Dysregulated TFR and TFH cells correlate with B-cell differentiation and antibody production in autoimmune hepatitis

Follicular helper T (TFH) cell provides germinal centre (GC) B cell with critical signals for autoantibody production in the immunopathogenesis and progression of autoimmune hepatitis (AIH). However, the immunoregulatory functions of follicular regulatory T (TFR) cell in AIH are still unclear. The numbers of circulating TFR/TFH cells were measured in AIH patients. Moreover, we established experimental autoimmune hepatitis (EAH) model to examine the function of TFR cells on B-cell differentiation and autoantibody production in vivo and vitro. AIH patients had significantly increased numbers of TFH cells and decreased numbers of TFR cells as well as imbalanced TFR/TFH-type cytokines (IL-10, TGF-β1 and IL-21) compared with healthy controls (HCs). In addition, TFR cell numbers negatively correlated with TFH cell numbers. Also, serum hypergammaglobulinaemia (IgG and IgM) concentration negatively correlated the levels of serum IL-21, but positively correlated with the levels of serum IL-10 in AIH patients. Furthermore, in comparison with control group, significantly higher frequencies of spleen TFR cells but lower frequencies of spleen TFH cells were detected in the EAH group. Further analysis found that TFR cells simultaneously express the phenotypic characteristics of Treg and TFH cells, but exercise as negative regulators of autoantibody production in vitro culture. Our findings demonstrated that dysregulated between TFR and TFH cells might cause excessive production of autoantibodies and destruction of the immune homeostasis, leading to the immunopathological process in AIH.     

Novel aspects of parenchymal–mesenchymal interactions: from cell types to molecules and beyond

Mesenchymal stem or stromal cells (MSCs) were initially isolated from the bone marrow and received their name on the basis of their ability to differentiate into multiple lineages such as bone, cartilage, fat and muscle. However, more recent studies suggest that MSCs residing in perivascular compartments of the small and large blood vessels play a regulatory function supporting physiologic and pathologic responses of parenchymal cells, which define the functional representation of an organ or tissue. MSCs secrete or express factors that reach neighbouring parenchymal cells via either a paracrine effect or a direct cell‐to‐cell interaction promoting functional activity, survival and proliferation of the parenchymal cells. Previous concept of ‘epithelial–stromal’ interactions can now be widened. Given that MSC can also support hematopoietic, neuronal and other non‐epithelial parenchymal lineages, terms ‘parenchymal–stromal’ or ‘parenchymal–mesenchymal’ interactions may better describe the supportive or ‘trophic’ functions of MSC. Importantly, in many cases, MSCs specifically provide supportive microenvironment for the most primitive stem or progenitor populations and therefore can play a role as ‘stem/progenitor niche’ forming cells. So far, regulatory roles of MSCs have been reported in many tissues. In this review article, we summarize the latest studies that focused on the supportive function of MSC. This thread of research leads to a new perspective on the interactions between parenchymal and mesenchymal cells and justifies a principally novel approach for regenerative medicine based on co‐application of MSC and parenchymal cell for the most efficient tissue repair. Copyright © 2013 John Wiley & Sons, Ltd.     

Adenosine production: a common path for mesenchymal stem-cell and regulatory T-cell-mediated immunosuppression

Adenosine is an important molecule that exerts control on the immune system, by signaling through receptors lying on the surface of immune cells. This nucleotide is produced, in part, by the action of the ectoenzymes CD39 and CD73. Interestingly, these proteins are expressed on the cell surface of regulatory T-cells (Tregs) and mesenchymal stromal cells (MSCs)—two cell populations that have emerged as potential therapeutic tools in the field of cell therapy. In fact, the production of adenosine constitutes a mechanism used by both cell types to control the immune response. Recently, great scientific progress was obtained regarding the role of adenosine in the inflammatory environment. In this context, the present review focuses on the advances related to the impact of adenosine production over the immune modulatory activity of Tregs and MSCs, and how this nucleotide controls the biological functions of these cells. Finally, we mention the main challenges and hurdles to bring such molecule to clinical settings.     

The inhibition of T-cells proliferation by mouse mesenchymal stem cells through the induction of p16INK4A-cyclin D1/cdk4 and p21waf1, p27kip1-cyclin E/cdk2 pathways

Mesenchymal stem cells (MSCs) have been shown to down-regulate T-cell responses. However, the mechanisms underlying remain unknown. In this study, we report that BALB/c bone marrow-derived MSCs inhibit the proliferation of allogeneic T-cells in mixed lymphocyte reactions (MLR), This inhibition is dependent on cell-cell contact, and do not induce apoptosis. Furthermore, cell-cycle analyses reveal that T-cells, in the presence of MSCs, are arrested in the G0/G1 phase through. The blockage of phosphorylation of retinoblastoma protein (Rb), mediated by the p16(INK4A)-cyclin D1/cdk4 complex and p21(waf1), p27(kip1)-cyclin E/cdk2 complex pathway. Our results suggest that MSCs may perform a crucial function in the maintenance of immune homeostasis, via direct regulation of the clonal expansion of activated T-cells. The novel T-cell regulatory mechanism exhibited by MSCs may prove useful in a variety of therapeutic applications.     

Mast Cells Mobilize Myeloid-Derived Suppressor Cells and Treg Cells in Tumor Microenvironment via IL-17 Pathway in Murine Hepatocarcinoma Model

Tumor immunosuppression is commonly braided with chronic inflammation during tumor development. However, the relationship between immunosuppression and inflammation in tumor microenvironment is still unclear. We have demonstrated that mast cells are accumulated and exacerbate the inflammation and immunosuppression in tumor microenvironment via SCF/c-kit signaling pathway. Here, we further elucidate the underlying mechanism, which involves both myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells. Our data showed that mast cells mobilized the infiltration of MDSCs to tumor and induced the production of IL-17 by MDSCs; MDSCs-derived IL-17 indirectly attracted Treg cells, enhanced their suppressor function, and induced the IL-9 production by Treg cells; in turn, IL-9 strengthened the survival and protumor effect of mast cells in tumor microenvironment. Our findings disclose a closed loop among mast cells, MDSCs and Treg cells in tumor microenvironment, which provides a new insight into the paralleled developments of inflammation and immunosuppression in tumor microenvironment. Based on these findings, we propose that targeting tumor inflammation might be a potential strategy to reverse the immunosuppression of tumor microenvironment, thus facilitating cancer immunotherapy.     

Clinical Significance of sIL-2R Levels in B-Cell Lymphomas

Elevated soluble interleukin-2 receptor (sIL-2R) in sera is observed in patients with malignant lymphoma (ML). Therefore, sIL-2R is commonly used as a diagnostic and prognostic marker for ML, but the mechanisms responsible for the increase in sIL-2R levels in patients with B-cell lymphomas have not yet been elucidated. We first hypothesized that lymphoma cells expressing IL-2R and some proteinases such as matrix metalloproteinases (MMPs) in the tumor microenvironment can give rise to increased sIL-2R in sera. However, flow cytometric studies revealed that few lymphoma cells expressed IL-2R α chain (CD25) in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL), and most CD25-expressing cells in the tumor were T-cells. Distinct correlations between CD25 expression on B-lymphoma cells and sIL-2R levels were not observed. We then confirmed that MMP-9 plays an important role in producing sIL-2R in functional studies. Immunohistochemical (IHC) analysis also revealed that MMP-9 is mainly derived from tumor-associated macrophages (TAMs). We therefore evaluated the number of CD68 and CD163 positive macrophages in the tumor microenvironment using IHC analysis. A positive correlation between the levels of sIL-2R in sera and the numbers of CD68 positive macrophages in the tumor microenvironment was confirmed in FL and extranodal DLBCL. These results may be useful in understanding the pathophysiology of B-cell lymphomas.     

Role of mesenchymal stromal cell-derived extracellular vesicles in tumour microenvironment

Stromal cells, deriving from mesenchymal stromal cells (MSCs), are crucial component of tumour microenvironment and represent key regulators of tumour processes. MSCs can be recruited to the tumour environment and interact with many cellular elements, thus influencing tumour biology. Cell-to-cell communication is in part mediated by the release of extracellular vesicle (EVs). EVs can induce significant molecular changes in recipient cells, delivering bioactive molecules. In this review, we describe the MSC-derived EVs content and discuss their role in different processes related to cancer biology. Furthermore, we summarize chemical or biological EVs modifications aiming to develop more efficient antitumor therapies.     

间充质干细胞与肿瘤形成的研究进展

间充质干细胞(Mesenchymal stem cells,MSCs)具有独特的免疫调节作用、自我更新和跨胚层多向分化的潜能,存在于许多组织中并活跃地向组织损伤部位迁移,参与伤口修复。在对肿瘤的信号发生反应后,MSCs不断被招募并成为肿瘤微环境的成分。肿瘤相关MSCs(Tumor-associated MSCs, TA-MSCs)在肿瘤发生、促进、进展和转移中有重要作用。本文对MSCs在调节肿瘤细胞的存活、增殖、迁移、药物抵抗中如何发挥作用,以及MSCs对肿瘤微环境免疫状态的影响作一综述。我们强调了MSCs和其他肿瘤基质细胞之间的复杂关系,特别是炎症细胞可以改变肿瘤微环境的免疫状态,以期通过对TA-MSCs进一步的研究来取得对不同肿瘤类型和肿瘤进展不同阶段中肿瘤相关MSCs功能的更好的理解,并优化MSCs来得到更有效和安全的MSCs为基础的肿瘤治疗。MSCs已被有效用于治疗慢性炎性疾病和慢性损伤,因此,其机制方面的研究还有利于在其他疾病中合理利用MSCs从而达到疾病治疗的目的。     

p73 is expressed in human thymic epithelial cells.

The thymus is a heterogeneous immune organ in which immature T-cells develop and eventually specialize to make certain immune responses of their own. Among various types of stromal cells in the thymus, thymic epithelial cells (TECs) have a crucially important function for presenting self-antigens and secreting cytokines to thymocytes for their maturation into T-cells. In this study we show that the p73 gene, a homologue of the tumor suppressor gene p53, was expressed in the nucleus of the human TEC in vivo and in TEC lines in vitro. Because p73 has the capacity to be a transactivator like p53, it may contribute to T-cell development in the context of TEC biology as regulated in the cell cycle and apoptosis.     

Lymphocyte Activation Gene 3 (LAG-3) Modulates the Ability of CD4 T-cells to Be Suppressed In Vivo

Lymphocyte Activation Gene – 3 (LAG-3) is an immune checkpoint molecule that regulates both T-cell activation and homeostasis. However, the molecular mechanisms underlying LAG-3’s function are generally unknown. Using a model in which LAG-3 blockade or absence reliably augmented homeostatic proliferation in vivo, we found that IL-2 and STAT5 are critical for LAG-3 function. Similarly, LAG-3 blockade was ineffective in the absence of regulatory T-cells (Treg), suggesting an important role for LAG-3 in either the responsiveness of conventional T-cells (Tconv) to regulation, or a relative defect in the ability of LAG-3 KO regulatory T-cells (Treg) to suppress the proliferation of Tconv. In this model, LAG-3 KO Treg suppressed proliferation in a manner fairly similar to wild-type (WT) Treg, but LAG-3 KO Tconv were relatively resistant to suppression. Further studies also identified a role for LAG-3 in the induction/expansion of Treg. Finally, we found that LAG-3 blockade (or knockout) led to a relative skewing of naïve CD4 T-cells toward a T_H1 phenotype both in vitro and in in vivo. Together, these data suggest that LAG-3 expression on Tconv cells makes them more susceptible to Treg based suppression, and also regulates the development of a T_H1 T-cell response.     

Fetal Liver Stromal Cells Support Blast Growth in Transient Abnormal Myelopoiesis in Down Syndrome Through GM‐CSF

Transient abnormal myelopoiesis (TAM) in neonates with Down syndrome, which spontaneously resolves within several weeks or months after birth, may represent a very special form of leukemia arising in the fetal liver (FL). To explore the role of the fetal hematopoietic microenvironment in the pathogenesis of TAM, we examined the in vitro influences of stromal cells of human FL and fetal bone marrow (FBM) on the growth of TAM blasts. Both FL and FBM stromal cells expressed mesenchymal cell antigens (vimentin, α‐smooth muscle actin, CD146, and nestin), being consistent with perivascular cells/mesenchymal stem cells that support hematopoietic stem cells. In addition, a small fraction of the FL stromal cells expressed an epithelial marker, cytokeratin 8, indicating that they could be cells in epithelial‐mesenchymal transition (EMT). In the coculture system, stromal cells of the FL, but not FBM, potently supported the growth of TAM blast progenitors, mainly through humoral factors. High concentrations of hematopoietic growth factors were detected in culture supernatants of the FL stromal cells and a neutralizing antibody against granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) almost completely inhibited the growth‐supportive activity of the culture supernatants. These results indicate that FL stromal cells with unique characteristics of EMT cells provide a pivotal hematopoietic microenvironment for TAM blasts and that GM‐CSF produced by FL stromal cells may play an important role in the pathogenesis of TAM. J. Cell. Biochem. 115: 1176–1186, 2014. © 2014 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals, Inc.     

An Increase in CD3+CD4+CD25+ Regulatory T Cells after Administration of Umbilical Cord-Derived Mesenchymal Stem Cells during Sepsis

Sepsis remains an important cause of death worldwide, and vigorous immune responses during sepsis could be beneficial for bacterial clearance but at the price of collateral damage to self tissues. Mesenchymal stem cells (MSCs) have been found to modulate the immune system and attenuate sepsis. In the present study, MSCs derived from bone marrow and umbilical cord were used and compared. With a cecal ligation and puncture (CLP) model, the mechanisms of MSC-mediated immunoregulation during sepsis were studied by determining the changes of circulating inflammation-associated cytokine profiles and peripheral blood mononuclear cells 18 hours after CLP-induced sepsis. In vitro, bone marrow-derived MSCs (BMMSCs) and umbilical cord-derived MSCs (UCMSCs) showed a similar morphology and surface marker expression. UCMSCs had stronger potential for osteogenesis but lower for adipogenesis than BMMSCs. Compared with rats receiving PBS only after CLP, the percentage of circulating CD3+CD4+CD25+ regulatory T (Treg) cells and the ratio of Treg cells/T cells were elevated significantly in rats receiving MSCs. Further experiment regarding Treg cell function demonstrated that the immunosuppressive capacity of Treg cells from rats with CLP-induced sepsis was decreased, but could be restored by administration of MSCs. Compared with rats receiving PBS only after CLP, serum levels of interleukin-6 and tumor necrosis factor-α were significantly lower in rats receiving MSCs after CLP. There were no differences between BMMSCs and UCMSCs. In summary, this work provides the first in vivo evidence that administering BMMSCs or UCMSCs to rats with CLP-induced sepsis could increase circulating CD3+CD4+CD25+ Treg cells and Treg cells/T cells ratio, enhance Treg cell suppressive function, and decrease serum levels of interleukin-6 and tumor necrosis factor-α, suggesting the immunomodulatory association of Treg cells and MSCs during sepsis.     

Immunomodulation of mesenchymal stromal cells on regulatory T cells and its possible mechanism

Mesenchymal stromal cells (MSCs) and regulatory T cells (Tregs) have both garnered abundant interests from immunologists worldwide, as both MSCs and Tregs can be considered immunosuppressive in their own right. But a little attention has been paid to the impacts of MSCs on Tregs. To clarify the effects of MSCs on Tregs, we performed the coculture systems within MSCs and Tregs. We confirmed that MSC-exposed Tregs are capable of more immunosuppressive than Tregs without coculturing with MSCs. And this augmenting suppressive capacity was accompanied with an upregulation of programmed cell death 1 receptor (PD-1) on Tregs. Importantly, we found that cell viability of Tregs was excluded from the influences of MSCs. Finally, we showed that PD-1/B7-H1 interactions and IL-10 might be responsible for the enhanced suppressive capability of MSC-exposed Tregs. Further analysis revealed that PD-1/B7-H1 interactions were not responsible for the productions of IL-10 and TGF-β₁ in the MSC-Treg coculture systems; in contrast, IL-10 rather than TGF-β₁ played a role in the upregualtion of PD-1. Furthermore, this is the first explorative study to evaluate the immunomodulation of MSCs on the suppressive capacity of Tregs in MSC–Treg in vitro coculture setting.     

Optimization of in vitro expansion of human multipotent mesenchymal stromal cells for cell-therapy approaches: further insights in the search for a fetal calf serum substitute

There is great interest in mesenchymal stromal cells (MSCs) for cell-therapy and tissue engineering approaches. MSCs are currently expanded in vitro in the presence of fetal calf serum (FCS); however, FCS raises concerns when used in clinical grade preparations. The aim of this study was to evaluate whether MSCs expanded in medium supplemented with platelet-lysate (PL), already shown to promote MSC growth, are endowed with biological properties appropriate for cell-therapy approaches. We confirm previously published data showing that MSCs expanded in either FCS or PL display comparable morphology, phenotype, and differentiation capacity, while PL-MSCs were superior in terms of clonogenic efficiency and proliferative capacity. We further extended these data by investigating the immune-regulatory effect of MSCs on the alloantigen-specific immune response in mixed lymphocyte culture (MLC). We found that MSCs-PL are comparable to MSCs-FCS in their capacity to: (i) decrease alloantigen-induced cytotoxic activity; (ii) favor differentiation of CD4+ T-cell subsets expressing a Treg phenotype; (iii) increase early secretion of IL-10 in MLC supernatant, as well as induce a striking augmentation of IL-6 production. As compared with MSCs-PL, MSCs-FCS were more efficient in suppressing alloantigen-induced lymphocyte subset proliferation and reducing early IFNgamma-secretion. Resistance to spontaneous transformation into tumor cells of expanded MSCs was demonstrated by molecular karyotyping and maintenance of normal morphology/phenotype after prolonged in vitro culture. Our data support the immunological functional plasticity of MSCs and suggest that MSCs-PL can be used as an alternative to MSCs-FCS, although these latter cells might be more suitable for preventing/treating alloreactivity-related immune complications.     

Pancreatic cancer growth promoted by bone marrow mesenchymal stromal cell–derived IL-6 is reversed predominantly by IL-6 blockade

Pancreatic cancer is a highly lethal cancer characterized by local invasiveness, early metastasis, recurrence and high resistance to current therapies. Extensive stroma or desmoplasia is a key histological feature of the disease, and interactions between cancer and stromal cells are critical for pancreatic cancer development and progression. Mesenchymal stromal cells [MSCs] exhibit preferential tropism to primary and metastatic tumor sites and may either suppress or support tumor growth. Although MSCs represent a potential source of pancreatic cancer stroma, their contribution to pancreatic tumor growth remains poorly known. Here, we show that bone marrow MSCs significantly contribute to pancreatic cancer growth in vitro and in vivo. Furthermore, MSCs create a pro-carcinogenic microenvironment through the release of key factors mediating growth and angiogenesis, including interleukin (IL)-6, IL-8, vascular endothelial growth factor and activation of STAT3 signaling in tumor cells. IL-6 released by MSCs was largely responsible for the pro-tumorigenic effects of MSCs. Knockdown of IL-6 expression in MSCs by small interfering RNA (siRNA) abolished the MSC growth-promoting effect in vitro, reducing tumor cell proliferation and clonogenic potential. In addition, in a heterotopic nude mouse model of human pancreatic tumor xenografts, blockade of IL-6 with the anti-IL-6 receptor antibody, tocilizumab, or of its downstream effector STAT3 with the small molecule STAT3 inhibitor S3I-201, abrogated MSC-mediated tumor promotion and delayed tumor formation significantly. Our data demonstrate that MSCs promote pancreatic cancer growth, with IL-6 produced by MSCs playing a pivotal role.     

Dual role of mesenchymal stem/stromal cells and their cell-free extracellular vesicles in colorectal cancer

Colorectal cancer (CRC) is one of the main causes of cancer-related deaths. However, the surgical control of the CRC progression is difficult, and in most cases, the metastasis leads to cancer-related mortality. Mesenchymal stem/stromal cells (MSCs) with potential translational applications in regenerative medicine have been widely researched for several years. MSCs could affect tumor development through secreting exosomes. The beneficial properties of stem cells are attributed to their cell–cell interactions as well as the secretion of paracrine factors in the tissue microenvironment. For several years, exosomes have been used as a cell-free therapy to regulate the fate of tumor cells in a tumor microenvironment. This review discusses the recent advances and current understanding of assessing MSC-derived exosomes for possible cell-free therapy in CRC.     

Current Understanding of Cytotoxic T Lymphocyte Antigen-4 (CTLA-4) Signaling in T-Cell Biology and Disease Therapy

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an immune checkpoint molecule that is mainly expressed on activated T cells and regulatory T (Treg) cells that inhibits T-cell activation and regulates immune homeostasis. Due to the crucial functions of CTLA-4 in T-cell biology, CTLA-4-targeted immunotherapies have been developed for autoimmune disease as well as cancers. CTLA-4 is known to compete with CD28 to interact with B7, but some studies have revealed that its downstream signaling is independent of its ligand interaction. As a signaling domain of CTLA-4, the tyrosine motif plays a role in inhibiting T-cell activation. Recently, the lysine motif has been shown to be required for the function of Treg cells, emphasizing the importance of CTLA-4 signaling. In this review, we summarize the current understanding of CTLA-4 biology and molecular signaling events and discuss strategies to target CTLA-4 signaling for immune modulation and disease therapy.     

Protein kinase C zeta regulates interleukin-8-mediated stromal-derived factor-1 expression and migration of human mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) are bone marrow-derived cells with multipotent differentiation capability that are mobilized into the circulation in response to injury and localize to areas of tissue damage including solid tumors. They have the capacity to adopt a phenotype similar to carcinoma-associated fibroblasts (CAFs) and, like CAFs, promote tumor growth. The molecular communication between tumor cells and MSCs has not been well defined. However, MSCs have increased expression of the chemokine stromal-derived factor 1 (SDF-1) when exposed to conditioned medium from tumor cells. Additionally, SDF-1 has been shown to be important in the promotion of tumor growth by CAFs. These data suggest that the SDF-1 signaling axis is a key feature of the tumor microenvironment. In this report, we demonstrate that interleukin 8 (IL-8) induces an increase in SDF-1 expression by MSCs. The increase in SDF-1 expression in response to IL-8 is mediated by the activation of the protein kinase C (PKC) zeta isoform. In a functional assay, activation of PKC is required for in vitro MSC migration in response to tumor conditioned medium. These results indicate that IL-8-mediated SDF-1 production by MSCs requires PKC zeta activation. This signaling pathway provides insight into possible molecular targets for cancer therapy aimed at disrupting the interaction between components of the tumor microenvironment.