Remi Creusot

Deaf1 isoforms control the expression of genes encoding peripheral tissue antigens in the pancreatic lymph nodes during type 1 diabetes

Type 1 diabetes may result from a breakdown in peripheral tolerance that is partially controlled by the expression of peripheral tissue antigens (PTAs) in lymph nodes. Here we show that the transcriptional regulator Deaf1 controls the expression of genes encoding PTAs in the pancreatic lymph nodes (PLNs). The expression of canonical Deaf1 was lower, whereas that of an alternatively spliced variant was higher, during the onset of destructive insulitis in the PLNs of nonobese diabetic (NOD) mice. We identified an equivalent variant Deaf1 isoform in the PLNs of patients with type 1 diabetes. Both the NOD mouse and human Deaf1 variant isoforms suppressed PTA expression by inhibiting the transcriptional activity of canonical Deaf1. Lower PTA expression resulting from the alternative splicing of DEAF1 may contribute to the pathogenesis of type 1 diabetes.

Murine CD4+CD25+ Regulatory T Cells Fail to Undergo Chromatin Remodeling Across the Proximal Promoter Region of the IL-2 Gene

CD4+CD25+ regulatory T cells (Treg) acquire unique immunosuppressive properties while maintaining an anergy phenotype when activated in vitro under conditions that induce IL-2 production and proliferation in conventional CD4+ T cells. We investigated the mechanism underlying one component of this naturally anergic phenotype, the inability of the Treg cells to produce IL-2 following activation. Analysis of freshly isolated murine CD4+CD25+ Treg and conventional CD4+CD25− T cells following PMA/ionomycin stimulation demonstrated no differences in inducible AP-1 formation, an important transcriptional complex in regulating IL-2 gene expression. Although p38 MAPK and ERK1/2 protein kinases were phosphorylated with similar kinetics, we observed diminished activation of JNK in the CD4+CD25+ Treg cells. However, lentiviral-mediated reconstitution of the JNK pathway using a constitutively active construct did not overcome the block in IL-2 synthesis. Using a PCR-based chromatin accessibility assay we found that the minimal IL-2 promoter region of CD4+CD25+ Treg cells, unlike conventional CD4 T cells, did not undergo chromatin remodeling following stimulation, suggesting that the inability of CD4+CD25+ Treg cells to secrete IL-2 following activation is controlled at the chromatin level.

Tissue-targeted therapy of autoimmune diabetes using dendritic cells transduced to express IL-4 in NOD mice

A deficit in IL-4 production has been previously reported in both diabetic human patients and non-obese diabetic (NOD) mice. In addition, re-introducing IL-4 into NOD mice systemically, or as a transgene, led to a beneficial outcome in most studies. Here, we show that prediabetic, 12-week old female NOD mice have a deficit in IL-4 expression in the pancreatic lymph nodes (PLN) compared to age-matched diabetes-resistant NOD.B10 mice. By bioluminescence imaging, we demonstrated that the PLN was preferentially targeted by bone marrow-derived dendritic cells (DCs) following intravenous (IV) administration. Following IV injection of DCs transduced to express IL-4 (DC/IL-4) into 12-week old NOD mice, it was possible to significantly delay or prevent the onset of hyperglycemia. We then focused on the PLN to monitor, by microarray analysis, changes in gene expression induced by DC/IL-4 and observed a rapid normalization of the expression of many genes, that were otherwise under-expressed compared to NOD.B10 PLN. The protective effect of DC/IL-4 required both MHC and IL-4 expression by the DCs. Thus, adoptive cellular therapy, using DCs modified to express IL-4, offers an effective, tissue-targeted cellular therapy to prevent diabetes in NOD mice at an advanced stage of pre-diabetes, and may offer a safe approach to consider for treatment of high risk human pre-diabetic patients.

Lymphoid-tissue-specific homing of bone-marrow-derived dendritic cells.

Because of their potent immunoregulatory capacity, dendritic cells (DCs) have been exploited as therapeutic tools to boost immune responses against tumors or pathogens, or dampen autoimmune or allergic responses. Murine bone marrow-derived DCs (BM-DCs) are the closest known equivalent of the blood monocyte-derived DCs that have been used for human therapy. Current imaging methods have proven unable to properly address the migration of injected DCs to small and deep tissues in mice and humans. This study presents the first extensive analysis of BM-DC homing to lymph nodes (and other selected tissues) after intravenous and intraperitoneal inoculation. After intravenous delivery, DCs accumulated in the spleen, and preferentially in the pancreatic and lung-draining lymph nodes. In contrast, DCs injected intraperitoneally were found predominantly in peritoneal lymph nodes (pancreatic in particular), and in omentum-associated lymphoid tissue. This uneven distribution of BM-DCs, independent of the mouse strain and also observed within pancreatic lymph nodes, resulted in the uneven induction of immune response in different lymphoid tissues. These data have important implications for the design of systemic cellular therapy with DCs, and in particular underlie a previously unsuspected potential for specific treatment of diseases such as autoimmune diabetes and pancreatic cancer.

Redirecting cell-type specific cytokine responses with engineered interleukin-4 superkines

Cytokines dimerize their receptors, with binding of the “second chain” triggering signaling. In the interleukin (IL)-4/13 system, different cell types express varying levels of alternative second receptor chains (γc or IL-13Rα1), forming functionally distinct Type-I or Type-II complexes. We manipulated the affinity and specificity of second chain recruitment by human IL-4. A Type-I receptor-selective IL-4 ‘superkine’ with 3700-fold higher affinity for γc was 3-10 fold more potent than wild-type IL-4. Conversely, a variant with high affinity for IL-13Rα1 more potently activated cells expressing the Type-II receptor, and induced differentiation of dendritic cells from monocytes, implicating the Type-II receptor in this process. Superkines exhibited signaling advantages on cells with lower second chain levels. Comparative transcriptional analysis reveals that the superkines induce largely redundant gene expression profiles. Variable second chain levels can be exploited to redirect cytokines towards distinct cell subsets and elicit novel actions, potentially improving the selectivity of cytokine therapy.

It’s Time to Bring Dendritic Cell Therapy to Type 1 Diabetes

Type 1 diabetes (T1D) is an autoimmune disease that results from a deficient induction or maintenance of tolerance to islet β-cell antigens, allowing the eventual T-cell–mediated destruction of insulin-producing β-cells within the pancreatic islets (1). Under homeostatic conditions, immune tolerance is established by various subsets of antigen-presenting cells (APCs) with tolerance-inducing/maintaining (tolerogenic) functions and reinforced by other cells with suppressor and immunomodulatory properties. T-cell tolerance manifests itself through elimination (deletion), inactivation (anergy), or suppression of self-reactive T cells (Fig. 1 A ). These functions may be performed by a variety of tolerogenic APCs (Table 1), some of which have the ability to induce/boost regulatory T cells (Tregs) and/or B cells (Bregs). Genetic and environmental factors that vary among different individuals contribute to the development of T1D, in part by impacting mechanisms of tolerance (Fig. 1 B ). Therefore, a major goal for the prevention and/or reversal of T1D is to restore effective and durable tolerance to either prevent further destruction of remaining islet β-cells, help islet β-cell regeneration, or protect islet transplants and obviate the use of nonspecific immunosuppressive drugs. The approach reviewed here consists of developing a personalized therapy using the patient’s own cells manipulated to perform as tolerogenic APCs (Fig. 1 C ). In this review, we will explain why dendritic cells (DCs) are the tolerogenic cells of choice to fulfill the goal of restoring immune tolerance in T1D as part of a potentially powerful and safe cellular immunotherapy, the next generation of therapies (2). We will present the clinical considerations and challenges ahead and the plans to address them, taking into account lessons learned from animal models such as the NOD mouse. Figure 1 Tolerance to β-cell antigens: induction, loss, and restoration. A : Tolerance to β-cell antigens is induced and maintained by tolerogenic cells comprised of migratory DCs transporting antigens from peripheral to lymphoid …

Multimodality imaging of T-cell hybridoma trafficking in collagen-induced arthritic mice: image-based estimation of the number of cells accumulating in mouse paws

Appropriate targeting of therapeutic cells is essential in adoptive cellular gene therapy (ACGT). Imaging cell trafficking in animal models and patients will guide development of ACGT protocols. Collagen type II (C-II)-specific T cell hybridomas are transduced with a lentivirus carrying a triple fusion reporter gene (TFR) construct consisting of a fluorescent reporter gene (RG), a bioluminescent RG (hRluc), and a positron emission tomography (PET) RG. Collagen-induced arthritic (CIA) mice are scanned with a bioluminescence imaging camera before and after implantation of various known cell quantities in their paws. Linear regression analysis yields equations relating two parameters of image signal intensity in mice paws to the quantity of hRluc expressing cells in the paws. Afterward, trafficking of intravenously injected cells is studied by quantitative analysis of bioluminescence images. Comparison of the average cell numbers does not demonstrate consistently higher accumulation of T-cell hybridomas in the paws with higher inflammation scores, and injecting more cells does not cause increased accumulation. MicroPET images illustrate above background signal in the inflamed paws and chest areas of CIA mice. The procedures described in this study can be used to derive equations for cells expressing other bioluminescent RGs and in other animal models.

Reduced DEAF1 function during type 1 diabetes inhibits translation in lymph node stromal cells by suppressing Eif4g3

The transcriptional regulator deformed epidermal autoregulatory factor 1 (DEAF1) has been suggested to play a role in maintaining peripheral tolerance by controlling the transcription of peripheral tissue antigen genes in lymph node stromal cells (LNSCs). Here, we demonstrate that DEAF1 also regulates the translation of genes in LNSCs by controlling the transcription of the poorly characterized eukaryotic translation initiation factor 4 gamma 3 (Eif4g3) that encodes eIF4GII. Eif4g3 gene expression was reduced in the pancreatic lymph nodes of Deaf1-KO mice, non-obese diabetic mice, and type 1 diabetes patients, where functional Deaf1 is absent or diminished. Silencing of Deaf1 reduced Eif4g3 expression, but increased the expression of Caspase 3, a serine protease that degrades eIF4GII. Polysome profiling showed that reduced Eif4g3 expression in LNSCs resulted in the diminished translation of various genes, including Anpep, the gene for aminopeptidase N, an enzyme involved in fine-tuning antigen presentation on major histocompatibility complex (MHC) class II. Together these findings suggest that reduced DEAF1 function, and subsequent loss of Eif4g3 transcription may affect peripheral tissue antigen (PTA) expression in LNSCs and contribute to the pathology of T1D.

Inflammation and Hyperglycemia Mediate Deaf1 Splicing in the Pancreatic Lymph Nodes via Distinct Pathways During Type 1 Diabetes

Peripheral tolerance is partially controlled by the expression of peripheral tissue antigens (PTAs) in lymph node stromal cells (LNSCs). We previously identified a transcriptional regulator, deformed epidermal autoregulatory factor 1 (Deaf1), that can regulate PTA expression in LNSCs of the pancreatic lymph nodes (PLNs). During the pathogenesis of type 1 diabetes (T1D), Deaf1 is spliced to form the dominant-negative isoform Deaf1-Var1. Here we show that Deaf1-Var1 expression correlates with the severity of disease in NOD mice and is reduced in the PLNs of mice that do not develop hyperglycemia. Inflammation and hyperglycemia independently drive Deaf1 splicing through activation of the splicing factors Srsf10 and Ptbp2, respectively. Inflammation induced by injection of activated splenocytes increased Deaf1-Var1 and Srsf10, but not Ptbp2, in the PLNs of NOD.SCID mice. Hyperglycemia induced by treatment with the insulin receptor agonist S961 increased Deaf1-Var1 and Ptbp2, but not Srsf10, in the PLNs of NOD.B10 and NOD mice. Overexpression of PTBP2 and/or SRSF10 also increased human DEAF1-VAR1 and reduced PTA expression in HEK293T cells. These data suggest that during the progression of T1D, inflammation and hyperglycemia mediate the splicing of DEAF1 and loss of PTA expression in LNSCs by regulating the expression of SRSF10 and PTBP2.

NF-κB in DCs: it takes effort to be immature

The nuclear factor-κB (NF-κB) family of regulators is well known for mediating dendritic cell (DC) maturation—that is, the acquisition of the functions required for full activation of T cells. Paradoxically, a key member of this family, NF-κB1, is now also implicated in maintaining DCs in an immature state (pages 1663–1667).