Epigenetic control of multiple genes with a lentiviral vector encoding transcriptional repressors fused to compact zinc finger arrays.

Gene silencing without gene editing holds great potential for the development of safe therapeutic applications. Here, we describe a novel strategy to concomitantly repress multiple genes using zinc finger proteins fused to Krüppel-Associated Box repression domains (ZF-Rs). This was achieved via the optimization of a lentiviral system tailored for the delivery of ZF-Rs in hematopoietic cells.

Harnessing regulatory T cells to establish immune tolerance.

Engineered regulatory T (T) cells have emerged as precision therapeutics aimed at inducing immune tolerance while reducing the risks associated with generalized immunosuppression. This Viewpoint highlights the opportunities and challenges for engineered T cell therapies in treating autoimmune and other inflammatory diseases.

Production of therapeutic levels of human FIX-R338L by engineered B cells using GMP-compatible medium.

B cells can differentiate into plasmablast and plasma cells, capable of producing antibodies for decades. Gene editing using zinc-finger nucleases (ZFN) enables the engineering of B cells capable of secreting sustained and high levels of therapeutic proteins. In this study, we established an advanced good manufacturing practice-compatible culturing system characterized by robust and consistent expansion rate, high viability, and efficient B cell differentiation.

Group 1 ILCs regulate T cell-mediated liver immunopathology by controlling local IL-2 availability.

Group 1 innate lymphoid cells (ILCs), which comprise both natural killer (NK) cells and ILC1s, are important innate effectors that can also positively and negatively influence adaptive immune responses. The latter function is generally ascribed to the ability of NK cells to recognize and kill activated T cells. Here, we used multiphoton intravital microscopy in mouse models of hepatitis B to study the intrahepatic behavior of group 1 ILCs and their cross-talk with hepatitis B virus (HBV)-specific CD8 T cells.

Characterization of the Impact of Daclizumab Beta on Circulating Natural Killer Cells by Mass Cytometry.

Daclizumab beta is a humanized monoclonal antibody that binds to CD25 and selectively inhibits high-affinity IL-2 receptor signaling. As a former treatment for relapsing forms of multiple sclerosis (RMS), daclizumab beta induces robust expansion of the CD56 subpopulation of NK cells that is correlated with the drug's therapeutic effects. As NK cells represent a heterogeneous population of lymphocytes with a range of phenotypes and functions, the goal of this study was to better understand how daclizumab beta altered the NK cell repertoire to provide further insight into the possible mechanism(s) of action in RMS.

Investigating small molecules to inhibit germinal center kinase-like kinase (GLK/MAP4K3) upstream of PKCθ phosphorylation: Potential therapy to modulate T cell dependent immunity.

Germinal center kinase-like kinase (GLK, also known as MAP4K3) has been hypothesized to have an effect on key cellular activities, including inflammatory responses. GLK is required for activation of protein kinase C-θ (PKCθ) in T cells. Controlling the activity of T helper cell responses could be valuable for the treatment of autoimmune diseases.

An essential role for the IL-2 receptor in T cell function.

Regulatory T cells (T cells), which have abundant expression of the interleukin 2 receptor (IL-2R), are reliant on IL-2 produced by activated T cells. This feature indicates a key role for a simple network based on the consumption of IL-2 by T cells in their suppressor function. However, congenital deficiency in IL-2R results in reduced expression of the T cell lineage-specification factor Foxp3, which has confounded experimental efforts to understand the role of IL-2R expression and signaling in the suppressor function of T cells.

Circulating innate lymphoid cells are unchanged in response to DAC HYP therapy.

Innate lymphoid cells (ILCs) play an important role in immunity, inflammation, and tissue remodeling and their dysregulation is implicated in autoimmune and inflammatory disorders. We analyzed the impact of daclizumab, a humanized monoclonal anti-CD25 antibody, on circulating natural killer (NK) cells and ILCs in a cohort of multiple sclerosis patients. An increase in CD56(bright) NK cells and CD56(hi)CD16(intermediate) transitional NK cells was observed.

Discovery of biaryls as RORγ inverse agonists by using structure-based design.

RORγ plays a critical role in controlling a pro-inflammatory gene expression program in several lymphocyte lineages including T cells, γδ T cells, and innate lymphoid cells. RORγ-mediated inflammation has been linked to susceptibility to Crohn's disease, arthritis, and psoriasis. Thus inverse agonists of RORγ have the potential of modulating inflammation.

Anti-CD25 monoclonal antibody Fc variants differentially impact regulatory T cells and immune homeostasis.

Interleukin-2 (IL-2) is a critical regulator of immune homeostasis through its non-redundant role in regulatory T (Treg) cell biology. There is major interest in therapeutic modulation of the IL-2 pathway to promote immune activation in the context of tumour immunotherapy or to enhance immune suppression in the context of transplantation, autoimmunity and inflammatory diseases. Antibody-mediated targeting of the high-affinity IL-2 receptor α chain (IL-2Rα or CD25) offers a direct mechanism to target IL-2 biology and is being actively explored in the clinic.

Small molecule mediated inhibition of RORγ-dependent gene expression and autoimmune disease pathology in vivo.

Retinoic acid receptor-related orphan nuclear receptor γ (RORγ) orchestrates a pro-inflammatory gene expression programme in multiple lymphocyte lineages including T helper type 17 (Th17) cells, γδ T cells, innate lymphoid cells and lymphoid tissue inducer cells. There is compelling evidence that RORγ-expressing cells are relevant targets for therapeutic intervention in the treatment of autoimmune and inflammatory diseases. Unlike Th17 cells, where RORγ expression is induced under specific pro-inflammatory conditions, γδ T cells and other innate-like immune cells express RORγ in the steady state.

Discovery of biaryl carboxylamides as potent RORγ inverse agonists.

RORγt is a pivotal regulator of a pro-inflammatory gene expression program implicated in the pathology of several major human immune-mediated diseases. Evidence from mouse models demonstrates that genetic or pharmacological inhibition of RORγ activity can block the production of pathogenic cytokines, including IL-17, and convey therapeutic benefit. We have identified and developed a biaryl-carboxylamide series of RORγ inverse agonists via a structure based design approach.

Discovery of novel pyrazole-containing benzamides as potent RORγ inverse agonists.

The nuclear receptor RORγ plays a central role in controlling a pro-inflammatory gene expression program in several lymphocyte lineages including TH17 cells. RORγ-dependent inflammation has been implicated in the pathogenesis of several major autoimmune diseases and thus RORγ is an attractive target for therapeutic intervention in these diseases. Starting from a lead biaryl compound 4a, replacement of the head phenyl moiety with a substituted aminopyrazole group resulted in a series with improved physical properties.

DMF, but not other fumarates, inhibits NF-κB activity in vitro in an Nrf2-independent manner.

Fumarate-containing pharmaceuticals are potent therapeutic agents that influence multiple cellular pathways. Despite proven clinical efficacy, there is a significant lack of data that directly defines the molecular mechanisms of action of related, yet distinct fumarate compounds. We systematically compared the impact of dimethyl fumarate (DMF), monomethyl fumarate (MMF) and a mixture of monoethyl fumarate salts (Ca(++), Mg(++), Zn(++); MEF) on defined cellular responses.

In vivo maintenance of human regulatory T cells during CD25 blockade.

Regulatory T cells (Tregs) mediate immune tolerance to self and depend on IL-2 for homeostasis. Treg deficiency, dysfunction, and instability are implicated in the pathogenesis of numerous autoimmune diseases. There is considerable interest in therapeutic modulation of the IL-2 pathway to treat autoimmunity, facilitate transplantation tolerance, or potentiate tumor immunotherapy.

The defect in T-cell regulation in NOD mice is an effect on the T-cell effectors.

FoxP3(+) regulatory T cells (Tregs) protect against autoimmunity, type 1 diabetes (T1D) in particular, prompting the hypothesis that a deficiency in Tregs is a critical determinant of diabetes susceptibility in NOD mice. However, tests of this hypothesis have yielded contradictory results. We confirmed that NOD mice, compared with reference strains, do not have a primary deficit in Treg numbers in the lymphoid organs, whether in prediabetic mice of any age or in animals with recent-onset diabetes.

Regulatory T cell-derived interleukin-10 limits inflammation at environmental interfaces.

The regulatory T (Treg) cells restrain immune responses through suppressor-function elaboration that is dependent upon expression of the transcription factor Foxp3. Despite a critical role for Treg cells in maintaining lympho-myeloid homeostasis, it remains unclear whether a single mechanism or multiple mechanisms of Treg cell-mediated suppression are operating in vivo and how redundant such mechanisms might be. Here we addressed these questions by examining the role of the immunomodulatory cytokine IL-10 in Treg cell-mediated suppression.

Expansion and function of Foxp3-expressing T regulatory cells during tuberculosis.

Mycobacterium tuberculosis (Mtb) frequently establishes persistent infections that may be facilitated by mechanisms that dampen immunity. T regulatory (T reg) cells, a subset of CD4(+) T cells that are essential for preventing autoimmunity, can also suppress antimicrobial immune responses. We use Foxp3-GFP mice to track the activity of T reg cells after aerosol infection with Mtb.

Altering the distribution of Foxp3(+) regulatory T cells results in tissue-specific inflammatory disease.

CD4(+)Foxp3(+) regulatory T cells (T reg) are essential for maintaining self-tolerance, but their functional mechanisms and sites of action in vivo are poorly defined. We examined the homing receptor expression and tissue distribution of T reg cells in the steady state and determined whether altering their distribution by removal of a single chemokine receptor impairs their ability to maintain tissue-specific peripheral tolerance. We found that T reg cells are distributed throughout all nonlymphoid tissues tested, and are particularly prevalent in the skin, where they express a unique CCR4(+)CD103(hi) phenotype.

Foxp3-dependent programme of regulatory T-cell differentiation.

Regulatory CD4+ T cells (Tr cells), the development of which is critically dependent on X-linked transcription factor Foxp3 (forkhead box P3), prevent self-destructive immune responses. Despite its important role, molecular and functional features conferred by Foxp3 to Tr precursor cells remain unknown. It has been suggested that Foxp3 expression is required for both survival of Tr precursors as well as their inability to produce interleukin (IL)-2 and independently proliferate after T-cell-receptor engagement, raising the possibility that such 'anergy' and Tr suppressive capacity are intimately linked.

Characterization of Foxp3+CD4+CD25+ and IL-10-secreting CD4+CD25+ T cells during cure of colitis.

CD4+CD25+ regulatory T cells can prevent and resolve intestinal inflammation in the murine T cell transfer model of colitis. Using Foxp3 as a marker of regulatory T cell activity, we now provide a comprehensive analysis of the in vivo distribution of Foxp3+CD4+CD25+ cells in wild-type mice, and during cure of experimental colitis. In both cases, Foxp3+CD4+CD25+ cells were found to accumulate in the colon and secondary lymphoid organs.

An intersection between the self-reactive regulatory and nonregulatory T cell receptor repertoires.

The relationship between the T cell receptor (TCR) repertoires used by self-reactive transcription factor Foxp3-positive (Foxp3(+)) CD4(+) regulatory T cells (T(reg) cells) and nonregulatory T cells with autoimmune potential is unclear. Here we found that the TCR repertoire of thymic T(reg) cells in TCRbeta-transgenic mice was diverse and was more similar to that of peripheral T(reg) cells than that of nonregulatory T cells, suggesting that thymic T(reg) cells make a substantial contribution to the peripheral T(reg) cell population. Activated T cells in Foxp3-deficient mice, which lack T(reg) cells, 'preferentially' used TCRs found in the TCR repertoire of T(reg) cells in Foxp3-sufficient TCRbeta-transgenic mice, suggesting that these self-reactive TCRs contribute to the pathology of Foxp3-deficient mice.

A function for interleukin 2 in Foxp3-expressing regulatory T cells.

Regulatory T cells (T(reg) cells) expressing the forkhead family transcription factor Foxp3 are critical mediators of dominant immune tolerance to self. Most T(reg) cells constitutively express the high-affinity interleukin 2 (IL-2) receptor alpha-chain (CD25); however, the precise function of IL-2 in T(reg) cell biology has remained controversial. To directly assess the effect of IL-2 signaling on T(reg) cell development and function, we analyzed mice containing the Foxp3(gfp) knock-in allele that were genetically deficient in either IL-2 (Il2(-/-)) or CD25 (Il2ra(-/-)).

Developmental regulation of Foxp3 expression during ontogeny.

Thymectomy of neonatal mice can result in the development of autoimmune pathology. It has been proposed that thymic output of regulatory T (T reg) cells is delayed during ontogeny and that the development of autoimmune disease in neonatally thymectomized mice is caused by the escape of self-reactive T cells before thymectomy without accompanying T reg cells. However, the kinetics of T reg cell production within the thymus during ontogeny has not been assessed.