A New Mesenchymal Stem Cell (MSC) Paradigm: Polarization into a Pro-Inflammatory MSC1 or an Immunosuppressive MSC2 Phenotype

BACKGROUND: Our laboratory and others reported that the stimulation of specific Toll-like receptors (TLRs) affects the immune modulating responses of human multipotent mesenchymal stromal cells (hMSCs). Toll-like receptors recognize "danger" signals, and their activation leads to profound cellular and systemic responses that mobilize innate and adaptive host immune cells. The danger signals that trigger TLRs are released following most tissue pathologies. Since danger signals recruit immune cells to sites of injury, we reasoned that hMSCs might be recruited in a similar way. Indeed, we found that hMSCs express several TLRs (e.g., TLR3 and TLR4), and that their migration, invasion, and secretion of immune modulating factors is drastically affected by specific TLR-agonist engagement. In particular, we noted diverse consequences on the hMSCs following stimulation of TLR3 when compared to TLR4 by our low-level, short-term TLR-priming protocol. PRINCIPAL FINDINGS: Here we extend our studies on the effect on immune modulation by specific TLR-priming of hMSCs, and based on our findings, propose a new paradigm for hMSCs that takes its cue from the monocyte literature. Specifically, that hMSCs can be polarized by downstream TLR signaling into two homogenously acting phenotypes we classify here as MSC1 and MSC2. This concept came from our observations that TLR4-primed hMSCs, or MSC1, mostly elaborate pro-inflammatory mediators, while TLR3-primed hMSCs, or MSC2, express mostly immunosuppressive ones. Additionally, allogeneic co-cultures of TLR-primed MSCs with peripheral blood mononuclear cells (PBMCs) predictably lead to suppressed T-lymphocyte activation following MSC2 co-culture, and permissive T-lymphocyte activation in co-culture with MSC1. SIGNIFICANCE: Our study provides an explanation to some of the conflicting reports on the net effect of TLR stimulation and its downstream consequences on the immune modulating properties of stem cells. We further suggest that MSC polarization provides a convenient way to render these heterogeneous preparations of cells more uniform while introducing a new facet to study, as well as provides an important aspect to consider for the improvement of current stem cell-based therapies

Disruption of CDK5 regulatory subunit 1, p35, limits immunosuppressive M2 macrophages while maintaining functional M1 macrophages

IntroductionMacrophage polarization into M1 or M2 phenotypes is a complex process influenced by various factors. However, existing literature and ongoing research support the view that Cyclin-Dependent Kinase 5 (CDK5) may play an important role in this process. CDK5 is a protein kinase that requires association with regulatory, co-activating proteins, p35 (CDK5R1) or p39 (CDK5R2), for functional activation.PurposeThis study investigated the role of the p35 protein in regulating M1 and M2 polarization.MethodsWe compared bone marrow derived macrophages from wild type (WT) and p35 knockout (KO) mice under both M1 (IFNγ + LPS) and M2 (IL4) conditions, differentiated with M-CSF or GM-CSF. The expression of surface markers (CD86, CD206), enzyme expression (Arginase-1 and iNOS), metabolism and antigen process and presentation were compared.ResultsWhile p35 had modest effect on phenotype during M1 or M2 polarization, p35 expression was important for Arginase1 induction after M2 polarization. The absence of p35 significantly increased glycolysis during M1 polarization, while it also enhanced mitochondrial oxidative phosphorylation in the context of M2 polarization. While p35 was important for antigen processing by M0 and M2, M1 were able to maintain capacity to process antigen albeit with a reduction due to decreased stability of peptide: MHC II complex.ConclusionWhile loss of p35 resulted in minor changes in phenotype, there were decreases in ARG-1 production and STAT3 phosphorylation, increased metabolism, and dramatically reduced antigen processing by M0, M1 or M2. The absence of p35 enhanced antigen uptake, but it had no effect on degradation of antigen, suggesting an inability to produce peptide: MHC II complexes in the absence of p35 in M0 and M2. In contrast, p35-deficient M1 maintained an ability to rapidly produce peptide: MHC II complexes but showed a reduction in the stability of these complexes on the surface. Our findings reveal a crucial role for p35 in regulating macrophage metabolism and antigen function, with implications for the development of novel therapeutic strategies

Toll-Like Receptor 3 and Suppressor of Cytokine Signaling Proteins Regulate CXCR4 and CXCR7 Expression in Bone Marrow-Derived Human Multipotent Stromal Cells

The use of bone marrow-derived human multipotent stromal cells (hMSC) in cell-based therapies has dramatically increased in recent years, as researchers have exploited the ability of these cells to migrate to sites of tissue injury, inflammation, and tumors. Our group established that hMSC respond to "danger" signals--by-products of damaged, infected or inflamed tissues--via activation of Toll-like receptors (TLRs). However, little is known regarding downstream signaling mediated by TLRs in hMSC.We demonstrate that TLR3 stimulation activates a Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 1 pathway, and increases expression of suppressor of cytokine signaling (SOCS) 1 and SOCS3 in hMSC. Our studies suggest that each of these SOCS plays a distinct role in negatively regulating TLR3 and JAK/STAT signaling. TLR3-mediated interferon regulatory factor 1 (IRF1) expression was inhibited by SOCS3 overexpression in hMSC while SOCS1 overexpression reduced STAT1 activation. Furthermore, our study is the first to demonstrate that when TLR3 is activated in hMSC, expression of CXCR4 and CXCR7 is downregulated. SOCS3 overexpression inhibited internalization of both CXCR4 and CXCR7 following TLR3 stimulation. In contrast, SOCS1 overexpression only inhibited CXCR7 internalization.These results demonstrate that SOCS1 and SOCS3 each play a functionally distinct role in modulating TLR3, JAK/STAT, and CXCR4/CXCR7 signaling in hMSC and shed further light on the way hMSC respond to danger signals

Isolation, expansion and function of cord blood natural killer cells. (P2226)

Abstract Rate of immune reconstitution (IR) directly correlates with the number of hematopoietic stem cells (HSC) infused and is particularly delayed in patients undergoing cord blood (CB) transplantation (CBT). Methods to increase the number of CB progenitors have the potential to accelerate IR after CBT. NK cells are the first lymphocyte subset to recover after HSC transplant (HCT) and are crucial in preventing early relapse. Unfortunately, NK cells in CB are low in number, incomplete in maturation and require activation for effective function. Here, we report a clinically relevant ex vivo expansion method that optimizes maturation as well as activation of CB NK cells. CB NK cells cultured 14-21 days with CB mononuclear cells, IL-2 and IL-15 result in a multi-log increase in cell number. Potential killer cell Ig-like receptor (KIR) ligand and adhesion molecule(s) responsible for NK cell cytotoxicity were examined prior to and after culture. Phenotypic analysis showed that the percent of NK cells expressing activating receptors and adhesion molecules increased (CD336 +39.11%) while inhibitory receptor expression decreased (CD159a -25.09%). Furthermore, cytotoxic function of CB NK cells against NK cell sensitive (K562) and resistant (MV4-11) cancer cells lines were enhanced after culture. The data suggest that this ex vivo expansion method of matured and activated NK cells is a potential clinically relevant method to prevent early relapse after CBT.</jats:p

Enhanced Cytotoxic Function of Natural Killer and CD3+CD56+ Cells in Cord Blood after Culture

AbstractRate of immune reconstitution directly correlates with the number of hematopoietic stem cells infused and is particularly delayed in patients undergoing cord blood (CB) transplantation (CBT). Methods to increase the number of CB natural killer (NK) cells have the potential to improve immune reconstitution after CBT. NK cells are the first lymphocyte population to recover after hematopoietic stem cells transplantation and are central to preventing early relapse and infection. CB NK cells are low in number and are known to be incomplete in maturation and require activation for effective function. Here, we report a clinically relevant ex vivo expansion method that increases the number of activated CB NK cells. We report a multilog increase in NK cell number when CB mononuclear cells are cocultured with IL-2 and IL-15. Furthermore, NK cells expressing activating receptors and adhesion molecules responsible for cytotoxicity increased throughout culture, whereas inhibitory receptor expression remained low. Additionally, cytotoxic function against various malignancies was significantly enhanced in cultured NK cells but not CD3+CD56+ cells. These data suggest that ex vivo expansion and activation of CB NK cells is a clinically feasible and relevant approach to prevent early infection and relapse after CBT

Mesenchymal stem cells as a novel vaccine platform

Vaccines are the most efficient and cost-effective means of preventing infectious disease. However, traditional vaccine approaches have thus far failed to provide protection against human immunodeficiency virus (HIV), tuberculosis, malaria and many other diseases. New approaches to vaccine development are needed to address some of these intractable problems. In this report, we review the literature identifying stimulatory effects of mesenchymal stem cells (MSC) on immune responses and explore the potential for MSC as a novel, universal vaccination platform. MSC are unique bone marrow-derived multipotent progenitor cells that are presently being exploited as gene therapy vectors for a variety of conditions, including cancer and autoimmune diseases. Although MSC are predominantly known for anti-inflammatory properties during allogeneic MSC transplant, there is evidence that MSC can actually promote adaptive immunity under certain settings. MSC have also demonstrated some success in anti-cancer therapeutic vaccines and anti-microbial prophylactic vaccines, as we report, for the first time, the ability of modified MSC to express and secrete a viral antigen that stimulates antigen-specific antibody production in vivo. We hypothesize that the unique properties of modified MSC may enable MSC to serve as an unconventional but innovative, vaccine platform. Such a platform would be capable of expressing hundreds of proteins, thereby generating a broad array of epitopes with correct post-translational processing, mimicking natural infection. By stimulating immunity to a combination of epitopes, it may be possible to develop prophylactic and even therapeutic vaccines to tackle major health problems including those of non-microbial and microbial origin, including cancer, or an infectious disease like HIV, where traditional vaccination approaches have failed