The lack of T cells in tumors is a major hurdle to successful immune checkpoint therapy (ICT). Therefore, therapeutic strategies promoting T cell recruitment into tumors are warranted to improve the treatment efficacy. Here, we report that Fc-optimized anti-cytotoxic T lymphocyte antigen 4 (CTLA-4) antibodies are potent remodelers of tumor vasculature that increase tumor-associated high endothelial venules (TA-HEVs), specialized blood vessels supporting lymphocyte entry into tumors. Mechanistically, this effect is dependent on the Fc domain of anti-CTLA-4 antibodies and CD4+ T cells and involves interferon gamma (IFNγ). Unexpectedly, we find that the human anti-CTLA-4 antibody ipilimumab fails to increase TA-HEVs in a humanized mouse model. However, increasing its Fc effector function rescues the modulation of TA-HEVs, promotes CD4+ and CD8+ T cell infiltration into tumors, and sensitizes recalcitrant tumors to programmed cell death protein 1 (PD-1) blockade. Our findings suggest that Fc-optimized anti-CTLA-4 antibodies could be used to reprogram tumor vasculature in poorly immunogenic cold tumors and improve the efficacy of ICT.

Epithelium-derived cytokines or alarmins, such as interleukin-33 (IL-33) and thymic stromal lymphopoietin (TSLP), are major players in type 2 immunity and asthma. Here, we demonstrate that TNF-like ligand 1A (TL1A) is an epithelial alarmin, constitutively expressed in alveolar epithelium at steady state in both mice and humans, which cooperates with IL-33 for early induction of IL-9high ILC2s during the initiation of allergic airway inflammation. Upon synergistic activation by IL-33 and TL1A, lung ILC2s acquire a transient IL-9highGATA3low "ILC9" phenotype and produce prodigious amounts of IL-9. A combination of large-scale proteomic analyses, lung intravital microscopy, and adoptive transfer of ILC9 cells revealed that high IL-9 expression distinguishes a multicytokine-producing state-of-activated ILC2s with an increased capacity to initiate IL-5-dependent allergic airway inflammation. Similar to IL-33 and TSLP, TL1A is expressed in airway basal cells in healthy and asthmatic human lungs. Together, these results indicate that TL1A is an epithelium-derived cytokine and an important cofactor of IL-33 in the airways.

Here, we present a protocol for flow cytometry analysis of endothelial cells (ECs) and CD8+ T cells in murine tumor models, at baseline and after cancer immuno-therapy with anti-PD-1/anti-CTLA-4 antibodies. We provide gating strategies for identification of specific cell subsets including ECs from tumor-associated high endothelial venules (TA-HEVs), stem-like, and terminally exhausted CD8+ T cells. This protocol represents a valuable tool for the analysis of rare subsets of tumor ECs and CD8+ T cells with critical roles in antitumor immunity.For complete details on the use and execution of this protocol, please refer to Asrir et al. (2022).

Recruitment of lymphocytes into tumors is critical for anti-tumor immunity and efficacious immunotherapy. We show in murine models that tumor-associated high endothelial venules (TA-HEVs) are major sites of lymphocyte entry into tumors at baseline and upon treatment with anti-PD-1/anti-CTLA-4 immune check-point blockade (ICB). TA-HEV endothelial cells (TA-HECs) derive from post-capillary venules, co-express MECA-79(+) HEV sialomucins and E/P-selectins, and are associated with homing and infiltration into tumors of various T cell subsets. Intravital microscopy further shows that TA-HEVs are the main sites of lymphocyte arrest and extravasation into ICB-treated tumors. Increasing TA-HEC frequency and maturation increases the proportion of tumor-infiltrating stem-like CD8(+) T cells, and ameliorates ICB efficacy. Analysis of tumor biopsies from 93 patients with metastatic melanoma reveals that TA-HEVs are predictive of better response and survival upon treatment with anti-PD-1/anti-CTLA-4 combination. These studies provide critical insights into the mechanisms governing lymphocyte trafficking in cancer immunity and immunotherapy.

Background Recent reports revealed that CD8+ T cell antitumor responses rely on a subset of stem-like CD8+ T cells during cancer immunity and immunotherapy. However, the origin of stem-like CD8+ T cells and the mechanisms controlling their trafficking are not well established. High endothelial venules (HEVs) are specialized blood vessels mediating lymphocyte trafficking in lymph nodes (LNs),1,2 and HEV-like blood vessels defined as tumor-associated HEVs (TA-HEVs) are important regulators of lymphocyte entry into tumors.3 Here, we investigated the role of LN-HEVs and TA-HEVs in the generation and migration of stem-like CD8+ T cells during spontaneous antitumor immunity and cancer immunotherapy. Methods In mouse syngeneic tumor models, we applied multidisciplinary approaches (immunofluorescence, flow cytometry, short-term homing assays and in vivo imaging) to identify the adhesion pathways used by LN-HEVs and TA-HEVs for CD8+ T cell trafficking. Then, using in vivo function-blocking antibodies targeting LN-HEVs and/or TA-HEVs, we defined their respective roles in the recruitment of naive CD44-PD-1- CD8+ T cells into tumor-draining lymph node and of CD44+PD-1+TCF-1high stem-like CD8+ T cells into tumor. Lastly, we blocked the function of LN-HEVs and/or TA-HEVs during combined anti-PD-1 plus anti-CTLA-4 cancer immunotherapy to analyze their contributions in treatment-induced CD8+ T cell responses. Results We found that naive and stem-like CD8+ T cells use distinct adhesion pathways to interact with LN-HEVs and TA-HEVs, respectively. Blocking naive CD8+ T cell adhesion to LN-HEVs and their subsequent entry into tumor-draining lymph node abrogated the generation of stem-like CD8+ T cells and prevented their accumulation in tumor, showing that peripheral and intratumoral responses are tightly related. In peripheral blood, circulating stem-like CD8+ T cells expressed adhesion molecules and chemokine receptors enabling interaction with TA-HEVs that mediated their entry into tumor. Finally, we found that checkpoint blockade therapy increased the number and activity of stem-like CD8+ T cells in tumor-draining lymph node and peripheral blood, suggesting that peripheral responses are involved during treatment. Accordingly, HEV-mediated trafficking in tumor, but also in tumor-draining lymph node, were required for expansion and differentiation of intratumoral stem-like CD8+ T cells during checkpoint blockade therapy. Conclusions In this study, we unveil the dual role of HEVs in tumor and periphery for CD8+ T cell responses during cancer immunity and immunotherapy, and provide mechanistic insights on the importance of peripheral responses during checkpoint blockade therapy. Our findings also strongly support the use of combined anti-PD-1 plus anti-CTLA-4 cancer immunotherapy in the neoadjuvant setting. Acknowledgements This work was supported by grants from Fondation ARC pour la Recherche sur le Cancer (PGA1 RF20180206911 to J-PG), Institut National du Cancer (INCa_2017-155 to J-PG). LB and EV were supported by fellowships from Fondation pour la Recherche Médicale (ECO201806006827, ECO202006011469, and FDT202106012889). References Moussion C, Girard JP. Dendritic cells control lymphocyte entry to lymph nodes through high endothelial venules. Nature. 2011;479:542–546. Blanchard L, Girard JP. High endothelial venules (HEVs) in immunity, inflammation and cancer. Angiogenesis. 2021;24:719–753. Asrir A*, Tardiveau C*, Coudert J*, Laffont R*, Blanchard L*, Bellard E, Veerman K, Bettini S, Lafouresse F, Vina E, Tarroux D, Roy S, Girault I, Molinaro I, Martins F, Scoazec JY, Ortega N, Robert C, Girard JP. Tumor-associated high endothelial venules mediate lymphocyte entry into tumors and predict response to PD-1 plus CTLA-4 combination immunotherapy. Cancer Cell. 2022;40:318–334. (* co-first authors) Ethics Approval This study obtained ethical approvals by the French Ministry of Research and the FRBT (C2EA-01) animal care committee (Projects APAFIS#12256-2017112021529157v3 and APAFIS#23416-2019122019025727v3).

In osteoclasts, Src controls podosome organization and bone degradation, which leads to an osteopetrotic phenotype in src(-/-) mice. Since this phenotype was even more severe in src(-/-)hck(-/-) mice, we examined the individual contribution of Hck in bone homeostasis. Compared to wt mice, hck(-/-) mice exhibited an osteopetrotic phenotype characterized by an increased density of trabecular bone and decreased bone degradation, although osteoclastogenesis was not impaired. Podosome organization and matrix degradation were found to be defective in hck(-/-) osteoclast precursors (preosteoclast) but were normal in mature hck(-/-) osteoclasts, probably through compensation by Src, which was specifically overexpressed in mature osteoclasts. As a consequence of podosome defects, the 3-dimensional migration of hck(-/-) preosteoclasts was strongly affected in vitro. In vivo, this translated by altered bone homing of preosteoclasts in hck(-/-) mice: in metatarsals of 1-wk-old mice, when bone formation strongly depends on the recruitment of these cells, reduced numbers of osteoclasts and abnormal developing trabecular bone were observed. This phenotype was still detectable in adults. In summmary, Hck is one of the very few effectors of preosteoclast recruitment described to date and thereby plays a critical role in bone remodeling.

IL-33 (previously known as NF from high endothelial venules) is an IL-1 family cytokine that signals through the ST2 receptor and drives cytokine production in mast cells, basophils, eosinophils, invariant NKT and NK cells, Th2 lymphocytes, and type 2 innate immune cells (natural helper cells, nuocytes, and innate helper 2 cells). Little is known about endogenous IL-33; for instance, the cellular sources of IL-33 in mouse tissues have not yet been defined. In this study, we generated an Il-33–LacZ gene trap reporter strain (Il-33Gt/Gt) and used this novel tool to analyze expression of endogenous IL-33 in vivo. We found that the Il-33 promoter exhibits constitutive activity in mouse lymphoid organs, epithelial barrier tissues, brain, and embryos. Immunostaining with anti–IL-33 Abs, using Il-33Gt/Gt (Il-33–deficient) mice as control, revealed that endogenous IL-33 protein is highly expressed in mouse epithelial barrier tissues, including stratified squamous epithelia from vagina and skin, as well as cuboidal epithelium from lung, stomach, and salivary gland. Constitutive expression of IL-33 was not detected in blood vessels, revealing the existence of species-specific differences between humans and mice. Importantly, IL-33 protein was always localized in the nucleus of producing cells with no evidence for cytoplasmic localization. Finally, strong expression of the Il-33–LacZ reporter was also observed in inflamed tissues, in the liver during LPS-induced endotoxin shock, and in the lung alveoli during papain-induced allergic airway inflammation. Together, our findings support the possibility that IL-33 may function as a nuclear alarmin to alert the innate immune system after injury or infection in epithelial barrier tissues.

Objective Inflammatory bowel diseases (IBD) have been intrinsically linked to a deregulated cytokine network, but novel therapeutic principles are urgently needed. Here we identify the interleukin (IL)-33 and its receptor ST2 as key negative regulators of wound healing and permeability in the colon of mice.Design Expression of IL-33 and ST2 was determined by qRT-PCR, ELISA, immunohistochemistry and western-blot analysis. Wild-type and St2(-/-) mice were used in wound healing experiments and in two experimental models of IBD triggered by 2,4,6-trinitrobenzene sulphonic acid or dextran sodium sulphate (DSS). Neutralisation of ST2 was performed by using a specific blocking antibody.Results Nuclear localisation and enhanced expression of IL-33 in myofibroblasts and enterocytes was linked to disease involvement independently of inflammation, while the expression of ST2 was primarily restricted to the colonic epithelia. In two experimental models of IBD, genetic ablation of ST2 significantly improved signs of colitis, while a sustained epithelial expression of the cyto-protective factor connexin-43 was observed in DSS-treated St2-deficient mice. Unexpectedly, absence of ST2 in non-hematopoietic cells was sufficient to protect against colitis. Consistently, specific inhibition of endogenous ST2-mediated signalling by treatment with neutralising antibody improved DSS-induced colitis. In addition, IL-33 treatment impaired epithelial barrier permeability in vitro and in vivo, whereas absence of ST2 enhanced wound healing response upon acute mechanical injury in the colon.Conclusions Our study unveiled a novel non-hematopoietic function of IL-33 in epithelial barrier function and wound healing. Therefore, blocking the IL-33/ST2 axis may represent an efficient therapy in IBD.

Novel therapeutic principles are urgently needed to cure inflammatory bowel diseases (IBD) that have been intrinsically linked to a deregulated inflammatory cytokine network. Here we identify the interleukin (IL)-1-like cytokine IL-33 and its receptor ST2 as novel negative regulators of wound healing in the colon of mice. Study in BL6 and IL-33R/ST2 deficient mice using dextran sodium sulfate (DSS) and 2,4,6-trinitrobenzene sulfonic acid (TNBS) colitis analyzing classical parameters of inflammation. Genetic ablation of ST2 protected the colon from chemical insults by dextran sodium sulfate (DSS) and 2,4,6-trinitrobenzene sulfonic acid (TNBS) that coincided with reduced acute pro-inflammatory response together with sustained expression of cytoprotective factors, including connexin-43. Likewise, administration of an anti-ST2 blocking antibody to mice treated by dextran sodium sulfate improved signs of morbidity, whereas recombinant IL-33 increased the risk for colitis. Consistently, the colonic mucosa of ST2-deficient mice was characterized by an enhanced wound healing response in a model of acute in vivo injury in the colon. In conclusion, we report here that the IL-33/ST2 axis plays a critical role in inflammation resolution by regulating self-renewal of the colonic epithelia, indicating neutralization of ST2 may represent a novel therapeutic target for effective treatment in IBD.

SUMMARY Long bone development depends on endochondral bone formation, a complex process requiring exquisite balance between hypertrophic cartilage (HC) formation and its ossification. Dysregulation of this process may result in skeletal dysplasias and heterotopic ossification. Endochondral ossification requires the precise orchestration of HC vascularization, extracellular matrix remodeling, and the recruitment of osteoclasts and osteoblasts. Matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEGF) and osteoclasts have all been shown to regulate endochondral ossification, but how their function interrelates is not known. We have investigated the functional relationship among these regulators of endochondral ossification, demonstrating that they have complementary but non-overlapping functions. MMP-9, VEGF and osteoclast deficiency all cause impaired growth plate ossification resulting in the accumulation of HC. VEGF mRNA and protein expression are increased at the MMP-9−/− growth plate, and VEGF activity contributes to endochondral ossification since sequestration of VEGF by soluble receptors results in further inhibition of growth plate vascularization and ossification. However, VEGF bioavailability is still limited in MMP-9 deficiency, as exogenous VEGF is able to rescue the MMP-9−/− phenotype, demonstrating that MMP-9 may partially, but not fully, regulate VEGF bioavailability. The organization of the HC extracellular matrix at the MMP-9−/− growth plate is altered, supporting a role for MMP-9 in HC remodeling. Inhibition of VEGF impairs osteoclast recruitment, whereas MMP-9 deficiency leads to an accumulation of osteoclasts at the chondro-osseous junction. Growth plate ossification in osteoclast-deficient mice is impaired in the presence of normal MMP-9 expression, indicating that other osteoclastic functions are also necessary. Our data delineate the complementary interplay between MMP-9, VEGF and osteoclast function that is necessary for normal endochondral bone formation and provide a molecular framework for investigating the molecular defects contributing to disorders of endochondral bone formation.

THAP1 is a sequence-specific DNA binding factor that regulates cell proliferation through modulation of target genes such as the cell cycle-specific gene RRM1. Mutations in the THAP1 DNA binding domain, an atypical zinc finger (THAP-zf), have recently been found to cause DYT6 dystonia, a neurological disease characterized by twisting movements and abnormal postures. In this study, we report that THAP1 shares sequence characteristics, in vivo expression patterns and protein partners with THAP3, another THAP-zf protein. Proteomic analyses identified HCF-1, a potent transcriptional coactivator and cell cycle regulator, and O-GlcNAc transferase (OGT), the enzyme that catalyzes the addition of O-GlcNAc, as major cellular partners of THAP3. THAP3 interacts with HCF-1 through a consensus HCF-1-binding motif (HBM), a motif that is also present in THAP1. Accordingly, THAP1 was found to bind HCF-1 in vitro and to associate with HCF-1 and OGT in vivo. THAP1 and THAP3 belong to a large family of HCF-1 binding factors since seven other members of the human THAP-zf protein family were identified, which harbor evolutionary conserved HBMs and bind to HCF-1. Chromatin immunoprecipitation (ChIP) assays and RNA interference experiments showed that endogenous THAP1 mediates the recruitment of HCF-1 to the RRM1 promoter during endothelial cell proliferation and that HCF-1 is essential for transcriptional activation of RRM1. Together, our findings suggest HCF-1 is an important cofactor for THAP1. Interestingly, our results also provide an unexpected link between DYT6 and DYT3 (X-linked dystonia-parkinsonism) dystonias because the gene encoding the THAP1/DYT6 protein partner OGT maps within the DYT3 critical region on Xq13.1.

Background: Interleukin-33 (IL-33) is an IL-1-like cytokine ligand for the IL-1 receptor-related protein ST2, that activates mast cells and Th2 lymphocytes, and induces production of Th2-associated cytokines in vivo. We initially discovered IL-33 as a nuclear factor (NF-HEV) abundantly expressed in high endothelial venules from lymphoid organs, that associates with chromatin and exhibits transcriptional regulatory properties. This suggested that, similarly to IL-1 alpha and chromatin-associated cytokine HMGB1, IL-33 may act as both a cytokine and a nuclear factor. Although the activity of recombinant IL-33 has been well characterized, little is known yet about the expression pattern of endogenous IL-33 in vivo.Methodology/Principal Findings: Here, we show that IL-33 is constitutively and abundantly expressed in normal human tissues. Using a combination of human tissue microarrays and IL-33 monoclonal and polyclonal antibodies, we found that IL-33 is a novel nuclear marker of the endothelium widely expressed along the vascular tree. We observed abundant nuclear expression of IL-33 in endothelial cells from both large and small blood vessels in most normal human tissues, as well as in human tumors. In addition to endothelium, we also found constitutive nuclear expression of IL-33 in fibroblastic reticular cells of lymphoid tissues, and epithelial cells of tissues exposed to the environment, including skin keratinocytes and epithelial cells of the stomach, tonsillar crypts and salivary glands.Conclusions/Significance: Together, our results indicate that, unlike inducible cytokines, IL-33 is constitutively expressed in normal human tissues. In addition, they reveal that endothelial cells and epithelial cells constitute major sources of IL-33 in vivo. Based on these findings, we speculate that IL-33 may function, similarly to the prototype 'alarmin' HMGB1, as an endogenous 'danger' signal to alert the immune system after endothelial or epithelial cell damage during trauma or infection.

Proteomics strategies based on nanoflow (nano-) LC-MS/MS allow the identification of hundreds to thousands of proteins in complex mixtures. When combined with protein isotopic labeling, quantitative comparison of the proteome from different samples can be achieved using these approaches. However, bioinformatics analysis of the data remains a bottleneck in large scale quantitative proteomics studies. Here we present a new software named Mascot File Parsing and Quantification (MFPaQ) that easily processes the results of the Mascot search engine and performs protein quantification in the case of isotopic labeling experiments using either the ICAT or SILAC (stable isotope labeling with amino acids in cell culture) method. This new tool provides a convenient interface to retrieve Mascot protein lists; sort them according to Mascot scoring or to user-defined criteria based on the number, the score, and the rank of identified peptides; and to validate the results. Moreover the software extracts quantitative data from raw files obtained by nano-LC-MS/MS, calculates peptide ratios, and generates a non-redundant list of proteins identified in a multisearch experiment with their calculated averaged and normalized ratio. Here we apply this software to the proteomics analysis of membrane proteins from primary human endothelial cells (ECs), a cell type involved in many physiological and pathological processes including chronic inflammatory diseases such as rheumatoid arthritis. We analyzed the EC membrane proteome and set up methods for quantitative analysis of this proteome by ICAT labeling. EC microsomal proteins were fractionated and analyzed by nano-LC-MS/MS, and database searches were performed with Mascot. Data validation and clustering of proteins were performed with MFPaQ, which allowed identification of more than 600 unique proteins. The software was also successfully used in a quantitative differential proteomics analysis of the EC membrane proteome after stimulation with a combination of proinflammatory mediators (tumor necrosis factor-alpha, interferon-gamma, and lymphotoxin alpha/beta) that resulted in the identification of a full spectrum of EC membrane proteins regulated by inflammation.

Recent studies indicate that IL-1α functions intracellularly in pathways independent of its cell surface receptors by translocating to the nucleus and regulating transcription. Similarly, the chromatin-associated protein HMGB1 acts as both a nuclear factor and a secreted proinflammatory cytokine. Here, we show that IL-33, an IL-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines, is an endothelium-derived, chromatin-associated nuclear factor with transcriptional repressor properties. We found that IL-33 is identical to NF-HEV, a nuclear factor preferentially expressed in high endothelial venules (HEV), that we previously characterized. Accordingly,in situhybridization demonstrated that endothelial cells constitute a major source of IL-33 mRNA in chronically inflamed tissues from patients with rheumatoid arthritis and Crohn's disease. Immunostaining with three distinct antisera, directed against the N-terminal part and IL-1-like C-terminal domain, revealed that IL-33 is a heterochromatin-associated nuclear factor in HEV endothelial cellsin vivo. Association of IL-33 with heterochromatin was also observed in human and mouse cells under living conditions. In addition, colocalization of IL-33 with mitotic chromatin was noted. Nuclear localization, heterochromatin-association, and targeting to mitotic chromosomes were all found to be mediated by an evolutionarily conserved homeodomain-like helix–turn–helix motif within the IL-33 N-terminal part. Finally, IL-33 was found to possess transcriptional repressor properties, associated with the homeodomain-like helix–turn–helix motif. Together, these data suggest that, similarly to IL1α and HMGB1, IL-33 is a dual function protein that may function as both a proinflammatory cytokine and an intracellular nuclear factor with transcriptional regulatory properties.

Endochondral bone formation is characterized by the progressive replacement of a cartilage anlagen by bone at the growth plate with a tight balance between the rates of chondrocyte proliferation, differentiation, and cell death. Deficiency of matrix metalloproteinase-9 (MMP-9) leads to an accumulation of late hypertrophic chondrocytes. We found that galectin-3, an in vitro substrate of MMP-9, accumulates in the late hypertrophic chondrocytes and their surrounding extracellular matrix in the expanded hypertrophic cartilage zone. Treatment of wild-type embryonic metatarsals in culture with full-length galectin-3, but not galectin-3 cleaved by MMP-9, mimicked the embryonic phenotype of Mmp-9 null mice, with an increased hypertrophic zone and decreased osteoclast recruitment. These results indicate that extracellular galectin-3 could be an endogenous substrate of MMP-9 that acts downstream to regulate hypertrophic chondrocyte death and osteoclast recruitment during endochondral bone formation. Thus, the disruption of growth plate homeostasis in Mmp-9 null mice links galectin-3 and MMP-9 in the regulation of the clearance of late chondrocytes through regulation of their terminal differentiation.

Endochondral ossification, the process by which most of the skeleton is formed, is a powerful system for studying various aspects of the biological response to degraded extracellular matrix (ECM). In addition, the dependence of endochondral ossification upon neovascularization and continuous ECM remodeling provides a good model for studying the role of the matrix metalloproteases (MMPs) not only as simple effectors of ECM degradation but also as regulators of active signal-inducers for the initiation of endochondral ossification. The daunting task of elucidating their specific role during endochondral ossification has been facilitated by the development of mice deficient for various members of this family. Here, we discuss the ECM and its remodeling as one level of molecular regulation for the process of endochondral ossification, with special attention to the MMPs.