ZEB1 shapes AML immunological niches, suppressing CD8 T cell activity while fostering Th17 cell expansion.

Acute myeloid leukemia (AML) progression is influenced by immune suppression induced by leukemia cells. ZEB1, a critical transcription factor in epithelial-to-mesenchymal transition, demonstrates immune regulatory functions in AML. Silencing ZEB1 in leukemic cells reduces engraftment and extramedullary disease in immune-competent mice, activating CD8 T lymphocytes and limiting Th17 cell expansion. ZEB1 in AML cells directly promotes Th17 cell development that, in turn, creates a self-sustaining loop and a pro-invasive phenotype, favoring transforming growth factor ß (TGF-ß), interleukin-23 (IL-23), and SOCS2 gene transcription. In bone marrow biopsies from AML patients, immunohistochemistry shows a direct correlation between ZEB1 and Th17. Also, the analysis of ZEB1 expression in larger datasets identifies two distinct AML groups, ZEB1high and ZEB1low, each with specific immunological and molecular traits. ZEB1high patients exhibit increased IL-17, SOCS2, and TGF-ß pathways and a negative association with overall survival. This unveils ZEB1's dual role in AML, entwining pro-tumoral and immune regulatory capacities in AML blasts.

High bone tumor burden to identify advanced non-small cell lung cancer patients with survival benefit upon bone targeted agents and immune checkpoint inhibitors.

BACKGROUND: Bone-targeted agents (BTA), such as denosumab (DN) and zoledronic acid (ZA), have historically reduced the risk of skeletal related events in cancer patients with bone metastases (BM), with no improvement in survival outcomes. In the immunotherapy era, BM have been associated with poor prognosis upon immune-checkpoint inhibitors (ICI). Currently, the impact of bone tumor burden on survival upon BTAs in advanced non-small cell lung cancer (aNSCLC) patients treated with ICI remains unknown. METHODS: Data from ICI-treated aNSCLC patients with BM (4/2013-5/2022) in one institution were retrospectively collected. BTA-ICI concurrent treatment was defined as BTA administration at any time before or within 90 days from ICI start. High bone tumor burden (HBTB) was defined as ≥ 3 sites of BM. Median OS (mOS) was estimated with Kaplan-Meier. Aikaike's information criterion (AIC) was used to select the best model for data analysis adjusted for clinical variables. RESULTS: Of 134 patients included, 51 (38 %) received BTA. At a mFU of 39.6 months (m), BTA-ICIs concurrent treatment did not significantly impact on mOS [8.3 m (95% CI 3.9-12.8) versus (vs) 6.8 m (95% CI 4.0-9.6) p = 0.36]; these results were confirmed after adjustment for clinical variables selected by AIC. A multivariate model showed a significant interaction between BTA use and HBTB or radiation therapy to BM. In subgroup analyses, only HBTB confirmed to be associated with significantly longer mOS [8.3 m (95% CI 2.4-14.2) vs 3.5 m (95% CI 2.9-4.1), p = 0.003] and mPFS [3.0 m (95% CI 1.6-4.4) vs 1.8 m (95% CI 1.6-2.0) p = 0.001] upon BTA-ICI concurrent treatment, with the most pronounced OS benefit observed for DN-ICI concurrent regimen [15.2 m (95% CI 0.1-30.7) vs 3.5 m (95% CI 2.9-4.1) p = 0.002]. CONCLUSIONS: In the immunotherapy era, HBTB can identify patients experiencing survival benefit with BTA, especially with DN-ICI combination. HBTB should be included as a stratification factor in the upcoming trials assessing BTA and ICI combinations in patients with aNSCLC and BM.

Extracellular nicotinamide phosphoribosyltransferase (eNAMPT) neutralization counteracts T cell immune evasion in breast cancer.

BACKGROUND: Nicotinamide phosphoribosyltransferase (NAMPT) is a key intracellular enzyme that participates in nicotinamide adenine dinucleotide (NAD) homeostasis as well as a released cytokine (eNAMPT) that is elevated in inflammatory conditions and in cancer. In patients with breast cancer, circulating eNAMPT is elevated and its plasma levels correlate with prognosis and staging. In light of this, we investigated the contribution of eNAMPT in triple negative mammary carcinoma progression by investigating the effect of its neutralization via a specific neutralizing monoclonal antibody (C269). METHODS: We used female BALB/c mice injected with 4T1 clone 5 cells and female C57BL6 injected with EO771 cells, evaluating tumoral size, spleen weight and number of metastases. We injected two times a week the anti-eNAMPT neutralizing antibody and we sacrificed the mice after 28 days. Harvested tumors were analyzed by histopathology, flow cytometry, western blot, immunohistochemistry, immunofluorescence and RNA sequencing to define tumor characteristics (isolating tumor infiltrating lymphocytes and tumoral cells) and to investigate the molecular mechanisms behind the observed phenotype. Moreover, we dissected the functional relationship between T cells and tumoral cells using three-dimensional (3D) co-cultures. RESULTS: The neutralization of eNAMPT with C269 led to decreased tumor size and reduced number of lung metastases. RNA sequencing and functional assays showed that eNAMPT controlled T-cell response via the programmed death-ligand 1/programmed cell death protein 1 (PD-L1/PD-1) axis and its neutralization led to a restoration of antitumoral immune responses. In particular, eNAMPT neutralization was able to activate CD8+IFNγ+GrzB+ T cells, reducing the immunosuppressive phenotype of T regulatory cells. CONCLUSIONS: These studies indicate for the first time eNAMPT as a novel immunotherapeutic target for triple negative breast cancer.

SCD5-dependent inhibition of SPARC secretion hampers metastatic spreading and favors host immunity in a TNBC murine model.

Dysregulated fatty acid metabolism interacts with oncogenic signals, thereby worsening tumor aggressiveness. The stearoyl-CoA desaturating enzymes, SCD1 and SCD5, convert of saturated fatty acids to monounsaturated fatty acids. While SCD1 is frequently overexpressed in tumor cells and has been widely studied, SCD5 has both limited expression and poor characterization. Here we evaluated, in vitro and in vivo, the effects of SCD5 overexpression in a metastatic clone of 4T1. The results showed SCD5-driven reprogramming of fatty acid metabolism, involving desaturation of stearic acid to oleic acid, which eventually blocked SPARC secretion. The latter event reduced the aggressiveness of the 4T1 subclone by decreasing the ECM deposition and reverting the Epithelial to Mesenchymal Transition (EMT) status. Variation of the fatty acid profile by SCD5-gene transduction or the direct administration oleic acid reduces the immune suppressive activity of myeloid cells and promoting granulocytic myeloid-derived suppressor cell maturation, eventually favoring T-cell activation. The less immunosuppressive microenvironment generated by SCD5 overexpression was enhanced in Sparc-KO mice, indicating that both extracellular and endogenous SPARC additively regulate myeloid cell-suppressive activities. Overall, our data sheds light on exploring the oleic acid-dependent inhibition of SPARC secretion as a possible mechanism to reduce breast cancer malignancy.

Neutrophil extracellular traps arm DC vaccination against NPM-mutant myeloproliferation.

Neutrophil extracellular traps (NETs) are web-like chromatin structures composed by dsDNA and histones, decorated with antimicrobial proteins. Their interaction with dendritic cells (DCs) allows DC activation and maturation toward presentation of NET-associated antigens. Differently from other types of cell death that imply protein denaturation, NETosis preserves the proteins localized onto the DNA threads for proper enzymatic activity and conformational status, including immunogenic epitopes. Besides neutrophils, leukemic cells can release extracellular traps displaying leukemia-associated antigens, prototypically mutant nucleophosmin (NPMc+) that upon mutation translocates from nucleolus to the cytoplasm localizing onto NET threads. We tested NPMc+ immunogenicity through a NET/DC vaccine to treat NPMc-driven myeloproliferation in transgenic and transplantable models. Vaccination with DC loaded with NPMc+ NET (NPMc+ NET/DC) reduced myeloproliferation in transgenic mice, favoring the development of antibodies to mutant NPMc and the induction of a CD8+ T-cell response. The efficacy of this vaccine was also tested in mixed NPMc/WT bone marrow (BM) chimeras in a competitive BM transplantation setting, where the NPMc+ NET/DC vaccination impaired the expansion of NPMc+ in favor of WT myeloid compartment. NPMc+ NET/DC vaccination also achieved control of an aggressive leukemia transduced with mutant NPMc, effectively inducing an antileukemia CD8 T-cell memory response.

CD40 Activity on Mesenchymal Cells Negatively Regulates OX40L to Maintain Bone Marrow Immune Homeostasis Under Stress Conditions.

Background: Within the bone marrow (BM), mature T cells are maintained under homeostatic conditions to facilitate proper hematopoietic development. This homeostasis depends upon a peculiar elevated frequency of regulatory T cells (Tregs) and immune regulatory activities from BM-mesenchymal stem cells (BM-MSCs). In response to BM transplantation (BMT), the conditioning regimen exposes the BM to a dramatic induction of inflammatory cytokines and causes an unbalanced T-effector (Teff) and Treg ratio. This imbalance negatively impacts hematopoiesis, particularly in regard to B-cell lymphopoiesis that requires an intact cross-talk between BM-MSCs and Tregs. The mechanisms underlying the ability of BM-MSCs to restore Treg homeostasis and proper B-cell development are currently unknown. Methods: We studied the role of host radio-resistant cell-derived CD40 in restoring Teff/Treg homeostasis and proper B-cell development in a murine model of BMT. We characterized the host cellular source of CD40 and performed radiation chimera analyses by transplanting WT or Cd40-KO with WT BM in the presence of T-reg and co-infusing WT or - Cd40-KO BM-MSCs. Residual host and donor T cell expansion and activation (cytokine production) and also the expression of Treg fitness markers and conversion to Th17 were analyzed. The presence of Cd40+ BM-MSCs was analyzed in a human setting in correlation with the frequency of B-cell precursors in patients who underwent HSCT and variably developed acute graft-versus-host (aGVDH) disease. Results: CD40 expression is nearly undetectable in the BM, yet a Cd40-KO recipient of WT donor chimera exhibited impaired B-cell lymphopoiesis and Treg development. Lethal irradiation promotes CD40 and OX40L expression in radio-resistant BM-MSCs through the induction of pro-inflammatory cytokines. OX40L favors Teff expansion and activation at the expense of Tregs; however, the expression of CD40 dampens OX40L expression and restores Treg homeostasis, thus facilitating proper B-cell development. Indeed, in contrast to dendritic cells in secondary lymphoid organs that require CD40 triggers to express OX40L, BM-MSCs require CD40 to inhibit OX40L expression. Conclusions: CD40+ BM-MSCs are immune regulatory elements within BM. Loss of CD40 results in uncontrolled T cell activation due to a reduced number of Tregs, and B-cell development is consequently impaired. GVHD provides an example of how a loss of CD40+ BM-MSCs and a reduction in B-cell precursors may occur in a human setting.

SPARC regulation of PMN clearance protects from pristane-induced lupus and rheumatoid arthritis.

The secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein with unexpected immunosuppressive function in myeloid cells. We investigated the role of SPARC in autoimmunity using the pristane-induced model of lupus that, in mice, mimics human systemic lupus erythematosus (SLE). Sparc -/- mice developed earlier and more severe renal disease, multi-organ parenchymal damage, and arthritis than the wild-type counterpart. Sparc +/- heterozygous mice showed an intermediate phenotype suggesting Sparc gene dosage in autoimmune-related events. Mechanistically, reduced Sparc expression in neutrophils blocks their clearance by macrophages, through defective delivery of don't-eat-me signals. Dying Sparc -/- neutrophils that escape macrophage scavenging become source of autoantigens for dendritic cell presentation and are a direct stimulation for γδT cells. Gene profile analysis of knee synovial biopsies from SLE-associated arthritis showed an inverse correlation between SPARC and key autoimmune genes. These results point to SPARC down-regulation as a leading event characterizing SLE and rheumatoid arthritis pathogenesis.

Intra-tumour heterogeneity of diffuse large B-cell lymphoma involves the induction of diversified stroma-tumour interfaces.

BACKGROUND: Intra-tumour heterogeneity in lymphoid malignancies encompasses selection of genetic events and epigenetic regulation of transcriptional programs. Clonal-related neoplastic cell populations are unsteadily subjected to immune editing and metabolic adaptations within different tissue microenvironments. How tissue-specific mesenchymal cells impact on the diversification of aggressive lymphoma clones is still unknown. METHODS: Combining in situ quantitative immunophenotypical analyses and RNA sequencing we investigated the intra-tumour heterogeneity and the specific mesenchymal modifications that are associated with A20 diffuse large B-cell lymphoma (DLBCL) cells seeding of different tissue microenvironments. Furthermore, we characterized features of lymphoma-associated stromatogenesis in human DLBCL samples using Digital Spatial Profiling, and established their relationship with prognostically relevant variables, such as MYC. FINDINGS: We found that the tissue microenvironment casts a relevant influence over A20 transcriptional landscape also impacting on Myc and DNA damage response programs. Extending the investigation to mice deficient for the matricellular protein SPARC, a stromal prognostic factor in human DLBCL, we demonstrated a different immune imprint on A20 cells according to stromal Sparc proficiency. Through Digital Spatial Profiling of 87 immune and stromal genes on human nodal DLBCL regions characterized by different mesenchymal composition, we demonstrate intra-lesional heterogeneity arising from diversified mesenchymal contextures and impacting on the stromal and immune milieu. INTERPRETATION: Our study provides experimental evidence that stromal microenvironment generates topological determinants of intra-tumour heterogeneity in DLBCL involving key transcriptional pathways such as Myc expression, damage response programs and immune checkpoints. FUNDING: This study has been supported by the Italian Foundation for Cancer Research (AIRC) (grants 15999 and 22145 to C. Tripodo) and by the University of Palermo.

SPARC Is a New Myeloid-Derived Suppressor Cell Marker Licensing Suppressive Activities.

Myeloid-derived suppressor cells (MDSC) are well-known key negative regulators of the immune response during tumor growth, however scattered is the knowledge of their capacity to influence and adapt to the different tumor microenvironments and of the markers that identify those capacities. Here we show that the secreted protein acidic and rich in cysteine (SPARC) identifies in both human and mouse MDSC with immune suppressive capacity and pro-tumoral activities including the induction of epithelial-to-mesenchymal transition (EMT) and angiogenesis. In mice the genetic deletion of SPARC reduced MDSC immune suppression and reverted EMT. Sparc-/- MDSC were less suppressive overall and the granulocytic fraction was more prone to extrude neutrophil extracellular traps (NET). Surprisingly, arginase-I and NOS2, whose expression can be controlled by STAT3, were not down-regulated in Sparc-/- MDSC, although less suppressive than wild type (WT) counterpart. Flow cytometry analysis showed equal phosphorylation of STAT3 but reduced ROS production that was associated with reduced nuclear translocation of the NF-kB p50 subunit in Sparc-/- than WT MDSC. The limited p50 in nuclei reduce the formation of the immunosuppressive p50:p50 homodimers in favor of the p65:p50 inflammatory heterodimers. Supporting this hypothesis, the production of TNF by Sparc-/- MDSC was significantly higher than by WT MDSC. Although associated with tumor-induced chronic inflammation, TNF, if produced at high doses, becomes a key factor in mediating tumor rejection. Therefore, it is foreseeable that an unbalance in TNF production could skew MDSC toward an inflammatory, anti-tumor phenotype. Notably, TNF is also required for inflammation-driven NETosis. The high level of TNF in Sparc-/- MDSC might explain their increased spontaneous NET formation as that we detected both in vitro and in vivo, in association with signs of endothelial damage. We propose SPARC as a new potential marker of MDSC, in both human and mouse, with the additional feature of controlling MDSC suppressive activity while preventing an excessive inflammatory state through the control of NF-kB signaling pathway.

Nicotinamide Phosphoribosyltransferase Acts as a Metabolic Gate for Mobilization of Myeloid-Derived Suppressor Cells.

Cancer induces alteration of hematopoiesis to fuel disease progression. We report that in tumor-bearing mice the macrophage colony-stimulating factor elevates the myeloid cell levels of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD salvage pathway, which acts as negative regulator of the CXCR4 retention axis of hematopoietic cells in the bone marrow. NAMPT inhibits CXCR4 through a NAD/Sirtuin 1-mediated inactivation of HIF1α-driven CXCR4 gene transcription, leading to mobilization of immature myeloid-derived suppressor cells (MDSC) and enhancing their production of suppressive nitric oxide. Pharmacologic inhibition or myeloid-specific ablation of NAMPT prevented MDSC mobilization, reactivated specific antitumor immunity, and enhanced the antitumor activity of immune checkpoint inhibitors. Our findings identify NAMPT as a metabolic gate of MDSC precursor function, providing new opportunities to reverse tumor immunosuppression and to restore clinical efficacy of immunotherapy in patients with cancer. SIGNIFICANCE: These findings identify NAMPT as a metabolic gate of MDSC precursor function, providing new opportunities to reverse tumor immunosuppression and to restore clinical efficacy of immunotherapy in cancer patients.

The P2X7 receptor modulates immune cells infiltration, ectonucleotidases expression and extracellular ATP levels in the tumor microenvironment.

In the tumor microenvironment (TME) ATP and its receptor P2X7 exert a pivotal influence on cancer growth and tumor-host interactions. Here we analyzed the different effect of P2X7 genetic deficiency versus its antagonism on response against P2X7-expressing implanted tumors. We focused on immune cell expression of ATP degrading enzymes CD39 and CD73 and in vivo measured TME's ATP. The immune infiltrate of tumors growing in P2X7 null mice shows a decrease in CD8+ cells and an increased number of Tregs, overexpressing the fitness markers OX40, PD-1, and CD73. A similar Treg phenotype is also present in the spleen of tumor-bearing P2X7 null mice and it is paralleled by a decrease in proinflammatory cytokines and an increase in TGF-ß. Differently, systemic administration of the P2X7 blocker A740003 in wild-type mice left unaltered the number of tumor-infiltrating CD8+ and Treg lymphocytes but increased CD4+ effector cells and decreased their expression of CD39 and CD73. P2X7 blockade did not affect spleen immune cell composition or ectonucleotidase expression but increased circulating INF-γ. Augmented CD73 in P2X7 null mice was mirrored by a decrease in TME ATP concentration and nucleotide reduced secretion from immune cells. On the contrary, TME ATP levels remained unaltered upon P2X7 antagonism, owing to release of ATP from cancerous cells and diminished ectonucleotidase expression by CD4+ and dendritic cells. These data point at P2X7 receptor as a key determinant of TME composition due to its combined action on immune cell infiltrate, ectonucleotidases, and ATP release.

Persistent Immune Stimulation Exacerbates Genetically Driven Myeloproliferative Disorders via Stromal Remodeling.

Systemic immune stimulation has been associated with increased risk of myeloid malignancies, but the pathogenic link is unknown. We demonstrate in animal models that experimental systemic immune activation alters the bone marrow stromal microenvironment, disarranging extracellular matrix (ECM) microarchitecture, with downregulation of secreted protein acidic and rich in cysteine (SPARC) and collagen-I and induction of complement activation. These changes were accompanied by a decrease in Treg frequency and by an increase in activated effector T cells. Under these conditions, hematopoietic precursors harboring nucleophosmin-1 (NPM1) mutation generated myeloid cells unfit for normal hematopoiesis but prone to immunogenic death, leading to neutrophil extracellular trap (NET) formation. NET fostered the progression of the indolent NPM1-driven myeloproliferation toward an exacerbated and proliferative dysplastic phenotype. Enrichment in NET structures was found in the bone marrow of patients with autoimmune disorders and in NPM1-mutated acute myelogenous leukemia (AML) patients. Genes involved in NET formation in the animal model were used to design a NET-related inflammatory gene signature for human myeloid malignancies. This signature identified two AML subsets with different genetic complexity and different enrichment in NPM1 mutation and predicted the response to immunomodulatory drugs. Our results indicate that stromal/ECM changes and priming of bone marrow NETosis by systemic inflammatory conditions can complement genetic and epigenetic events towards the development and progression of myeloid malignancy. Cancer Res; 77(13); 3685-99. ©2017 AACR.

ATP Release from Chemotherapy-Treated Dying Leukemia Cells Elicits an Immune Suppressive Effect by Increasing Regulatory T Cells and Tolerogenic Dendritic Cells.

Chemotherapy-induced immunogenic cell death can favor dendritic cell (DC) cross-priming of tumor-associated antigens for T cell activation thanks to the release of damage-associated molecular patterns, including ATP. Here, we tested the hypothesis that in acute myeloid leukemia (AML), ATP release, along with its well-known immune stimulatory effect, may also contribute to the generation of an immune suppressive microenvironment. In a cohort of AML patients, undergoing combined daunorubicin and cytarabine chemotherapy, a population of T regulatory cells (Tregs) with suppressive phenotype, expressing the immune checkpoint programmed cell death protein 1 (PD-1), was significantly increased. Moving from these results, initial in vitro data showed that daunorubicin was more effective than cytarabine in modulating DC function toward Tregs induction and such difference was correlated with the higher capacity of daunorubicin to induce ATP release from treated AML cells. DCs cultured with daunorubicin-treated AML cells upregulated indoleamine 2,3-dioxygenase 1 (IDO1), which induced anti-leukemia Tregs. These data were confirmed in vivo as daunorubicin-treated mice show an increase in extracellular ATP levels with increased number of Tregs, expressing PD-1 and IDO1+CD39+ DCs. Notably, daunorubicin failed to induce Tregs and tolerogenic DCs in mice lacking the ATP receptor P2X7. Our data indicate that ATP release from chemotherapy-treated dying cells contributes to create an immune suppressive microenvironment in AML.

Mesenchymal Transition of High-Grade Breast Carcinomas Depends on Extracellular Matrix Control of Myeloid Suppressor Cell Activity.

The extracellular matrix (ECM) contributes to the biological and clinical heterogeneity of breast cancer, and different prognostic groups can be identified according to specific ECM signatures. In high-grade, but not low-grade, tumors, an ECM signature characterized by high SPARC expression (ECM3) identifies tumors with increased epithelial-to-mesenchymal transition (EMT), reduced treatment response, and poor prognosis. To better understand how this ECM3 signature is contributing to tumorigenesis, we expressed SPARC in isogenic cell lines and found that SPARC overexpression in tumor cells reduces their growth rate and induces EMT. SPARC expression also results in the formation of a highly immunosuppressive microenvironment, composed by infiltrating T regulatory cells, mast cells, and myeloid-derived suppressor cells (MDSCs). The ability of SPARC to induce EMT depended on the localization and suppressive function of myeloid cells, and inhibition of the suppressive function MDSCs by administration of aminobisphosphonates could revert EMT, rendering SPARC-overexpressing tumor cells sensitive to Doxil. We conclude that that SPARC is regulating the interplay between MDSCs and the ECM to drive the induction of EMT in tumor cells.

Stromal niche communalities underscore the contribution of the matricellular protein SPARC to B-cell development and lymphoid malignancies.

Neoplastic B-cell clones commonly arise within secondary lymphoid organs (SLO). However, during disease progression, lymphomatous cells may also colonize the bone marrow (BM), where they localize within specialized stromal niches, namely the osteoblastic and the vascular niche, according to their germinal center- or extra-follicular-derivation, respectively. We hypothesized the existence of common stromal motifs in BM and SLO B-cell lymphoid niches involved in licensing normal B-cell development as well as in fostering transformed B lymphoid cells. Thus, we tested the expression of prototypical mesenchymal stromal cell (MSC) markers and regulatory matricellular proteins in human BM and SLO under physiologically unperturbed conditions and during B-cell lymphoma occurrence. We identified common stromal features in the BM osteoblastic niche and SLO germinal center (GC) microenvironments, traits that were also enriched within BM infiltrates of GC-associated B-cell lymphomas, suggesting that stromal programs involved in central and peripheral B-cell lymphopoiesis are also involved in malignant B-cell nurturing. Among factors co-expressed by stromal elements within these different specialized niches, we identified the pleiotropic matricellular protein secreted protein acidic and rich in cysteine (SPARC). The actual role of stromal SPARC in normal B-cell lymphopoiesis, investigated in Sparc-/- mice and BM chimeras retaining the Sparc-/- genotype in host stroma, demonstrated defective BM and splenic B-cell lymphopoiesis. Moreover, in the Trp53 knockout (KO) lymphoma model, p53-/-/Sparc-/- double-KO mice displayed impaired spontaneous splenic B-cell lymphomagenesis and reduced neoplastic clone BM infiltration in comparison with their p53-/-/Sparc+/+ counterparts. Our results are among the first to demonstrate the existence of common stromal programs regulating both the BM osteoblastic niche and the SLO GC lymphopoietic functions potentially fostering the genesis and progression of B-cell malignancies.

Defective stromal remodeling and neutrophil extracellular traps in lymphoid tissues favor the transition from autoimmunity to lymphoma.

Altered expression of matricellular proteins can become pathogenic in the presence of persistent perturbations in tissue homeostasis. Here, we show that autoimmunity associated with Fas mutation was exacerbated and transitioned to lymphomagenesis in the absence of SPARC (secreted protein acidic rich in cysteine). The absence of SPARC resulted in defective collagen assembly, with uneven compartmentalization of lymphoid and myeloid populations within secondary lymphoid organs (SLO), and faulty delivery of inhibitory signals from the extracellular matrix. These conditions promoted aberrant interactions between neutrophil extracellular traps and CD5(+) B cells, which underwent malignant transformation due to defective apoptosis under the pressure of neutrophil-derived trophic factors and NF-κB activation. Furthermore, this model of defective stromal remodeling during lymphomagenesis correlates with human lymphomas arising in a SPARC-defective environment, which is prototypical of CD5(+) B-cell chronic lymphocytic leukemia (CLL).

Tissue kallikrein is essential for invasive capacity of circulating proangiogenic cells.

RATIONALE: Homing of proangiogenic cells (PACs) is guided by chemoattractants and requires proteases to disrupt the extracellular matrix. The possibility that PAC recruitment involves an interaction between proteases and chemotactic factor receptors remains largely unexplored. OBJECTIVE: To determine the role of human tissue kallikrein (hK1) in PAC invasion and its dependency on kinin receptor signaling. METHODS AND RESULTS: Human mononuclear cells (MNCs) and culture-selected PACs express and release mature hK1 protein. HK1 gene (KLK1) silencing reduced PACs migratory, invasive, and proangiogenic activities. KLK1-knockout mouse bone marrow-derived MNCs showed similar impairments and were unable to support reparative angiogenesis in a mouse model of peripheral ischemia. Conversely, adenovirus-mediated KLK1 (Ad.KLK1) gene transfer enhanced PAC-associated functions, whereas the catalytically inactive variant R53H-KLK1 was ineffective. HK1-induced effects are mediated by a kinin B(2) receptor (B(2)R)-dependent mechanism involving inducible nitric oxide synthase and metalloproteinase-2 (MMP2). Lower hK1 protein levels were observed in PACs from type 2 diabetic (T2D) patients, whereas KLK1 mRNA levels were similar to those of healthy subjects, suggesting a post-transcriptional defect. Furthermore, B(2)R is normally expressed on T2D-PACs but remains uncoupled from downstream signaling. Importantly, whereas Ad.KLK1 alone could not restore T2D-PAC invasion capacity, combined KLK1 and B(2)R expression rescued the diabetic phenotype. CONCLUSIONS: This study reveals new interactive components of the PACs invasive machinery, acting via protease- and kinin receptor-dependent mechanisms.