Pellino3 targets the IRF7 pathway and facilitates autoregulation of TLR3- and viral-induced expression of type I interferons.

Toll-like receptors (TLRs) sense pathogen-associated molecules and respond by inducing cytokines and type I interferon. Here we show that genetic ablation of the E3 ubiquitin ligase Pellino3 augmented the expression of type I interferon but not of proinflammatory cytokines in response to TLR3 activation. Pellino3-deficient mice had greater resistance against the pathogenic and lethal effects of encephalomyocarditis virus (EMCV). TLR3 signaling induced Pellino3, which in turn interacted with and ubiquitinated TRAF6. This modification suppressed the ability of TRAF6 to interact with and activate IRF7, resulting in downregulation of type I interferon expression. Our findings highlight a new physiological role for Pellino3 and define a new autoregulatory network for controlling type I interferon expression.

Helminth antigens modulate human PBMCs, attenuating disease progression in a humanised mouse model of graft versus host disease.

Fasciola hepatica is a trematode worm that causes fascioliasis, a neglected tropical disease in humans and livestock. To gain insight into the host-parasite interactions that facilitate infection, we have investigated the immunomodulatory properties of the parasite's tegumental coat (FhTeg), a major antigen source that is sloughed off and renewed every 2-3 h as the worm migrates through host tissue. Using mouse models of infection, we have previously shown that FhTeg induces a novel phenotype of dendritic cells that induce anergic CD4+ T-cells. We proposed that this induced state of hyporesponsiveness characterised by suppression of cell proliferation and cytokine secretion was one mechanism by which F. hepatica prevented host protective immunity to support the parasite survival. To determine if the same mechanisms are utilised during human infections, we have now examined the interaction of FhTeg with human PBMCs. FhTeg binds to and modulates cytokine production in human PBMCs, in particular targeting the CD4+ population resulting in reduced levels of TNF, IL-2 and IFNγ and increased markers of anergy. Furthermore, the adoptive transfer of FhTeg stimulated PBMCs to a humanised model of acute graft versus host disease (GvHD) attenuated disease progression by increasing survival and reducing pathological scores. These mice also displayed a significant decrease in the total number of human CD4+ cells expressing TNF, IL-2 and IFNγ in the spleen, liver and lung. This study therefore concurs with evidence from ruminant and murine models of infection suggesting that anergic CD4+ T cells are associated with successful Fasciola hepatica infection and highlights an important role for FhTeg in contributing to the overall immunosuppressive effects of this parasite.

Differential effects of the cystic fibrosis lung inflammatory environment on mesenchymal stromal cells.

Growing evidence demonstrates that human mesenchymal stromal cells (MSCs) modify their in vivo anti-inflammatory actions depending on the specific inflammatory environment encountered. Understanding this better is crucial to refine MSC-based cell therapies for lung and other diseases. Using acute exacerbations of cystic fibrosis (CF) lung disease as a model, the effects of ex vivo MSC exposure to clinical bronchoalveolar lavage fluid (BALF) samples, as a surrogate for the in vivo clinical lung environment, on MSC viability, gene expression, secreted cytokines, and mitochondrial function were compared with effects of BALF collected from healthy volunteers. CF BALF samples that cultured positive for Aspergillus sp. (Asp) induced rapid MSC death, usually within several hours of exposure. Further analyses suggested the fungal toxin gliotoxin as a potential mediator contributing to CF BALF-induced MSC death. RNA sequencing analyses of MSCs exposed to either Asp+ or Asp- CF BALF samples identified a number of differentially expressed transcripts, including those involved in interferon signaling, antimicrobial gene expression, and cell death. Toxicity did not correlate with bacterial lung infections. These results suggest that the potential use of MSC-based cell therapies for CF or other lung diseases may not be warranted in the presence of Aspergillus.

Linocin and OmpW Are Involved in Attachment of the Cystic Fibrosis-Associated Pathogen Burkholderia cepacia Complex to Lung Epithelial Cells and Protect Mice against Infection.

Members of the Burkholderia cepacia complex (Bcc) cause chronic opportunistic lung infections in people with cystic fibrosis (CF), resulting in a gradual lung function decline and, ultimately, patient death. The Bcc is a complex of 20 species and is rarely eradicated once a patient is colonized; therefore, vaccination may represent a better therapeutic option. We developed a new proteomics approach to identify bacterial proteins that are involved in the attachment of Bcc bacteria to lung epithelial cells. Fourteen proteins were reproducibly identified by two-dimensional gel electrophoresis from four Bcc strains representative of two Bcc species: Burkholderia cenocepacia, the most virulent, and B. multivorans, the most frequently acquired. Seven proteins were identified in both species, but only two were common to all four strains, linocin and OmpW. Both proteins were selected based on previously reported data on these proteins in other species. Escherichia coli strains expressing recombinant linocin and OmpW showed enhanced attachment (4.2- and 3.9-fold) to lung cells compared to the control, confirming that both proteins are involved in host cell attachment. Immunoproteomic analysis using serum from Bcc-colonized CF patients confirmed that both proteins elicit potent humoral responses in vivo Mice immunized with either recombinant linocin or OmpW were protected from B. cenocepacia and B. multivorans challenge. Both antigens induced potent antigen-specific antibody responses and stimulated strong cytokine responses. In conclusion, our approach identified adhesins that induced excellent protection against two Bcc species and are promising vaccine candidates for a multisubunit vaccine. Furthermore, this study highlights the potential of our proteomics approach to identify potent antigens against other difficult pathogens.

Mesenchymal stem cell inhibition of T-helper 17 cell- differentiation is triggered by cell-cell contact and mediated by prostaglandin E2 via the EP4 receptor.

Mesenchymal stem cells (MSCs) inhibit T-cell activation and proliferation but their effects on individual T-cell-effector pathways and on memory versus naïve T cells remain unclear. MSC influence on the differentiation of naïve and memory CD4(+) T cells toward the Th17 phenotype was examined. CD4(+) T cells exposed to Th17-skewing conditions exhibited reduced CD25 and IL-17A expression following MSC co-culture. Inhibition of IL-17A production persisted upon re-stimulation in the absence of MSCs. These effects were attenuated when cell-cell contact was prevented. Th17 cultures from highly purified naïve- and memory-phenotype responders were similarly inhibited. Th17 inhibition by MSCs was reversed by indomethacin and a selective COX-2 inhibitor. Media from MSC/Th17 co-cultures contained increased prostaglandin E2 (PGE2) levels and potently suppressed Th17 differentiation in fresh cultures. MSC-mediated Th17 inhibition was reversed by a selective EP4 antagonist and was mimicked by synthetic PGE2 and a selective EP4 agonist. Activation-induced IL-17A secretion by naturally occurring, effector-memory Th17 cells from a urinary obstruction model was also inhibited by MSC co-culture in a COX-dependent manner. Overall, MSCs potently inhibit Th17 differentiation from naïve and memory T-cell precursors and inhibit naturally-occurring Th17 cells derived from a site of inflammation. Suppression entails cell-contact-dependent COX-2 induction resulting in direct Th17 inhibition by PGE2 via EP4.

Allogeneic mesenchymal stem cells: agents of immune modulation.

Adult mesenchymal stem cells possess a remarkably diverse array of immunosuppressive characteristics. The capacity to suppress the regular processes of allogeneic rejection, have allowed the use of tissue mismatched cells as therapeutic approaches in regenerative medicine and as agents of immune deviation. This review describes recent advances in understanding the mechanistic basis of mesenchymal stromal or stem cells (MSC) interaction with innate immunity. Particular emphasis is placed on the effect of Toll-like receptor signalling on MSC and a hypothesis that innate immune signals induce a 'licensing switch' in MSC is put forward. The mechanisms underlying MSC suppression of T cell responses and induction of regulatory populations are surveyed. Conflicting data regarding the influence of MSC on B cell function are outlined and discussed. Finally the limits to MSC mediated immune modulation are discussed with reference to the future clinical application of novel cell therapies.

IL-17A induces CCL28, supporting the chemotaxis of IgE-secreting B cells.

BACKGROUND: Atopic asthma is an allergic disease typically associated with T(H)2 cytokines. IL-17A is also associated with asthma, through the induction of chemokines. Mucosal CCL28 concentrations correlate with cellular recruitment to inflamed airways and support migration of IgA(+) B cells. Here, a link between IL-17A, CCL28 and IgE-secreting B cell chemotaxis is examined. METHODS: Primary human airway cells and the airway epithelial line A549 were used to characterize IL-17A receptor expression and the effect of IL-17A on CCL28 transcription and translation. B cells, differentiated to IgE+ cells ex vivo, were assessed for CCR10 surface expression and chemotaxis to CCL28 by flow cytometry, transwell migration and ELISpot. RESULTS: Human airway epithelium expressed both IL-17RA and IL-17RC, and was responsive to IL-17A stimulation. Cultured human IgE+ B cells expressed surface CCR10 and displayed CCR10-dependent chemotaxis towards recombinant CCL28. Enhanced levels of CCL28 were observed upon A549 cell incubation with IL-17A, and this up-regulation significantly increased the migration of IgE+ antibody-secreting B cells. The specificity of chemotaxis was confirmed by migration blockade in the presence of anti-CCL28 or anti-CCR10. CONCLUSIONS: This work identifies a novel chemokine for the migration of IgE+ B cells, in addition to characterizing induction of CCL28 by IL-17A. Taken together the results presented here propose a new role for IL-17A in the allergic airways, linking this cytokine with the recruitment of IgE+ antibody-secreting B cells, via the induction of CCL28. These observations justify further in vivo studies of larger cohorts.

Adeno-associated virus serotype 2 induces cell-mediated immune responses directed against multiple epitopes of the capsid protein VP1.

Adeno-associated virus serotype 2 (AAV-2) has been developed as a gene therapy vector. Antibody and cell-mediated immune responses to AAV-2 or AAV-2-transfected cells may confound the therapeutic use of such vectors in clinical practice. In one of the most detailed examinations of AAV-2 immunity in humans to date, cell-mediated and humoral immune responses to AAV-2 were characterized from a panel of healthy blood donors. The extent of AAV-2-specific antibody in humans was determined by examination of circulating AAV-2-specific total IgG levels in plasma from 45 normal donors. Forty-one donors were seropositive and responses were dominated by IgG1 and IgG2 subclasses. Conversely, AAV-2-specific IgG3 levels were consistently low in all donors. Cell-mediated immune recall responses were detectable in nearly half the population studied. In vitro restimulation with AAV-2 of peripheral blood mononuclear cell cultures from 16 donors elicited gamma interferon (IFN-gamma) (ten donors), interleukin-10 (IL-10) (eight donors) and interleukin-13 (IL-13) (four donors) responses. Using a series of overlapping peptides derived from the sequence of the VP1 viral capsid protein, a total of 59 candidate T-cell epitopes were identified. Human leukocyte antigen characterization of donors revealed that the population studied included diverse haplotypes, but that at least 17 epitopes were recognized by multiple donors and could be regarded as immunodominant. These data indicate that robust immunological memory to AAV-2 is established. The diversity of sequences recognized suggests that attempts to modify the AAV-2 capsid, as a strategy to avoid confounding immunity, will not be feasible.

The Necrobiology of Mesenchymal Stromal Cells Affects Therapeutic Efficacy.

Rapid progress is occurring in understanding the mechanisms underlying mesenchymal stromal cell (MSC)-based cell therapies (MSCT). However, the results of clinical trials, while demonstrating safety, have been varied in regard to efficacy. Recent data from different groups have shown profound and significant influences of the host inflammatory environment on MSCs delivered systemically or through organ-specific routes, for example intratracheal, with subsequent actions on potential MSC efficacies. Intriguingly in some models, it appears that dead or dying cells or subcellular particles derived from them, may contribute to therapeutic efficacy, at least in some circumstances. Thus, the broad cellular changes that accompany MSC death, autophagy, pre-apoptotic function, or indeed the host response to these processes may be essential to therapeutic efficacy. In this review, we summarize the existing literature concerning the necrobiology of MSCs and the available evidence that MSCs undergo autophagy, apoptosis, transfer mitochondria, or release subcellular particles with effector function in pathologic or inflammatory in vivo environments. Advances in understanding the role of immune effector cells in cell therapy, especially macrophages, suggest that the reprogramming of immunity associated with MSCT has a weighty influence on therapeutic efficacy. If correct, these data suggest novel approaches to enhancing the beneficial actions of MSCs that will vary with the inflammatory nature of different disease targets and may influence the choice between autologous or allogeneic or even xenogeneic cells as therapeutics.

Murine mesenchymal stem cells suppress dendritic cell migration, maturation and antigen presentation.

Mesenchymal stem cells (MSC) possess a wide range of immunosuppressive functions. Among these is the ability to inhibit CD4+ T cell proliferation. Dendritic cells (DC) play a role in initiating cell-mediated immunity; however, the immunosuppressive influence of MSC on professional antigen presenting cells remains unclear. DC exposed to TNF-alpha and cultured with murine MSC failed to show regular upregulation of maturation markers. Similarly, the presence of MSC abrogated the capacity of ovalbumin-pulsed DC to support antigen specific CD4+ T cell proliferation, or for DC to display an MHC class II- peptide complex recognizable by specific antibody. Interestingly, culture of MSC with DC resulted in reduced expression of CCR7 by DC following stimulation. Likewise, DC matured in the presence of MSC, showed significantly less migration to CCL19. In contrast, murine MSC prevented loss of expression of the tissue anchoring protein E-cadherin by DC. Modulation of DC maturation and function was not permanent and could be restored after removal of MSC. These data demonstrate that MSC modulate the three cardinal features of DC maturation, providing the first demonstration of MSC interference with DC migration.

A possible role for protein synthesis, extracellular signal-regulated kinase, and brain-derived neurotrophic factor in long-term spatial memory retention in the water maze.

Hippocampal protein synthesis is dependent upon a number of different molecular and cellular mechanisms that act together to make previously labile memories more stable and resistant to disruption. Both brain-derived neurotrophic factor (BDNF) and extracellular signal-regulated kinase (ERK) are known to play an important role in protein synthesis-dependent memory consolidation, via the mitogen-activated protein-kinase (MAP-K) signaling pathway during the transcription phase of protein synthesis. The current study investigates the influence of protein synthesis inhibition (PSI) by cycloheximide on spatial learning and memory. In an initial experiment, the authors utilized two doses of cycloheximide (0.5 mg/kg and 1.0 mg/kg, intraperitoneally) to determine the dose at which long-term (>24 hours) memories are impaired. A second experiment was designed to investigate the effect of PSI on the formation of cue-platform associations in the watermaze, and on BDNF and ERK expression in the hippocampus. At the higher dose (1.0 mg/kg) cycloheximide resulted in impaired retention of the water maze. BDNF and ERK expression was also down-regulated in animals injected with this dose of cycloheximide. Our results demonstrate a role of protein synthesis in spatial memory retention, along with a possible relationship between protein synthesis and hippocampal BDNF/ERK expression.

IFN-gamma and TNF-alpha differentially regulate immunomodulation by murine mesenchymal stem cells.

Murine mesenchymal stem cells (MSC) have the ability to inhibit allogeneic immune responses. Two different mechanisms, either cell contact-dependent or independent, have been proposed to account for this immunosuppression. The focus of this study was to elucidate the involvement of soluble suppressive factors secreted by murine MSC in an inflammatory setting, and their role in MSC immunomodulation. In a non-inflammatory environment, bone marrow derived murine MSC constitutively expressed low levels of COX-2, PGE-2, TGF-beta1 and HGF, but not IL-10, PD-1, PD-L1 or PD-L2. These MSC were able to significantly reduce alloantigen driven proliferation in mixed lymphocyte reactions as well as mitogen driven proliferation. The pro-inflammatory cytokines IFN-gamma and TNF-alpha did not ablate MSC mediated immunosuppression. MSC expression of PGE-2, IDO and PD-L1 was differentially regulated by these cytokines. COX-2 and PGE-2 expression by MSC were upregulated by both IFN-gamma and TNF-alpha, and using a biochemical inhibitor this was shown to have an essential, non-redundant role in modulating alloantigen-driven proliferation. However, the surface expression of PD-L1 was induced by IFN-gamma but not TNF-alpha and similarly functional IDO expression was only induced by IFN-gamma stimulation. Blocking studies using neutralising antibodies and biochemical antagonists revealed that while PD-L1 induction was not essential, IDO expression was a prerequisite for IFN-gamma mediated MSC immunomodulation. These data demonstrate that murine MSC expression of immunomodulatory factors dramatically changes in a pro-inflammatory environment and that IFN-gamma in particular has an important role in regulating MSC immunomodulatory factor expression.

Inflammation of the respiratory tract is associated with CCL28 and CCR10 expression in a murine model of allergic asthma.

Mouse models and in vitro cell culture were used to examine airway expression of the mucosal chemokine CCL28. Low levels of constitutively expressed mRNA were observed in transformed murine epithelial cells, but high levels could be induced by stimulation. Cytokines that signal through NF-kappaB, including IL-1beta and TNF-alpha or via JAK-STAT pathway including oncostatin M induced CCL28 in airway epithelial cells in vitro. Immunohistochemistry of murine airway tissue revealed that constitutive expression of CCL28 protein in vivo was low and not ubiquitous. However, abundant expression was detected in epithelia and lymphoid aggregates following allergic sensitization and challenge with ovalbumin. This was accompanied by increased detection of cells expressing CCR10 protein and mRNA in inflamed airways. Taken together, these data support a role for CCL28 in contributing to allergen driven airway pathologies, show that proinflammatory cytokines can induce this signal and suggest a role for CCR10 expressing cells in airway inflammation.

Interleukin-10 (IL-10) but not Lipopolysaccharide (LPS) produces increased motor activity and abnormal exploratory patterns while impairing spatial learning in Balb/c mice.

Lipopolysaccharide (LPS) is a potent endotoxin, which produces "sickness behaviours" including loss of weight, loss of interest in food and decreased exploration. LPS has also been shown in some studies to cause deficits in various learning and memory abilities, while in others these LPS-induced learning impairments have been attributed to performance-related deficits rather than learning deficits per se. Here, we use the novelty-preference paradigm, a task that minimises performance-related factors such as motivation, in an attempt to extract and examine the effects of LPS on spatial learning. In addition, some studies have indicated that the anti-inflammatory cytokine Interleukin-10 (IL-10) can alleviate some of the symptoms induced by LPS. Here, we also examine the effect of IL-10 on feeding, motor and learning behaviours. We demonstrate that a single injection of LPS does produce a lack of interest in food and weight loss; LPS, however, does not impair habituation in the novelty-preference paradigm. Furthermore, co-injection of IL-10 with LPS does not attenuate the LPS-induced effects of weight loss and lack of food intake. Interestingly, a single injection of IL-10 produces abnormal patterns of exploration, a general increase in activity and abnormal patterns of habituation.

Human mesenchymal stromal cells exert HGF dependent cytoprotective effects in a human relevant pre-clinical model of COPD.

Bone-marrow derived mesenchymal stromal cells (MSCs) have potent immunomodulatory and tissue reparative properties, which may be beneficial in the treatment of inflammatory diseases such as COPD. This study examined the mechanisms by which human MSCs protect against elastase induced emphysema. Using a novel human relevant pre-clinical model of emphysema the efficacy of human MSC therapy and optimal cell dose were investigated. Protective effects were examined in the lung through histological examination. Further in vivo experiments examined the reparative abilities of MSCs after tissue damage was established and the role played by soluble factors secreted by MSCs. The mechanism of MSC action was determined in using shRNA gene knockdown. Human MSC therapy and MSC conditioned media exerted significant cytoprotective effects when administered early at the onset of the disease. These protective effects were due to significant anti-inflammatory, anti-fibrotic and anti-apoptotic mechanisms, mediated in part through MSC production of hepatocyte growth factor (HGF). When MSC administration was delayed, significant protection of the lung architecture was observed but this was less extensive. MSC cell therapy was more effective than MSC conditioned medium in this emphysema model.

Hepatocyte Growth Factor Is Required for Mesenchymal Stromal Cell Protection Against Bleomycin-Induced Pulmonary Fibrosis.

: The incidence of idiopathic pulmonary fibrosis is on the rise and existing treatments have failed to halt or reverse disease progression. Mesenchymal stromal cells (MSCs) have potent cytoprotective effects, can promote tissue repair, and have demonstrated efficacy in a range of fibrotic lung diseases; however, the exact mechanisms of action remain to be elucidated. Chemical antagonists and short hairpin RNA knockdown were used to identify the mechanisms of action used by MSCs in promoting wound healing, proliferation, and inhibiting apoptosis. Using the bleomycin induced fibrosis model, the protective effects of early or late MSC administration were examined. The role for hepatocyte growth factor (HGF) in MSC protection against bleomycin lung injury was examined using HGF knockdown MSC. Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling assay was performed on ex vivo lung sections to examine the effects of MSC on apoptosis. MSC conditioned media (CM) enhanced wound closure and inhibited apoptosis of pulmonary cells in vitro. HGF was required for MSC CM enhancement of epithelial cell proliferation and inhibition of apoptosis. In contrast, MSC required COX-2 for CM to inhibit fibroblast proliferation. In a murine model, early administration of MSC protected against bleomycin induced lung fibrosis and correlated with reduced levels of the proinflammatory cytokine interleukin-1ß, reduced levels of apoptosis, and significantly increased levels of HGF. These protective effects were in part mediated by MSC derived HGF as HGF knockdown MSC were unable to protect against fibrosis in vivo. These findings delineate the mechanisms of MSC protection in a preclinical model of fibrotic lung disease. SIGNIFICANCE: The mechanisms used by mesenchymal stromal cells (MSCs) in mediating protective effects in chronic models of lung disease are not understood and remain to be elucidated. These findings from in vitro studies highlight an important role for the MSC-derived soluble factors hepatocyte growth factor (HGF) and prostaglandin E2 in promoting wound healing and inhibiting apoptosis. Furthermore, this study translates these findings demonstrating an important role for HGF in the protective effects mediated by MSC in vivo in the bleomycin model. These findings support a targeted approach to enhancing MSC therapy for fibrotic disease and highlight the importance of timing of MSC therapy.

Immunogenicity of adult mesenchymal stem cells: lessons from the fetal allograft.

Herein we review recent data that support host tolerance of allogeneic adult mesenchymal stem cells (MSC). Evidence is emerging that donor MSC deploy a very powerful array of mechanisms that allow escape from host allogeneic responses. These mechanisms include limited expression of alloantigen by the stem cell and cell contact-dependent and -independent mechanisms. MSC modulate host dendritic cell and T cell function, promoting induction of suppressor or regulatory T cells. These effects are complemented by the induction of divisional arrest anergy in T cells and by stem cell production of soluble immunomodulatory factors, including interleukin-10, transforming growth factor-beta, prostaglandin E2, and hepatocyte growth factor. In addition, MSC express the enzyme indoleamine 2,3-dioxygenase, which creates a tryptophan-depleted milieu that promotes immunosuppression. We propose that these observations show striking similarity to emerging data on the maternal acceptance of the fetal allograft. This comparison suggests new approaches to determine the contribution of different mechanisms to the successful use of MSC in regenerative medicine.

Whole-cell pertussis vaccine protects against Bordetella pertussis exacerbation of allergic asthma.

The prevalence of asthma and allergic disease has increased in many countries and there has been speculation that immunization promotes allergic sensitization. Bordetella pertussis infection exacerbates allergic asthmatic responses. We investigated whether whole-cell pertussis vaccine (Pw) enhanced or prevented B. pertussis induced exacerbation of allergic asthma. Groups of mice were immunized with Pw, infected with B. pertussis and/or sensitized to ovalbumin. Immunological, pathological and physiological changes were measured to assess the impact of Pw immunization on immune deviation and airway function. Pw immunization modulated ovalbumin-specific serum IgE production, and reduced local and systemic IL-13 and other cytokine responses to sensitizing allergen. Histopathological examination revealed Pw immunization reduced the severity of airway pathology and decreased bronchial hyperreactivity to methacholine exposure. Pw does not enhance airway IL-13 and consequently does not enhance but protects against the exacerbation of allergic responses. We find no evidence of Pw contributing to allergic asthma, but rather provide evidence of a mechanism whereby whole-cell pertussis vaccination has a protective role.

Mesenchymal stromal cells protect against caspase 3-mediated apoptosis of CD19(+) peripheral B cells through contact-dependent upregulation of VEGF.

The immune suppressive and anti-inflammatory capabilities of bone marrow-derived mesenchymal stromal cells (MSCs) represent an innovative new tool in regenerative medicine and immune regulation. The potent immune suppressive ability of MSC over T cells, dendritic cells, and natural killer cells has been extensively characterized, however, the effect of MSC on B cell function has not yet been clarified. In this study, the direct effect of MSC on peripheral blood B cell function is defined and the mechanism utilized by MSC in enhancing B cell survival in vitro identified. Human MSC supported the activation, proliferation, and survival of purified CD19(+) B cells through a cell contact-dependent mechanism. These effects were not mediated through B cell activating factor or notch signaling. However, cell contact between MSC and B cells resulted in increased production of vascular endothelial growth factor (VEGF) by MSC facilitating AKT phosphorylation within the B cell and inhibiting caspase 3-mediated apoptosis. Blocking studies demonstrated that this cell contact-dependent effect was not dependent on signaling through CXCR4-CXCL12 or through the epidermal growth factor receptor (EGFR). These results suggest that direct cell contact between MSC and B cells supports B cell viability and function, suggesting that MSC may not represent a suitable therapy for B cell-mediated disease.