Katarzyna Karwacz

Dynamic regulatory network controlling Th17 cell differentiation

Despite their importance, the molecular circuits that control the differentiation of naive T cells remain largely unknown. Recent studies that reconstructed regulatory networks in mammalian cells have focused on short-term responses and relied on perturbation-based approaches that cannot be readily applied to primary T cells. Here we combine transcriptional profiling at high temporal resolution, novel computational algorithms, and innovative nanowire-based perturbation tools to systematically derive and experimentally validate a model of the dynamic regulatory network that controls the differentiation of mouse TH17 cells, a proinflammatory T-cell subset that has been implicated in the pathogenesis of multiple autoimmune diseases. The TH17 transcriptional network consists of two self-reinforcing, but mutually antagonistic, modules, with 12 novel regulators, the coupled action of which may be essential for maintaining the balance between TH17 and other CD4+ T-cell subsets. Our study identifies and validates 39 regulatory factors, embeds them within a comprehensive temporal network and reveals its organizational principles; it also highlights novel drug targets for controlling TH17 cell differentiation.

HIV-1 Lentiviral Vector Immunogenicity Is Mediated by Toll-Like Receptor 3 (TLR3) and TLR7

ABSTRACT Lentiviral vectors are promising vaccine vector candidates that have been tested extensively in preclinical models of infectious disease and cancer immunotherapy. They are also used in gene therapy clinical trials both for the ex vivo modification of cells and for direct in vivo injection. It is therefore critical to understand the mechanism(s) by which such vectors might stimulate the immune system. We evaluated the effect of lentiviral vectors on myeloid dendritic cells (DC), the main target of lentiviral transduction following subcutaneous immunization. The activation of DC cultures was independent of the lentiviral pseudotype but dependent on cell entry and reverse transcription. In vivo-transduced DC also displayed a mature phenotype, produced tumor necrosis factor alpha (TNF-α), and stimulated naive CD8+ T cells. The lentiviral activation of DC was Toll-like receptor (TLR) dependent, as it was inhibited in TRIF/MyD88 knockout (TRIF/MyD88−/−) DC. TLR3−/− or TLR7−/− DC were less activated, and reverse transcription was important for the activation of TLR7−/− DC. Moreover, lentivirally transduced DC lacking TLR3 or TLR7 had an impaired capacity to induce antigen-specific CD8+ T-cell responses. In conclusion, we demonstrated TLR-dependent DC activation by lentiviral vectors, explaining their immunogenicity. These data allow the rational development of strategies to manipulate the hosts immune response to the transgene.

PD-L1 co-stimulation contributes to ligand-induced T cell receptor down-modulation on CD8(+) T cells

T cell receptor (TCR) down‐modulation after antigen presentation is a fundamental process that regulates TCR signal transduction. Current understanding of this process is that intrinsic TCR/CD28 signal transduction leads to TCR down‐modulation. Here, we show that the interaction between programmed cell death 1 ligand 1 (PD‐L1) on dendritic cells (DCs) and programmed death 1 (PD‐1) on CD8 T cells contributes to ligand‐induced TCR down‐modulation. We provide evidence that this occurs via Casitas B‐lymphoma (Cbl)‐b E3 ubiquitin ligase up‐regulation in CD8 T cells. Interference with PD‐L1/PD‐1 signalling markedly inhibits TCR down‐modulation leading to hyper‐activated, proliferative CD8 T cells as assessed in vitro and in vivo in an arthritis model. PD‐L1 silencing accelerates anti‐tumour immune responses and strongly potentiates DC anti‐tumour capacities, when combined with mitogen‐activated kinase (MAPK) modulators that promote DC activation.

Nonintegrating Lentivector Vaccines Stimulate Prolonged T-Cell and Antibody Responses and Are Effective in Tumor Therapy

ABSTRACT Lentiviral vectors (lentivectors) are effective for stimulation of cell-mediated and humoral immunity following subcutaneous and intramuscular immunization. However, lentivector genome integration carries a risk of perturbation of host gene expression. Here, we demonstrate that lentivectors with multiple mutations that prevent integration are also effective immunogens. First, systemic CD8+ T-cell responses to the model antigen ovalbumin were detected following subcutaneous injection of nonintegrating lentivectors. Transfer of transgenic OT1 T cells demonstrated that antigen presentation persisted for at least 30 days. Furthermore, an enhanced CD8+ T-cell response, peaking at 7 days, was stimulated by coexpression of p38 MAP kinase or an NF-κB activator from the same vector. Second, we demonstrated systemic CD8+ T-cell and antibody responses to the secreted hepatitis B virus (HBV) surface antigen expressed from a nonintegrating lentivector injected intramuscularly. The induction, specificity, and kinetics of antibody production closely mimicked those of natural HBV infection. In this case, both the vector genome and the immune response were maintained for at least 2 months. Together, our data indicate that nonintegrating lentivectors can be employed to generate effective vaccines.

Selective ERK Activation Differentiates Mouse and Human Tolerogenic Dendritic Cells, Expands Antigen-Specific Regulatory T Cells, and Suppresses Experimental Inflammatory Arthritis

Objective Most therapeutic treatments for autoimmune arthritis rely on immunosuppressive drugs, which have side effects. Although a previous study by our group showed that specific ERK activation suppressed immune responses, its application in a therapeutic setting has never been tested. The aim of the present study was to define the ERK-dependent immunosuppressive mechanisms and to apply selective ERK activation for the treatment of experimental inflammatory arthritis. Methods A constitutively active ERK activator was coexpressed with a model antigen using lentivectors. Immunosuppressive mechanisms were characterized at the level of dendritic cell (DC) function, differentiation of antigen-specific Treg cells, and inhibition of inflammatory T cells. Administration of the ERK activator with antigen as a strategy to suppress inflammatory arthritis was tested in an experimental mouse model. Results Selective ERK activation induced mouse and human DCs to secrete bioactive transforming growth factor β, a process required for suppression of T cell responses and differentiation of antigen-specific Treg cells. Treg cells strongly proliferated after antigen reencounter in inflammatory conditions, and these cells exhibited antigen-dependent suppressive activities. Inflammatory arthritis was effectively inhibited through antigen-specific mechanisms. Importantly, this strategy did not rely on identification of the initiating arthritogenic antigen. Equivalent mechanisms were demonstrated in human monocyte–derived DCs, setting the scene for a possible rapid translation of this approach to patients with rheumatoid arthritis. Conclusion This strategy of selective ERK activation resulted in an effective therapeutic protocol, with substantial advantages over DC or T cell vaccination.

PD-L1 co-stimulation, ligand-induced TCR down-modulation and anti-tumor immunotherapy

PD-1 engagement on the surface of effector T cells strongly suppresses their cytotoxic function, which constitutes a major obstacle for T cell-mediated anti-tumor activities. Surprisingly, PD-1 is strongly upregulated in T cells, engaging its ligand PD-L1 during antigen presentation. However, our recent published data may provide an explanation for this apparent contradiction.

Critical role of IRF1 and BATF in forming chromatin landscape during type 1 regulatory cell differentiation

Type 1 regulatory T cells (Tr1 cells) are induced by interleukin-27 (IL-27) and have critical roles in the control of autoimmunity and resolution of inflammation. We found that the transcription factors IRF1 and BATF were induced early on after treatment with IL-27 and were required for the differentiation and function of Tr1 cells in vitro and in vivo. Epigenetic and transcriptional analyses revealed that both transcription factors influenced chromatin accessibility and expression of the genes required for Tr1 cell function. IRF1 and BATF deficiencies uniquely altered the chromatin landscape, suggesting that these factors serve a pioneering function during Tr1 cell differentiation.

Differentiation and Characterization of Tr1 Cells.

Regulatory T cell–mediated suppression serves as a pivotal mechanism of negative regulation of immune‐mediated inflammation. Type 1 regulatory T cells (Tr1 cells) are an important subset of CD4+ T cells that prevent excessive inflammatory responses and maintain immune tolerance. The anti‐inflammatory role of Tr1 cells is mediated in part by their production of interleukin 10 (IL‐10), which dampens the function of both antigen‐presenting cells and antigen‐specific effector T cells. Additionally, Tr1 cells can kill effector and myeloid cells through the perforin‐granzyme B pathway. Adoptive transfer of in vitro differentiated Tr1 cells can be used to suppress autoimmune tissue inflammation in vivo. This unit describes the in vitro stimulation of naïve murine CD4+ T cells using IL‐27 to generate IL‐10–producing Tr1 cells.

Lentiviral vector followed by protein immunisation breaks tolerance against the self‐antigen Her1 and results in lung cancer immunotherapy

Lung cancer remains a leading cause of cancer mortality, and so the aim of the present study was to develop a therapeutic vaccine protocol.