Disruption of CD47-SIRPα signaling restores inflammatory function in tumor-associated myeloid-derived suppressor cells.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous immune population with diverse immunosuppressive functions in solid tumors. Here, we explored the role of the tumor microenvironment in regulating MDSC differentiation and immunosuppressive properties via signal-regulatory protein alpha (SIRPα)/CD47 signaling. In a murine melanoma model, we observed progressive increases in monocytic MDSCs and monocyte-derived dendritic cells that exhibited potent T cell-suppressive capabilities. These adaptations could be recapitulated in vitro by exposing hematopoietic stem cells to tumor-derived factors. Engagement of CD47 with SIRPα on myeloid cells reduced their phagocytic capability, enhanced expression of immune checkpoints, increased reactive oxygen species production, and suppressed T cell proliferation. Perturbation of SIRPα signaling restored phagocytosis and antigen presentation by MDSCs, which was accompanied by renewed T cell activity and delayed tumor growth in multiple solid cancers. These data highlight that therapeutically targeting myeloid functions in combination with immune checkpoint inhibitors could enhance anti-tumor immunity.

Modeling Structural Elements and Functional Responses to Lymphatic-Delivered Cues in a Murine Lymph Node on a Chip.

Lymph nodes (LNs) are organs of the immune system, critical for maintenance of homeostasis and initiation of immune responses, yet there are few models that accurately recapitulate LN functions in vitro. To tackle this issue, an engineered murine LN (eLN) has been developed, replicating key cellular components of the mouse LN; incorporating primary murine lymphocytes, fibroblastic reticular cells, and lymphatic endothelial cells. T and B cell compartments are incorporated within the eLN that mimic LN cortex and paracortex architectures. When challenged, the eLN elicits both robust inflammatory responses and antigen-specific immune activation, showing that the system can differentiate between non specific and antigen-specific stimulation and can be monitored in real time. Beyond immune responses, this model also enables interrogation of changes in stromal cells, thus permitting investigations of all LN cellular components in homeostasis and different disease settings, such as cancer. Here, how LN behavior can be influenced by murine melanoma-derived factors is presented. In conclusion, the eLN model presents a promising platform for in vitro study of LN biology that will enhance understanding of stromal and immune responses in the murine LN, and in doing so will enable development of novel therapeutic strategies to improve LN responses in disease.

An Affordable Approach of Mesenchymal Stem Cell Therapy in Treating Perianal Fistula Treatment.

The application of stem cells to treat perianal fistula due to Crohn's disease has attracted a lot of interest in recent decades. Though still a popular procedure, the existing surgical methods may be an ideal form of therapy since the recurrence rate is high, which affects the quality of life badly. Stem cell therapy offers to be a better solution in treating PF, but the utilisation is often restricted because of the manufacturing cost. Hence in this review, the selection of suitable cell sources, the use of bioreactors and preconditioning MSCs as well as modified stem cells will be discussed for a more affordable as compared with the current MSC therapy towards PF. We anticipate that exploring these approaches may give a complete picture in understanding stem cells in order to make them effective and affordable for long-term therapeutic applications.

T2B or not to B: Calming neutrophils offshore.

In this issue of JEM, Podstawka et al. (2021. J. Exp. Med.https://doi.org/10.1084/jem.20210409) show that B cells can limit neutrophil responses within the lung microvasculature by marginating and acting on marginated neutrophils. This study provides a new view of B cells and reveals a novel mechanism of cell-mediated intravascular regulation.

Stromal-driven and Amyloid ß-dependent induction of neutrophil extracellular traps modulates tumor growth.

Tumors consist of cancer cells and a network of non-cancerous stroma. Cancer-associated fibroblasts (CAF) are known to support tumorigenesis, and are emerging as immune modulators. Neutrophils release histone-bound nuclear DNA and cytotoxic granules as extracellular traps (NET). Here we show that CAFs induce NET formation within the tumor and systemically in the blood and bone marrow. These tumor-induced NETs (t-NETs) are driven by a ROS-mediated pathway dependent on CAF-derived Amyloid ß, a peptide implicated in both neurodegenerative and inflammatory disorders. Inhibition of NETosis in murine tumors skews neutrophils to an anti-tumor phenotype, preventing tumor growth; reciprocally, t-NETs enhance CAF activation. Mirroring observations in mice, CAFs are detected juxtaposed to NETs in human melanoma and pancreatic adenocarcinoma, and show elevated amyloid and ß-Secretase expression which correlates with poor prognosis. In summary, we report that CAFs drive NETosis to support cancer progression, identifying Amyloid ß as the protagonist and potential therapeutic target.

Stromal regulation of tumor-associated lymphatics.

Recent advances have identified a growing array of roles played by lymphatics in the tumor microenvironment, from providing a route of metastasis to immune modulation. The tumor microenvironment represents an exceptionally complex, dynamic niche comprised of a diverse mixture of cancer cells and normal host cells termed the stroma. This review discusses our current understanding of stromal elements and how they regulate lymphatic growth and functional properties in the tumor context.

Comparative adhesive and migratory properties of mesenchymal stem cells from different tissues.

BACKGROUND: Mesenchymal stem cells (MSC) are used in therapy, often by injection into the blood. OBJECTIVE: We aimed to compare the adhesive and migratory properties of MSC from umbilical cords (UCMSC), bone marrow (BMMSC) or trabecular bone (TBMSC), which might influence delivery to injured tissue. METHODS: MSC were perfused through glass capillaries coated with matrix proteins, collagen or fibronectin, or albumin. Adherent cells were counted microscopically and their spreading analysed over time. MSC migration through 8 µm pore filters coated with the same proteins was analysed. RESULTS: The number of MSC adhering to collagen was greater than fibronectin, decreased as wall shear rate increased from 17 to 70 s-1, and was in the order UCMSC>BMMSC>TBMSC. Conversely, spreading was more effective on fibronectin and was in the order BMMSC>TBMSC≥UCMSC. Migration was promoted by coating the lower surface of filters with either matrix protein, with UCMSC migrating more efficiently than BMMSC. CONCLUSIONS: MSC show origin-dependent variations in their efficiency of capture from flow and subsequent spreading or ability to migrate on matrix proteins. UCMSC showed most efficient capture from flow, which was followed by less spreading, but more rapid migration. These responses might be associated with more effective delivery from the circulation into damaged tissue.

Mesenchymal Stem Cells as Endogenous Regulators of Inflammation.

This chapter discusses the regulatory role of endogenous mesenchymal stem cells (MSC) during an inflammatory response. MSC are a heterogeneous population of multipotent cells that normally contribute towards tissue maintenance and repair but have garnered significant scientific interest for their potent immunomodulatory potential. It is through these physicochemical interactions that MSC are able to exert an anti-inflammatory response on neighbouring stromal and haematopoietic cells. However, the impact of the chronic inflammatory environment on MSC function remains to be determined. Understanding the relationship of MSC between resolution of inflammation and autoimmunity will both offer new insights in the use of MSC as a therapeutic, and also their involvement in the pathogenesis of inflammatory disorders.

Origin-Specific Adhesive Interactions of Mesenchymal Stem Cells with Platelets Influence Their Behavior After Infusion.

We investigated the adhesive behavior of mesenchymal stem cells (MSC) in blood, which might influence their fate when infused as therapy. Isolated human bone marrow MSC (BMMSC) or umbilical cord MSC (UCMSC) adhered efficiently from flow to the matrix proteins, collagen, or fibronectin, but did not adhere to endothelial selectins. However, when suspended in blood, BMMSC no longer adhered to collagen, while UCMSC adhered along with many aggregated platelets. Neither MSC adhered to fibronectin from flowing blood, although the fibronectin surface did become coated with a platelet monolayer. UCMSC induced platelet aggregation in platelet rich plasma, and caused a marked drop in platelet count when mixed with whole human or mouse blood in vitro, or when infused into mice. In contrast, BMMSC did not activate platelets or induce changes in platelet count. Interestingly, isolated UCMSC and BMMSC both adhered to predeposited platelets. The differences in behavior in blood were attributable to expression of podoplanin (an activating ligand for the platelet receptor CLEC-2), which was detected on UCMSC, but not BMMSC. Thus, platelets were activated when bound to UCMSC, but not BMMSC. Platelet aggregation by UCMSC was inhibited by recombinant soluble CLEC-2, and UCMSC did not cause a reduction in platelet count when mixed with blood from mice deficient in CLEC-2. We predict that both MSC would carry platelets in the blood, but their interaction with vascular endothelium would depend on podoplanin-induced activation of the bound platelets. Such interactions with platelets might target MSC to damaged tissue, but could also be thrombotic. Stem Cells 2018;36:1062-1074.

Cancer-associated fibroblasts induce antigen-specific deletion of CD8 + T Cells to protect tumour cells.

Tumours have developed strategies to interfere with most steps required for anti-tumour immune responses. Although many populations contribute to anti-tumour responses, tumour-infiltrating cytotoxic T cells dominate, hence, many suppressive strategies act to inhibit these. Tumour-associated T cells are frequently restricted to stromal zones rather than tumour islands, raising the possibility that the tumour microenvironment, where crosstalk between malignant and "normal" stromal cells exists, may be critical for T cell suppression. We provide evidence of direct interactions between stroma and T cells driving suppression, showing that cancer-associated fibroblasts (CAFs) sample, process and cross-present antigen, killing CD8+ T cells in an antigen-specific, antigen-dependent manner via PD-L2 and FASL. Inhibitory ligand expression is observed in CAFs from human tumours, and neutralisation of PD-L2 or FASL reactivates T cell cytotoxic capacity in vitro and in vivo. Thus, CAFs support T cell suppression within the tumour microenvironment by a mechanism dependent on immune checkpoint activation.

Identification of a transitional fibroblast function in very early rheumatoid arthritis.

OBJECTIVES: Synovial fibroblasts actively regulate the inflammatory infiltrate by communicating with neighbouring endothelial cells (EC). Surprisingly, little is known about how the development of rheumatoid arthritis (RA) alters these immunomodulatory properties. We examined the effects of phase of RA and disease outcome (resolving vs persistence) on fibroblast crosstalk with EC and regulation of lymphocyte recruitment. METHODS: Fibroblasts were isolated from patients without synovitis, with resolving arthritis, very early RA (VeRA; symptom ≤12 weeks) and established RA undergoing joint replacement (JRep) surgery. Endothelial-fibroblast cocultures were formed on opposite sides of porous filters. Lymphocyte adhesion from flow, secretion of soluble mediators and interleukin 6 (IL-6) signalling were assessed. RESULTS: Fibroblasts from non-inflamed and resolving arthritis were immunosuppressive, inhibiting lymphocyte recruitment to cytokine-treated endothelium. This effect was lost very early in the development of RA, such that fibroblasts no longer suppressed recruitment. Changes in IL-6 and transforming growth factor beta 1 (TGF-ß1) signalling appeared critical for the loss of the immunosuppressive phenotype. In the absence of exogenous cytokines, JRep, but not VeRA, fibroblasts activated endothelium to support lymphocyte. CONCLUSIONS: In RA, fibroblasts undergo two distinct changes in function: first a loss of immunosuppressive responses early in disease development, followed by the later acquisition of a stimulatory phenotype. Fibroblasts exhibit a transitional functional phenotype during the first 3 months of symptoms that contributes to the accumulation of persistent infiltrates. Finally, the role of IL-6 and TGF-ß1 changes from immunosuppressive in resolving arthritis to stimulatory very early in the development of RA. Early interventions targeting 'pathogenic' fibroblasts may be required in order to restore protective regulatory processes.

Adipogenic Differentiation of Mesenchymal Stem Cells Alters Their Immunomodulatory Properties in a Tissue-Specific Manner.

Chronic inflammation is associated with formation of ectopic fat deposits that might represent damage-induced aberrant mesenchymal stem cell (MSC) differentiation. Such deposits are associated with increased levels of inflammatory infiltrate and poor prognosis. Here we tested the hypothesis that differentiation from MSC to adipocytes in inflamed tissue might contribute to chronicity through loss of immunomodulatory function. We assessed the effects of adipogenic differentiation of MSC isolated from bone marrow or adipose tissue on their capacity to regulate neutrophil recruitment by endothelial cells and compared the differentiated cells to primary adipocytes from adipose tissue. Bone marrow derived MSC were immunosuppressive, inhibiting neutrophil recruitment to TNFα-treated endothelial cells (EC), but MSC-derived adipocytes were no longer able to suppress neutrophil adhesion. Changes in IL-6 and TGFß1 signalling appeared critical for the loss of the immunosuppressive phenotype. In contrast, native stromal cells, adipocytes derived from them, and mature adipocytes from adipose tissue were all immunoprotective. Thus disruption of normal tissue stroma homeostasis, as occurs in chronic inflammatory diseases, might drive "abnormal" adipogenesis which adversely influences the behavior of MSC and contributes to pathogenic recruitment of leukocytes. Interestingly, stromal cells programmed in native fat tissue retain an immunoprotective phenotype. Stem Cells 2017;35:1636-1646.

Biomimetic Peptides for the Treatment of Cancer.

Cancer remains one of the leading causes of death worldwide, indicating that current cancer therapies are ineffective. Therefore, new treatments with high specificity and low toxicity are needed. Cancerous cells can be distinguished from normal cells based on expression of key proteins, namely surface proteins, scaffold proteins and signaling molecules. Moreover, cancer cells communicate with the tumor microenvironment consisting of a heterogenous population of cells, extracellular matrix components and soluble factors such as cytokines/chemokines and growth factors. Most therapeutic interventions have been designed to specifically target these proteins of interest. Biomimetic peptides (BPs) are artificially designed peptides that imitate the action of parent proteins or peptides. BPs can be classified into at least three types based on their target molecule: BPs that target (i) cell-surface molecules, (ii) intracellular molecules, and (iii) cancer cell-tumor microenvironment interactions. In this review, we analyze/discuss the current strategies for targeting tumors using BPs.

Comparative Ability of Mesenchymal Stromal Cells from Different Tissues to Limit Neutrophil Recruitment to Inflamed Endothelium.

Mesenchymal stromal cells (MSC) are tissue-resident stromal cells capable of modulating immune responses, including leukocyte recruitment by endothelial cells (EC). However, the comparative potency of MSC from different sources in suppressing recruitment, and the necessity for close contact with endothelium remain uncertain, although these factors have implications for use of MSC in therapy. We thus compared the effects of MSC isolated from bone marrow, Wharton's jelly, and trabecular bone on neutrophil recruitment to cytokine-stimulated EC, using co-culture models with different degrees of proximity between MSC and EC. All types of MSC suppressed neutrophil adhesion to inflamed endothelium but not neutrophil transmigration, whether directly incorporated into endothelial monolayers or separated from them by thin micropore filters. Further increase in the separation of the two cell types tended to reduce efficacy, although this diminution was least for the bone marrow MSC. Immuno-protective effects of MSC were also diminished with repeated passage; with BMMSC, but not WJMSC, completing losing their suppressive effect by passage 7. Conditioned media from all co-cultures suppressed neutrophil recruitment, and IL-6 was identified as a common bioactive mediator. These results suggest endogenous MSC have a homeostatic role in limiting inflammatory leukocyte infiltration in a range of tissues. Since released soluble mediators might have effects locally or remotely, infusion of MSC into blood or direct injection into target organs might be efficacious, but in either case, cross-talk between EC and MSC appears necessary.

Mesenchymal Stem Cell Therapy for Autoimmune Disease: Risks and Rewards.

Mesenchymal stem cells (MSC) possess a range of immunomodulatory properties which they exert through soluble mediators and through direct cell-cell contact. Due to these immune regulatory properties, the safety and clinical efficacy of MSC treatment has been tested in a number of autoimmune disorders. In this review we analyze the current data from early phase trials into Crohn's disease, systemic lupus erythematosus, and rheumatoid arthritis. In general, no adverse side effects were observed in patients treated with MSC; however, their clinical efficacy is difficult to interpret. Systemic or site-specific administration of MSC has been reported to exert potent immunomodulatory effects in 7 of the 11 trials discussed. Nonetheless, the mechanism(s) by which MSC exert their regulatory effects in vivo remain largely unknown. We discuss potential limitations or safety concerns associated with MSC therapy, including the heterogeneity of MSC and their potential contribution to disease pathogenesis, which need to be considered when designing future clinical trials, along with the need to standardize trial design. Although we are bridging the translational gap between scientific observations on MSC function and clinical applications for therapy, our understanding of basic MSC biology is still limited. Despite these issues, large, double-blinded, multicenter clinical trials are already underway. Further research into the endogenous function of MSC is required to elucidate the mechanism by which therapeutic MSC are acting.

Analyzing the effects of stromal cells on the recruitment of leukocytes from flow.

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro "vascular" models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment. Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNFα) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy. In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNFα-stimulated endothelium. Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.