Translating Treg Therapy in Humanized Mice.

Regulatory T cells (Treg) control immune cell function as well as non-immunological processes. Their far-reaching regulatory activities suggest their functional manipulation as a means to sustainably and causally intervene with the course of diseases. Preclinical tools and strategies are however needed to further test and develop interventional strategies outside the human body. "Humanized" mouse models consisting of mice engrafted with human immune cells and tissues provide new tools to analyze human Treg ontogeny, immunobiology, and therapy. Here, we summarize the current state of humanized mouse models as a means to study human Treg function at the molecular level and to design strategies to harness these cells for therapeutic purposes.

Interferon-Beta Therapy of Multiple Sclerosis Patients Improves the Responsiveness of T Cells for Immune Suppression by Regulatory T Cells.

Multiple sclerosis (MS) is an inflammatory autoimmune disease characterized by imbalanced immune regulatory networks, and MS patient-derived T effector cells are inefficiently suppressed through regulatory T cells (Treg), a phenomenon known as Treg resistance. In the current study we investigated T cell function in MS patients before and after interferon-beta therapy. We compared cytokine profile, responsiveness for Treg-mediated suppression ex vivo and evaluated reactivity of T cells in vivo using a humanized mouse model. We found that CD4+ and CD8+ T cells of therapy-naive MS patients were resistant to Treg-mediated suppression. Treg resistance is associated with an augmented IL-6 production, enhanced IL-6 receptor expression, and increased PKB/c-Akt phosphorylation. These parameters as well as responsiveness of T cells to Treg-mediated suppression were restored after interferon-beta therapy of MS patients. Following transfer into immunodeficient mice, MS T cells induced a lethal graft versus host disease (GvHD) and in contrast to T cells of healthy volunteers, this aggressive T cell response could not be controlled by Treg, but was abolished by anti-IL-6 receptor antibodies. However, magnitude and lethality of GvHD induced by MS T cells was significantly decreased after interferon-beta therapy and the reaction was prevented by Treg activation in vivo. Our data reveals that interferon-beta therapy improves the immunoregulation of autoaggressive T effector cells in MS patients by changing the IL-6 signal transduction pathway, thus restoring their sensitivity to Treg-mediated suppression.

FTY720 (fingolimod) treatment tips the balance towards less immunogenic antigen-presenting cells in patients with multiple sclerosis.

OBJECTIVE: We aimed to clarify whether fingolimod has direct effects on antigen-presenting cells in multiple sclerosis patients. METHODS: Frequency and phenotype of directly ex vivo dendritic cells and monocytes were analyzed in 43 individuals, including fingolimod-treated and untreated multiple sclerosis patients as well as healthy subjects. These cells were further stimulated with lipopolysaccharide to determine functional effects of fingolimod treatment. RESULTS: Absolute numbers of CD1c+ dendritic cells and monocytes were not significantly reduced in fingolimod-treated patients indicating that fingolimod did not block the migration of antigen-presenting cells to peripheral blood. CD86 was upregulated on CD1c+ dendritic cells and thus their activation was not impaired under fingolimod treatment. Quantitative analyses of gene transcription in cells and protein content in supernatants from ex vivo CD1c+ dendritic cells and monocytes, however, showed lower secretion of TNFα, IL1-ß and IL-6 upon lipopolysaccharide-stimulation. These results could be matched with CD4+MOG-specific transgenic T cells exhibiting reduced levels of TNFα and IFN-γ but not IL-4 upon stimulation with murine dendritic cells loaded with MOG, when treated with fingolimod. CONCLUSIONS: Our data indicate that fingolimod - apart from trapping lymphocytes in lymph nodes - exerts its disease-modulating activity by rebalancing the immune tolerance networks by modulation of antigen-presenting cells.

Cladribine exerts an immunomodulatory effect on human and murine dendritic cells.

Cladribine is a purine nucleoside analog developed to treat lymphoid malignancies. Reported therapeutic benefits for the autoimmune disease multiple sclerosis indicate additional immunomodulatory effects beyond the well-characterized cytotoxic activity causing lymphopenia. Here, we demonstrate that cladribine reduces the secretion of inflammatory cytokines and chemokines by murine and human dendritic cells, the most potent antigen-presenting cells. This compound also modulates the expression of the activation markers CD86 and MHC II. Furthermore, cladribine affects the T cell priming capacity of dendritic cells, resulting in reduced induction of interferon-γ- and tumor necrosis factor-α-producing T cells and increased induction of interleukin-10-producing T cells. These effects, observed at cladribine concentrations in the therapeutically relevant range of serum steady-state concentrations for leukemia and multiple sclerosis, confirm the immunomodulatory activity of cladribine.

Kinetics of IL-6 production defines T effector cell responsiveness to regulatory T cells in multiple sclerosis.

In multiple sclerosis (MS) autoaggressive T effector cells (Teff) are not efficiently controlled by regulatory T cells (Treg) but the underlying mechanisms are incompletely understood. Proinflammatory cytokines are key factors facilitating Teff activity in chronic inflammation. Here we investigated the influence of IL-6 on Treg sensitivity of Teff from therapy-naïve MS patients with or without active disease. Compared to healthy volunteers and independent of disease course CD4(+) and especially CD8(+) MS-Teff were insensitive against functional active Treg from healthy controls. This unresponsiveness was caused by accelerated production of IL-6, elevated IL-6 receptor expression and phosphorylation of protein kinase B (PKB)/c-Akt in MS-Teff. In a positive feedback loop, IL-6 itself induced its accelerated synthesis and enhanced phosphorylation of PKB/c-Akt that finally mediated Treg resistance. Furthermore, accelerated IL-6 release especially by CD8(+) Teff prevented control of surrounding Teff, described here as "bystander resistance". Blockade of IL-6 receptor signaling or direct inhibition of PKB/c-Akt phosphorylation restored Treg responsiveness of Teff and prevented bystander resistance. In Teff of healthy controls (HC) exogenous IL-6 also changed the kinetics of IL-6 production and induced Treg unresponsiveness. This modulation was only transient in Teff from healthy volunteers, whereas accelerated IL-6 production in MS-Teff maintained also in absence of IL-6. Hence, we showed that the kinetics of IL-6 production instead of elevated IL-6 levels defines the Teff responsiveness in early Treg-T cell communication in MS independent of their disease course and propose IL-6 and associated PKB/c-Akt activation as effective therapeutic targets for modulation of Teff activity in MS.

Subclinical CNS inflammation as response to a myelin antigen in humanized mice.

Multiple sclerosis is a demyelinating autoimmune disease of the CNS. Its animal model experimental autoimmune encephalomyelitis is commonly induced by active immunization with myelin antigens. To investigate human immune responses against myelin antigens in vivo we established a new subclinical experimental autoimmune encephalomyelitis model in humanized mice. NOD/Scidγc⁻/⁻ animals were transferred with peripheral blood mononuclear cells from healthy human donors and immunized with myelin antigens in complete Freund's adjuvant and antigen-pulsed autologous dendritic cells. Human T cells recovered from these animals reacted specifically to the soluble domain of myelin oligodendrocyte glycoprotein and secreted proinflammatory cytokines. Furthermore, immunized animals developed subclinical CNS inflammation with infiltrating CD4⁺ and CD8⁺ T cells and production of encephalitogenic cytokines. Thus, this model of myelin-induced CNS inflammation by human T cells may allow testing of new human-specific therapeuticals for multiple sclerosis.

Costimulatory molecules on immunogenic versus tolerogenic human dendritic cells.

Dendritic cells (DC) are sentinels of immunity, essential for homeostasis of T cell-dependent immune responses. Both functions of DC, initiation of antigen-specific T cell immunity and maintenance of tissue-specific tolerance originate from distinct stages of differentiation, immunogenic versus tolerogenic. Dependent on local micro milieu and inflammatory stimuli, tissue resident immature DC with functional plasticity differentiate into tolerogenic or immunogenic DC with stable phenotypes. They efficiently link innate and adaptive immunity and are ideally positioned to modify T cell-mediated immune responses. Since the T cell stimulatory properties of DC are significantly influenced by their expression of signal II ligands, it is critical to understand the impact of distinct costimulatory pathways on DC function. This review gives an overview of functional different human DC subsets with unique profiles of costimulatory molecules and outlines how different costimulatory pathways together with the immunosuppressive cytokine IL-10 bias immunogenic versus tolerogenic DC functions. Furthermore, we exemplarily describe protocols for the generation of two well-defined monocyte-derived DC subsets for their clinical use, immunogenic versus tolerogenic.