Katharine A. Whartenby

Mesenchymal stem cells: Emerging mechanisms of immunomodulation and therapy.

Mesenchymal stem cells (MSCs) are a pleiotropic population of cells that are self-renewing and capable of differentiating into canonical cells of the mesenchyme, including adipocytes, chondrocytes, and osteocytes. They employ multi-faceted approaches to maintain bone marrow niche homeostasis and promote wound healing during injury. Biomedical research has long sought to exploit their pleiotropic properties as a basis for cell therapy for a variety of diseases and to facilitate hematopoietic stem cell establishment and stromal reconstruction in bone marrow transplantation. Early results demonstrated their usage as safe, and there was little host response to these cells. The discovery of their immunosuppressive functions ushered in a new interest in MSCs as a promising therapeutic tool to suppress inflammation and down-regulate pathogenic immune responses in graft-versus-host and autoimmune diseases such as multiple sclerosis, autoimmune diabetes, and rheumatoid arthritis. MSCs produce a large number of soluble and membrane-bound factors, some of which inhibit immune responses. However, the full range of MSC-mediated immune-modulation remains incompletely understood, as emerging reports also reveal that MSCs can adopt an immunogenic phenotype, stimulate immune cells, and yield seemingly contradictory results in experimental animal models of inflammatory disease. The present review describes the large body of literature that has been accumulated on the fascinating biology of MSCs and their complex effects on immune responses.

NFAT Binding and Regulation of T Cell Activation by the Cytoplasmic Scaffolding Homer Proteins

T cell receptor (TCR) and costimulatory receptor (CD28) signals cooperate in activating T cells, although understanding of how these pathways are themselves regulated is incomplete. We found that Homer2 and Homer3, members of the Homer family of cytoplasmic scaffolding proteins, are negative regulators of T cell activation. This is achieved through binding of nuclear factor of activated T cells (NFAT) and by competing with calcineurin. Homer-NFAT binding was also antagonized by active serine-threonine kinase AKT, thereby enhancing TCR signaling via calcineurin-dependent dephosphorylation of NFAT. This corresponded with changes in cytokine expression and an increase in effector-memory T cell populations in Homer-deficient mice, which also developed autoimmune-like pathology. These results demonstrate a further means by which costimulatory signals are regulated to control self-reactivity.

Signal Transduction Inhibition of APCs Diminishes Th17 and Th1 Responses in Experimental Autoimmune Encephalomyelitis

IL-17- and IFN-γ-secreting T cells play an important role in autoimmune responses in multiple sclerosis and the model system experimental autoimmune encephalomyelitis (EAE). Dendritic cells (DCs) in the periphery and microglia in the CNS are responsible for cytokine polarization and expansion of this T cell subset. Our results indicate that in vivo administration of a signal transduction inhibitor that targets DCs to mice with EAE led to a decrease in CNS infiltration of pathogenic Ag-specific T cells. Since this approach does not target T cells directly, we assessed the effects on the APCs that are involved in generating the T cell responses. Since in EAE and multiple sclerosis, both microglia and peripheral DCs are likely to contribute to disease, we utilized a bone marrow chimera system to distinguish between these two populations. These studies show that peripheral DCs are the primary target but that microglia are also modestly affected by CEP-701, as numbers and activation states of the cells in the CNS are decreased after therapy. Our results also showed a decrease in secretion of TNF-α, IL-6, and IL-23 by DCs as well as a decrease in expression of costimulatory molecules. We further determined that levels of phospho-Stat1, Stat3, Stat5, and NF-κB, which are signaling molecules that have been implicated in these pathways, were decreased. Thus, use of this class of signal transduction inhibitors may represent a novel method to treat autoimmunity by dampening the autoreactive polarizing condition driven by DCs.

Human CD34+ Hematopoietic Stem/Progenitor Cells Express High Levels of FLIP and Are Resistant to Fas‐Mediated Apoptosis

We sought to determine whether lympho‐hematopoietic stem‐progenitor cells (HSC) from human placental/umbilical cord blood (CB) or adult mobilized blood (PBSC) are sensitive to Fas‐induced apoptosis. Human CD34+ cells from CB or PBSC were cultured in serum‐free medium, with or without hematopoietic growth factors (FKT: FLT‐3 ligand [FL], KIT ligand [KL], and thrombopoietin [TPO]), and with or without soluble Fas ligand (sFasL) or agonistic anti‐Fas antibody. After 5‐48 hours of culture, cells were assessed for viability and stained with Annexin V and 7‐Aminoactinomycin D for apoptosis analysis by fluorescence‐activated cell sorting. Cultured cells were also assessed by in vitro hematopoietic colony‐forming cell (CFC) and in vivo nonobese diabetic/severe combined immunodeficient mouse engraftment potential (SEP) assays. Levels of Fas, FLICE inhibitory protein (FLIP), and Caspase 8 mRNA in CD34+ cells were determined by real‐time quantitative polymerase chain reaction. Expression of FLIP was confirmed by Western blotting. No decrease in viability, CFC, or SEP was observed in CB or PBSC CD34+ cells cultured in the presence of sFasL or agonistic anti‐Fas antibody. Human CB and mobilized PBSC CD34+ cells expressed high levels of FLIP, low ratios of Caspase 8:FLIP, and low levels of Fas. Thus, human CB and PBSC CD34+ HSC were resistant to Fas pathway agonists. High‐level expression of FLIP likely provides one level of protection of CD34+ cells from Fas‐mediated apoptosis.

Feedback inhibition of calcineurin and Ras by a dual inhibitory protein Carabin

Feedback regulation of adaptive immunity is a fundamental mechanism for controlling the overall output of different signal transduction pathways, including that mediated by the T-cell antigen receptor (TCR). Calcineurin and Ras are known to have essential functions during T-cell activation. However, how the calcineurin signalling pathway is terminated in the process is still largely unknown. Although several endogenous inhibitors of calcineurin have been reported, none fulfils the criteria of a feedback inhibitor, as their expression is not responsive to TCR signalling. Here we identify an endogenous inhibitor of calcineurin, named Carabin, which also inhibits the Ras signalling pathway through its intrinsic Ras GTPase-activating protein (GAP) activity. Expression of Carabin is upregulated on TCR signalling in a manner that is sensitive to inhibitors of calcineurin, indicating that Carabin constitutes part of a negative regulatory loop for the intracellular TCR signalling pathway. Knockdown of Carabin by short interfering RNA led to a significant enhancement of interleukin-2 production by antigen-specific T cells in vitro and in vivo. Thus, Carabin is a negative feedback inhibitor of the calcineurin signalling pathway that also mediates crosstalk between calcineurin and Ras.

Kv1.3 Deletion Biases T Cells toward an Immunoregulatory Phenotype and Renders Mice Resistant to Autoimmune Encephalomyelitis

Increasing evidence suggests ion channels have critical functions in the differentiation and plasticity of T cells. Kv1.3, a voltage-gated K+ channel, is a functional marker and a pharmacological target for activated effector memory T cells. Selective Kv1.3 blockers have been shown to inhibit proliferation and cytokine production by human and rat effector memory T cells. We used Kv1.3 knockout (KO) mice to investigate the mechanism by which Kv1.3 blockade affects CD4+ T cell differentiation during an inflammatory immune-mediated disease. Kv1.3 KO animals displayed significantly lower incidence and severity of myelin oligodendrocyte glycoprotein (MOG) peptide-induced experimental autoimmune encephalomyelitis. Kv1.3 was the only KV channel expressed in MOG 35–55-specific CD4+ T cell blasts, and no KV current was present in MOG-specific CD4+ T cell-blasts from Kv1.3 KO mice. Fewer CD4+ T cells migrated to the CNS in Kv1.3 KO mice following disease induction, and Ag-specific proliferation of CD4+ T cells from these mice was impaired with a corresponding cell-cycle delay. Kv1.3 was required for optimal expression of IFN-γ and IL-17, whereas its absence led to increased IL-10 production. Dendritic cells from Kv1.3 KO mice fully activated wild-type CD4+ T cells, indicating a T cell-intrinsic defect in Kv1.3 KO mice. The loss of Kv1.3 led to a suppressive phenotype, which may contribute to the mechanism by which deletion of Kv1.3 produces an immunotherapeutic effect. Skewing of CD4+ T cell differentiation toward Ag-specific regulatory T cells by pharmacological blockade or genetic suppression of Kv1.3 might be beneficial for therapy of immune-mediated diseases such as multiple sclerosis.

Cutting Edge: The Transcription Factor Kruppel-Like Factor 4 Regulates the Differentiation of Th17 Cells Independently of RORγt

Th17 cells play a significant role in inflammatory and autoimmune responses. Although a number of molecular pathways that contribute to the lineage differentiation of T cells have been discovered, the mechanisms by which lineage commitment occurs are not fully understood. Transcription factors play a key role in driving T cells toward specific lineages. We have identified a role for the transcription factor Kruppel-like factor (KLF) 4 in the development of IL-17–producing CD4+ T cells. KLF4 was required for the production of IL-17, and further, chromatin immunoprecipation analysis demonstrated binding of KLF4 to the IL-17 promoter, indicating a direct effect on the regulation of IL-17. Further, KLF4-deficient T cells upregulated expression of retinoic acid-related orphan receptor γt similar to wild-type during the polarization process toward Th17, suggesting that these two transcription factors are regulated independently.

Mesenchymal Stem Cells Differentially Modulate Effector CD8+ T Cell Subsets and Exacerbate Experimental Autoimmune Encephalomyelitis

Mesenchymal stem cells (MSC) have emerged as a promising candidate for inflammatory suppression and disease amelioration, especially of neuro‐inflammatory diseases such as multiple sclerosis (MS). Auto‐reactive CD4+ and CD8+ T cells acquire pathogenic IFNγ‐producing‐ (Type I) and IL‐17A‐producing‐ (Type 17) effector phenotypes in MS and its animal model experimental autoimmune encephalomyelitis (EAE). Although MSC have been extensively demonstrated to suppress pathogenic effector CD4+ T cells and CD4+ T cell‐mediated EAE, surprisingly few studies have addressed their modulation of effector CD8+ T cells represented in MS or their impact on CD8+ T cell‐mediated EAE. We find that MSC differentially modulate CD8+ T cell development depending on effector T cell subtype. MSC drive activated low‐IFNγ producers toward an enhanced high‐IFNγ Tc1‐like phenotype but strongly inhibit the production of IL‐17A and Tc17 polarization in vitro. These observations are underscored by differential MSC modulation of T cell activation, proliferation, and signature transcription factor up‐regulation. In addition, effector CD8+ T cells co‐cultured with MSC exhibited increased production of IL‐2, a molecule known to enhance IFNγ, yet suppress IL‐17A, production. Based on these in vitro effects on CD8+ T cells, we next evaluated their impact on the severity of EAE. To better evaluate CD8+ T cells, we immunized mice with MOG37‐50, which is a CD8‐targeted epitope. Our results revealed a worsening of disease, consistent with their in vitro stimulation of Tc1 cells. These findings highlight the emerging duality of MSC in immune modulation and provide implications for their future use in immune‐related diseases. Stem Cells 2014;32:2744–2755

Functional Blockade of the Voltage-gated Potassium Channel Kv1.3 Mediates Reversion of T Effector to Central Memory Lymphocytes through SMAD3/p21cip1 Signaling

Background: The role of Kv1.3 in regulating T cell differentiation and memory is incompletely understood. Results: A dominant negative mutation of Kv1.3 mediates reversion of TEM into TCM through SMAD3-dependent cell cycle changes. Conclusion: Signaling through Kv1.3 is a mechanism by which TEM may revert to TCM. Significance: These findings suggest a novel role for Kv1.3 in T cell differentiation and memory responses. The maintenance of T cell memory is critical for the development of rapid recall responses to pathogens, but may also have the undesired side effect of clonal expansion of T effector memory (TEM) cells in chronic autoimmune diseases. The mechanisms by which lineage differentiation of T cells is controlled have been investigated, but are not completely understood. Our previous work demonstrated a role of the voltage-gated potassium channel Kv1.3 in effector T cell function in autoimmune disease. In the present study, we have identified a mechanism by which Kv1.3 regulates the conversion of T central memory cells (TCM) into TEM. Using a lentiviral-dominant negative approach, we show that loss of function of Kv1.3 mediates reversion of TEM into TCM, via a delay in cell cycle progression at the G2/M stage. The inhibition of Kv1.3 signaling caused an up-regulation of SMAD3 phosphorylation and induction of nuclear p21cip1 with resulting suppression of Cdk1 and cyclin B1. These data highlight a novel role for Kv1.3 in T cell differentiation and memory responses, and provide further support for the therapeutic potential of Kv1.3 specific channel blockers in TEM-mediated autoimmune diseases.

Inhibition of activation-induced death of dendritic cells and enhancement of vaccine efficacy via blockade of MINOR.

Activation of dendritic cells (DCs) leads to cell maturation, which is accompanied by a regulated pattern of gene expression changes. Two significant and contradictory consequences of DC activation are that, although activation is necessary for maximal T-cell stimulation, it also leads to the initiation of gene expression that results ultimately in cell death. We have identified a gene, MINOR (mitogen-inducible nuclear orphan receptor), that becomes highly up-regulated on activation and whose expression leads to apoptosis in mature DCs. MINOR is a member of the Nur77 family of nuclear orphan receptors, which includes Nur77 and Nurr1. Although Nur77 and Nurr1 are expressed in macrophages and DCs, their expression levels do not change on DC activation. We thus tested the hypothesis that induction of MINOR would lead to an activation-induced cell death in DCs and that its inhibition would increase the lifespan of DCs and improve their vaccine efficacy. To block natural expression of MINOR by DCs, we generated a lentiviral vector that expresses a small interfering RNA. Our results indicate that blockade of MINOR expression dramatically decreases apoptosis in DCs and suggest that this approach may be a novel means to improve the potency of ex vivo-generated DC vaccines.