein kinase C-delta (PKCδ) in regulating bone resorption by affecting cathepsin K exocytosis. Importantly, PKCδ deficiency does not perturb formation of the ruffled border or trafficking of lysosomal vesicles containing the vacuolar-ATPase (v-ATPase). Mechanistically, we find that cathepsin K exocytosis is controlled by PKCδ through modulation of the actin bundling protein myristoylated alanine-rich C-kinase substrate (MARCKS). The relevance of our finding is emphasized in vivo because PKCδ-/- mice exhibit increased bone mass and are protected from pathological bone loss in a model of experimental postmenopausal osteoporosis. Collectively, our data provide novel mechanistic insights into the pathways that selectively promote secretion of cathepsin K lysosomes independently of ruffled border formation, providing evidence of the presence of multiple mechanisms that regulate lysosomal exocytosis in osteoclasts., (Copyright © 2012 American Society for Bone and Mineral Research.)

Blockade of osteoclast (OC) activity efficiently decreases tumor burden as well as associated bone erosion in immune-compromised animals bearing human osteolytic cancers. In this study, we showed that modulation of antitumor T-cell responses alters tumor growth in bone, regardless of OC status, by using genetic and pharmacologic models. PLCγ2(-/-) mice, with dysfunctional OCs and impaired dendritic cell (DC)-mediated T-cell activation, had increased bone tumor burden despite protection from bone loss. In contrast, Lyn(-/-) mice, with more numerous OCs and a hyperactive myeloid population leading to increased T-cell responses, had reduced tumor growth in bone despite enhanced osteolysis. The unexpected tumor/bone phenotype observed in PLCγ2(-/-) and Lyn(-/-) mice was transplantable, suggesting the involvement of an immune component. Consistent with this hypothesis, T-cell activation diminished skeletal metastasis whereas T-cell depletion enhanced it, even in the presence of zoledronic acid, a potent antiresorptive agent. Importantly, injection of antigen-specific wild-type cytotoxic CD8(+) T cells in PLCγ2(-/-) mice or CD8(+) T-cell depletion in Lyn(-/-) mice normalized tumor growth in bone. Our findings show the important contribution of CD8(+) T cells in the regulation of bone metastases regardless of OC status, thus including T cells as critical regulators of tumor growth in bone., (©2011 AACR.)

A number of matrix metalloproteinases (MMPs), proteins important in the balance of bone remodeling, play a critical role both in cancer metastasis and in bone matrix turnover associated with the presence of cancer cells in bone. Here, we report the synthesis and biological evaluation of a new class of MMP inhibitors characterized by a bisphosphonate function as the zinc binding group. Since the bisphosphonate group is also implicated in osteoclast inhibition and provides a preferential affinity to biological apatite, the new molecules can be regarded as bone-seeking medicinal agents. Docking experiments were performed to clarify the mode of binding of bisphosphonate inhibitors in the active site of MMP-2. The most promising of the studied bisphosphonates showed nanomolar inhibition against MMP-2 and resulted in potent inhibition of osteoclastic bone resorption in vitro., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Identifying common signaling pathways to bone and immune system may lead to better therapeutic approaches in diseases such as inflammatory arthritis. In this context, PLCgamma2 seems to be a promising target. PLCgamma2 modulates bone homeostasis by affecting osteoclast recruitment and function. Via its catalytic activity and the adapter domains, PLCgamma2 controls RANKL and alphavbeta3 integrin-dependent signaling pathways in the resorbing cell. Thus, mice lacking PLCgamma2 are osteopetrotic. PLCgamma2 also regulates neutrophil degranulation after beta2 integrin-dependent attachment. Indeed PLCgamma2(-/-) mice are protected from K/BxN serum transfer arthritis, which is known to require neutrophil activation. These studies position PLCgamma2 as a critical regulator of the cellular and molecular mechanisms occurring in bone and immune cells during autoimmune inflammation.

Background: Dendritic cells (DCs) are highly specialized cells, which capture antigen in peripheral tissues and migrate to lymph nodes, where they dynamically interact with and activate T cells. Both migration and formation of DC-T cell contacts depend on cytoskeleton plasticity. However, the molecular bases governing these events have not been completely defined., Methodology/principal Findings: Utilizing a T cell-dependent model of arthritis, we find that PLCgamma2-/- mice are protected from local inflammation and bone erosion. PLCgamma2 controls actin remodeling in dendritic cells, thereby affecting their capacity to prime T cells. DCs from PLCgamma2-/- mice mature normally, however they lack podosomes, typical actin structures of motile cells. Absence of PLCgamma2 impacts both DC trafficking to the lymph nodes and migration towards CCL21. The interaction with T cells is also affected by PLCgamma2 deficiency. Mechanistically, PLCgamma2 is activated by CCL21 and modulates Rac activation. Rac1/2-/- DCs also lack podosomes and do not respond to CCL21. Finally, antigen pulsed PLCgamma2-/- DCs fail to promote T cell activation and induce inflammation in vivo when injected into WT mice. Conversely, injection of WT DCs into PLCgamma2-/- mice rescues the inflammatory response but not focal osteolysis, confirming the importance of PLCgamma2 both in immune and bone systems., Conclusions/significance: This study demonstrates a critical role for PLCgamma2 in eliciting inflammatory responses by regulating actin dynamics in DCs and positions the PLCgamma2 pathway as a common orchestrator of bone and immune cell functions during arthritis.

The chemokine receptor CXCR3 is involved in cell trafficking dysregulation associated with several inflammatory conditions, including autoimmune and viral diseases. Downregulation of CXCR3, through binding with its ligand CXCL11 (I-TAC), represents a key mechanism in lymphocyte recruitment. Determination of circulating I-TAC can provide useful information in the investigation of inflammatory/infectious conditions. The existing commercial kit does not measure CXCL11/I-TAC in complex matrices, such as human plasma and serum, as reliably as in in vitro-generated cell culture supernatants. We here describe means which lead to an improvement of CXCL11/I-TAC measurement in human plasma and serum.

Integrin engagement induces a cascade of signaling pathways that include tyrosine phosphorylation of numerous proteins that lead to modulation of the actin cytoskeleton. Src is a major intracellular mediator of integrin-dependent functions, but the mechanism(s) by which Src is regulated in response to integrin signals is not fully understood. Here, we demonstrate an important role for phospholipase C gamma 2 (PLCgamma2) in Src activation in the osteoclast. Through analysis of primary cells from PLCgamma2(-/-) mice, PLCgamma2 was found to be an important regulator of alpha(v)beta(3) integrin-mediated bone osteoclast cell adhesion, migration, and bone resorption. Adhesion-induced PYK2 and Src phosphorylation is decreased in the absence of PLCgamma2, and the interaction of Src with beta(3) integrin and PYK2 is dramatically reduced. Importantly, PLCgamma2 was found to be required for proper localization of Src to the sealing actin ring, and this function required both its catalytic activity and adapter domains. Based on these results, we propose that PLCgamma2 influences Src activation by mediating the localization of Src to the integrin complex and thereby regulating integrin-mediated functions in the osteoclast.

Oxidative burst, a critical antimicrobial mechanism of neutrophils, involves the rapid generation and release of reactive oxygen intermediates (ROIs) by the NADPH oxidase complex. Genetic mutations in an NADPH oxidase subunit, gp91 (also referred to as NOX2), are associated with chronic granulomatous disease (CGD), which is characterized by recurrent and life-threatening microbial infections. To combat such infections, ROIs are produced by neutrophils after stimulation by integrin-dependent adhesion to the ECM in conjunction with stimulation from inflammatory mediators, or microbial components containing pathogen-associated molecular patterns. In this report, we provide genetic evidence that both the Vav family of Rho GTPase guanine nucleotide exchange factors (GEFs) and phospholipase C-gamma2 (PLC-gamma2) are critical mediators of adhesion-dependent ROI production by neutrophils in mice. We also demonstrated that Vav was critically required for neutrophil-dependent host defense against systemic infection by Staphylococcus aureus and Pseudomonas aeruginosa, 2 common pathogens associated with fatal cases of hospital-acquired pneumonia. We identified a molecular pathway in which Vav GEFs linked integrin-mediated signaling with PLC-gamma2 activation, release of intracellular Ca2+ cations, and generation of diacylglycerol to control assembly of the NADPH oxidase complex and ROI production by neutrophils. Taken together, our data indicate that integrin-dependent signals generated during neutrophil adhesion contribute to the activation of NADPH oxidase by a variety of distinct effector pathways, all of which require Vav.