Ph.D. Projects


Engineering NKT2-inducing dendritic cells to restore immune regulation and prevent autoimmune diabetes in NOD mice
We recently demonstrated that defective immune regulation in NOD mice that spontaneously develop autoimmune diabetes is due to impaired differentiation of IL-4/IL-10-secreting NKT2 cells (Baev D.V. et al. J. Immunol. 2008). This impairment is due to defective co-stimulatory signals that are normally provided to NKT cells by myeloid dendritic cells (DC) and defective in NOD mice. Indeed, mature DC of NOD mice fail to express the SLAM molecule and to engage the homotypic SLAM-SLAM interaction with NKT cells. This interaction is crucial to generate the intracellular cascade that leads to GATA-3 up-regulation within the NKT cell and to IL-4/IL-10 secretion. In addition, myeloid DC of NOD mice secrete higher level of IL-12 compared to DC of non-autoimmune mice (unpublished data). IL-12 is a critical cytokine that promote secretion of IFN-γ by NKT cells and inhibit their differentiation towards a regulatory IL-4/IL-10-secreting phenotype. Aim of this project is to engineer NOD DC that express the SLAM molecule and do not secrete IL-12 with the final goal to selectively induce the differentiation of regulatory NKT2 cells. To this aim, DC of NOD mice will be transfected with a SLAM-containing retroviral vector (that was generated in the laboratory of Prof. K. Nichols, Children’s Hospital of Philadelphia, USA). The IL-12-secretion by NOD DC will be blocked by gene silencing with an RNA interference approach and a lentiviral vector expressing the microRNA for IL-12p40 under an ubiquitous promoter (PGK). The capacity of the engineered NOD DC to induce regulatory NKT2 differentiation will be evaluated in vitro and in vivo. In addition, transduced DC will be injected into diabetic NOD mice and analyzed for their survival, homing within pancreatic islets and lymph nodes and capacity to avert autoimmune diabetes.


Analysis of intestinal immune regulation in Type 1 Diabetic patients
The gut is home of several immune regulatory cell subsets that contribute to maintain peripheral tolerance towards self-tissues and to prevent autoimmune diseases such as Type 1 Diabetes. Specifically, the gut is the preferential site for the extrathymic development of FoxP3+ Treg cells induced by tolerogenic dendritic cells (CD11c+CD103+) that reside in the intestinal lamina propria (Coombes JL. and F. Powrie, Nat. Rev. Immunol. 2008). Other regulatory T cells such as FoxP3- regulatory T cells that exert their suppressive activity through release of IL-10 (Tr1 cells) or TGF-β (Th3 cells) preferentially reside in the GALT (gut associated lymphoid tissue). Considering the key role of regulatory T cells for maintenance of peripheral tolerance and prevention of autoimmune diseases such as Type 1 Diabetes, it is clear that an alteration of Treg cell development in the gut could contribute to the pathogenesis of T1D. The objective of this project is to study the immune regulatory cell compartment in the gut of T1D patients and to assess whether a genetic or diet-induced alteration of intestinal immune regulation underlies the pathogenesis of T1D. We started collaboration with the Department of Internal Medicine (Prof. E. Bosi) and the Department of Surgery (Prof. C. Socci) at HSR who provide us with a large number of jejunum biopsies and intestinal tissues from T1D patients and controls. The aims of this project are:
 

  • Comparative Multiparameter Cytofluorimetric analysis and microarray analysis on FACS sorted cells of the frequency and phenotype of different regulatory T cell subsets such as FoxP3+ Treg cells (CD4+CD25+FoxP3+CD127-GITR+LAG-3+CTLA-4+), FoxP3- Tr1 cells (CD4+FoxP3-IL-10+) and Th3 cells in the intestine of T1D patients and healthy controls.
  • Evaluation of the functional properties (suppressive capacity and cytokine secretion profile) of the different regulatory T cells subsets in the intestine of T1D patients and controls.
  • Assessment of the capacity of intestinal tolerogenic DC from T1D patients and controls to induce the extrathymic differentiation of CD4+CD25+FoxP3+ Treg cells.