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. We showed that an optimal design of the lentiviral backbone is crucial to multiplex up to three ZF-Rs or two ZF-Rs and a chimeric antigen receptor. ZF-R expression had no impact on the integrity and functionality of transduced cells. Furthermore, gene repression in ZF-R-expressing T cells was highly efficient in vitro and in vivo during the entire monitoring period (up to 10 weeks), and it was accompanied by epigenetic remodeling events. Finally, we described an approach to improve ZF-R specificity to illustrate the path toward the generation of ZF-Rs with a safe clinical profile. In conclusion, we successfully developed an epigenetic-based cell engineering approach for concomitant modulation of multiple gene expressions that bypass the risks associated with DNA editing.

Harnessing regulatory T cells to establish immune tolerance.

Engineered regulatory T (Treg) 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 Treg 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 in vitro good manufacturing practice-compatible culturing system characterized by robust and consistent expansion rate, high viability, and efficient B cell differentiation. Using this process, an optimized B cell editing protocol was developed by combining ZFN/adeno-associated virus 6 technology to achieve site-specific insertion of the human factor IX R338L Padua into the silent TRAC locus. In vitro analysis revealed high levels of secreted human immunoglobulins and human factor IX-Padua. Following intravenous infusion in a mouse model, human plasma cells were detected in spleen and bone marrow, indicating successful and potentially long-term engraftment in vivo. Moreover, high levels of human immunoglobin and therapeutic levels of human factor IX-Padua were detected in mouse plasma, correlating with 15% of normal human factor IX activity. These data suggest that the proposed process promotes the production of functional and differentiated engineered B cells. In conclusion, this study represents an important step toward the development of a manufacturing platform for potential B cell-derived therapeutic products.

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. We found that hepatocellular antigen recognition by effector CD8+ T cells triggered a prominent increase in the number of hepatic NK cells and ILC1s. Group 1 ILCs colocalized and engaged in prolonged interactions with effector CD8+ T cells undergoing hepatocellular antigen recognition; however, they did not induce T cell apoptosis. Rather, group 1 ILCs constrained CD8+ T cell proliferation by controlling local interleukin-2 (IL-2) availability. Accordingly, group 1 ILC depletion, or genetic removal of their IL-2 receptor a chain, considerably increased the number of intrahepatic HBV-specific effector CD8+ T cells and the attendant immunopathology. Together, these results reveal a role for group 1 ILCs in controlling T cell-mediated liver immunopathology by limiting local IL-2 concentration and have implications for the treatment of chronic HBV infection.

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 CD56bright 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. We used mass cytometry to evaluate expression patterns of NK cell markers and provide a comprehensive assessment of the NK cell repertoire in individuals with RMS treated with daclizumab beta or placebo over the course of 1 year. Treatment with daclizumab beta significantly altered the NK cell repertoire compared to placebo treatment. As previously reported, daclizumab beta significantly increased expression of CD56 on total NK cells. Within the CD56bright NK cells, treatment was associated with multiple phenotypic changes, including increased expression of NKG2A and NKp44, and diminished expression of CD244, CD57, and NKp46. These alterations occurred broadly across the CD56bright population, and were not associated with a specific subset of CD56bright NK cells. While the changes were less dramatic, CD56dim NK cells responded distinctly to daclizumab beta treatment, with higher expression of CD2 and NKG2A, and lower expression of FAS-L, HLA-DR, NTB-A, NKp30, and Perforin. Together, these data indicate that the expanded CD56bright NK cells share features of both immature and mature NK cells. These findings show that daclizumab beta treatment is associated with unique changes in NK cells that may enhance their ability to kill autoreactive T cells or to exert immunomodulatory functions.

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. This approach circumvents previous unsuccessful approaches to target PKCθ directly. The use of structure based drug design, aided by the first crystal structure of GLK, led to the discovery of several inhibitors that demonstrate potent inhibition of GLK biochemically and in relevant cell lines.

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

Regulatory T cells (Treg 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 Treg cells in their suppressor function. However, congenital deficiency in IL-2R results in reduced expression of the Treg 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 Treg cells. Using genetic gain- and loss-of-function approaches, we found that capture of IL-2 was dispensable for the control of CD4+ T cells but was important for limiting the activation of CD8+ T cells, and that IL-2R-dependent activation of the transcription factor STAT5 had an essential role in the suppressor function of Treg cells separable from signaling via the T cell antigen receptor.

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. No significant change in total ILCs or major ILC subpopulations was observed. These results refine our understanding of the impact of daclizumab on innate lymphoid cell populations.

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. Our goal was to optimize two RORγ inverse agonists: T0901317 from literature and 1 that we obtained from internal screening. We used information from internal X-ray structures to design two libraries that led to a new biaryl series.

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. In mouse models, the rat anti-mouse CD25 clone PC61 has been used extensively to investigate the biology of IL-2 and Treg cells; however, there has been controversy and conflicting data on the exact in vivo mechanistic function of PC61. Engineering antibodies to alter Fc/Fc receptor interactions can significantly alter their in vivo function. In this study, we re-engineered the heavy chain constant region of an anti-CD25 monoclonal antibody to generate variants with highly divergent Fc effector function. Using these anti-CD25 Fc variants in multiple mouse models, we investigated the in vivo impact of CD25 blockade versus depletion of CD25(+) Treg cells on immune homeostasis. We report that immune homeostasis can be maintained during CD25 blockade but aberrant T-cell activation prevails when CD25(+) Treg cells are actively depleted. These results clarify the impact of PC61 on Treg cell biology and reveal an important distinction between CD25 blockade and depletion of CD25(+) Treg cells. These findings should inform therapeutic manipulation of the IL-2 pathway by targeting the high-affinity IL-2R.

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. Small molecule mediated disruption of RORγ function in cells with pre-existing RORγ transcriptional complexes represents a significant and challenging pharmacological hurdle. We present data demonstrating that a novel, selective and potent small molecule RORγ inhibitor can block the RORγ-dependent gene expression programme in both Th17 cells and RORγ-expressing γδ T cells as well as a disease-relevant subset of human RORγ-expressing memory T cells. Importantly, systemic administration of this inhibitor in vivo limits pathology in an innate lymphocyte-driven mouse model of psoriasis.

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. We demonstrate that DMF inhibited NF-κB-driven cytokine production and nuclear translocation of p65 and p52 in an Nrf2-independent manner. Equivalent doses of MMF and MEF did not affect NF-κB signaling. These results highlight a key difference in the biological impact of related, yet distinct fumarate compounds.

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. Daclizumab is a humanized mAb that binds the IL-2 receptor a subunit (IL-2R a or CD25) and prevents IL-2 binding. In this study, we investigated the effect of daclizumab-mediated CD25 blockade on Treg homeostasis in patients with relapsing-remitting multiple sclerosis. We report that daclizumab therapy caused an ~50% decrease in Tregs over a 52-wk period. Remaining FOXP3+ cells retained a demethylated Treg-specific demethylated region in the FOXP3 promoter, maintained active cell cycling, and had minimal production of IL-2, IFN- g, and IL-17. In the presence of daclizumab, IL-2 serum concentrations increased and IL-2R bg signaling induced STAT5 phosphorylation and sustained FOXP3 expression. Treg declines were not associated with daclizumab-related clinical benefit or cutaneous adverse events. These results demonstrate that Treg phenotype and lineage stability can be maintained in the face of CD25 blockade.

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. We report that during tuberculosis, T reg cells proliferate in the pulmonary lymph nodes (pLNs), change their cell surface phenotype, and accumulate in the pLNs and lung at a rate parallel to the accumulation of effector T cells. In the Mtb-infected lung, T reg cells accumulate in high numbers in all sites where CD4(+) T cells are found, including perivascular/peribronchiolar regions and within lymphoid aggregates of granulomas. To determine the role of T reg cells in the immune response to tuberculosis, we generated mixed bone marrow chimeric mice in which all cells capable of expressing Foxp3 expressed Thy1.1. When T reg cells were depleted by administration of anti-Thy1.1 before aerosol infection with Mtb, we observed approximately 1 log less of colony-forming units of Mtb in the lungs. Thus, after aerosol infection, T reg cells proliferate and accumulate at sites of infection, and have the capacity to suppress immune responses that contribute to the control of 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. T reg cell expression of CCR4 and CD103 is induced by antigen-driven activation within subcutaneous lymph nodes, and accumulation of T reg cells in the skin and lung airways is impaired in the absence of CCR4 expression. Mice with a complete loss of CCR4 in the T reg cell compartment develop lymphocytic infiltration and severe inflammatory disease in the skin and lungs, accompanied by peripheral lymphadenopathy and increased differentiation of skin-tropic CD4(+)Foxp3(+) T cells. Thus, selectively altering T reg cell distribution in vivo leads to the development of tissue-specific inflammatory disease.

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. Here we show, by dissociating Foxp3-dependent features from those induced by the signals preceding and promoting its expression in mice, that the latter signals include several functional and transcriptional hallmarks of Tr cells. Although its function is required for Tr cell suppressor activity, Foxp3 to a large extent amplifies and fixes pre-established molecular features of Tr cells, including anergy and dependence on paracrine IL-2. Furthermore, Foxp3 solidifies Tr cell lineage stability through modification of cell surface and signalling molecules, resulting in adaptation to the signals required to induce and maintain Tr cells. This adaptation includes Foxp3-dependent repression of cyclic nucleotide phosphodiesterase 3B, affecting genes responsible for Tr cell homeostasis.

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. Importantly, Foxp3+ cells were present at increased density in colon samples from patients with ulcerative colitis or Crohn's disease, suggesting similarities in the behavior of murine and human regulatory cells under inflammatory conditions. Cure of murine colitis was dependent on the presence of IL-10, and IL-10-producing CD4+CD25+ T cells were enriched within the colon during cure of colitis and also under steady state conditions. Our data indicate that although CD4+CD25+ T cells expressing Foxp3 are present within both lymphoid organs and the colon, subsets of IL-10-producing CD4+CD25+ T cells are present mainly within the intestinal lamina propria suggesting compartmentalization of the regulatory T cell response at effector sites.

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. Our analyses suggest that T(reg) cells and potentially pathogenic autoimmune T cells use overlapping pools of self-reactive TCRs.

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. We demonstrate that the development of Foxp3-expressing T reg cells is substantially delayed relative to nonregulatory thymocytes during ontogeny. Based on our data, we speculate that induction of Foxp3 in developing thymocytes and, thus, commitment to the T reg cell lineage is facilitated by a signal largely associated with the thymic medulla.