CD5L/AIM Regulates Lipid Biosynthesis and Restrains Th17 Cell Pathogenicity.

Th17 cells play a critical role in host defense against extracellular pathogens and tissue homeostasis but can induce autoimmunity. The mechanisms implicated in balancing "pathogenic" and "non-pathogenic" Th17 cell states remain largely unknown. We used single-cell RNA-seq to identify CD5L/AIM as a regulator expressed in non-pathogenic, but not in pathogenic Th17 cells. Although CD5L does not affect Th17 differentiation, it is a functional switch that regulates the pathogenicity of Th17 cells. Loss of CD5L converts non-pathogenic Th17 cells into pathogenic cells that induce autoimmunity. CD5L mediates this effect by modulating the intracellular lipidome, altering fatty acid composition and restricting cholesterol biosynthesis and, thus, ligand availability for Rorγt, the master transcription factor of Th17 cells. Our study identifies CD5L as a critical regulator of the Th17 cell functional state and highlights the importance of lipid metabolism in balancing immune protection and disease induced by T cells.

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.

Th17 cell master transcription factor RORC2 regulates HIV-1 gene expression and viral outgrowth.

Among CD4+ T cells, T helper 17 (Th17) cells are particularly susceptible to HIV-1 infection and are depleted from mucosal sites, which causes damage to the gut barrier, resulting in a microbial translocation-induced systemic inflammation, a hallmark of disease progression. Furthermore, a proportion of latently infected Th17 cells persist long term in the gastrointestinal lymphatic tract where a low-level HIV-1 transcription is observed. This residual viremia contributes to chronic immune activation. Thus, Th17 cells are key players in HIV pathogenesis and viral persistence. It is, however, unclear why these cells are highly susceptible to HIV-1 infection. Th17 cell differentiation depends on the expression of the master transcriptional regulator RORC2, a retinoic acid-related nuclear hormone receptor that regulates specific transcriptional programs by binding to promoter/enhancer DNA. Here, we report that RORC2 is a key host cofactor for HIV replication in Th17 cells. We found that specific inhibitors that bind to the RORC2 ligand-binding domain reduced HIV replication in CD4+ T cells. The depletion of RORC2 inhibited HIV-1 infection, whereas its overexpression enhanced it. RORC2 was also found to promote HIV-1 gene expression by binding to the nuclear receptor responsive element in the HIV-1 long terminal repeats (LTR). In treated HIV-1 patients, RORC2+ CD4 T cells contained more proviral DNA than RORC2- cells. Pharmacological inhibition of RORC2 potently reduced HIV-1 outgrowth in CD4+ T cells from antiretroviral-treated patients. Altogether, these results provide an explanation as to why Th17 cells are highly susceptible to HIV-1 infection and suggest that RORC2 may be a cell-specific target for HIV-1 therapy.

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.

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

BACKGROUND: Lung cancer remains a leading cause of cancer mortality, and so the aim of the present study was to develop a therapeutic vaccine protocol. METHODS: We constructed a lentiviral vector (LV) expressing the extracellular domain (ECD) of murine Her1, an antigen associated with poor prognosis in lung cancer. RESULTS: A single LV injection, followed by two Her1 protein boosts, was effective in reducing the metastatic burden of Lewis lung carcinoma in mice. The Her1 LV immunisation generated CD8+ T cells that recognised Her1 ECD presented by dendritic cells, and that also homed to Her1-expressing tumours. Protein boosting further increased the CD8+ T cell response and generated anti-Her1 antibodies; in the antibody response, Her1 LV priming increased Th1-dependent immunoglobulin G2c production. CONCLUSIONS: The ability of this vaccine protocol to break both T cell and B cell tolerance to a self-antigen likely explains its effectiveness.

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 ß, 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.

HIV-1 lentiviral vector immunogenicity is mediated by Toll-like receptor 3 (TLR3) and TLR7.

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-alpha), 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 host's immune response to the transgene.

Nonintegrating lentivector vaccines stimulate prolonged T-cell and antibody responses and are effective in tumor therapy.

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-kappaB 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.

An IL-27-Driven Transcriptional Network Identifies Regulators of IL-10 Expression across T Helper Cell Subsets.

Interleukin-27 (IL-27) is an immunoregulatory cytokine that suppresses inflammation through multiple mechanisms, including induction of IL-10, but the transcriptional network mediating its diverse functions remains unclear. Combining temporal RNA profiling with computational algorithms, we predict 79 transcription factors induced by IL-27 in T cells. We validate 11 known and discover 5 positive (Cebpb, Fosl2, Tbx21, Hlx, and Atf3) and 2 negative (Irf9 and Irf8) Il10 regulators, generating an experimentally refined regulatory network for Il10. We report two central regulators, Prdm1 and Maf, that cooperatively drive the expression of signature genes induced by IL-27 in type 1 regulatory T cells, mediate IL-10 expression in all T helper cells, and determine the regulatory phenotype of colonic Foxp3+ regulatory T cells. Prdm1/Maf double-knockout mice develop spontaneous colitis, phenocopying ll10-deficient mice. Our work provides insights into IL-27-driven transcriptional networks and identifies two shared Il10 regulators that orchestrate immunoregulatory programs across T helper cell subsets.

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.

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.

On the Mechanism of T cell receptor down-modulation and its physiological significance.

Effective, long-lasting immune responses largely depend upon T cell reponses. Antigen-specific T lymphocytes are activated and differentiate into effector T cells after antigen presentation by professional antigen presenting cells (APCs). However, T cell responses are tightly regulated to prevent T cell hyperactivation which may end up in autoimmune pathology. One of these regulatory mechanisms is ligand-induced TCR down-modulation, a process by which TCRs are removed from the T cell surface shortly after engagement with their cognate antigenic peptide associated to MHC molecules on the APC. TCR down-modulation is a complicated process. Here we briefly describe the three main models that attempt to clarify this mechanism in the context of T cell activation and function.

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.