Rare Diseases: don't stop the research

Project location: ITALY
Project start date: January 2001 - Project end date: January 2002
Project number: 2001-15
Beneficiary: ARMR

Study of immunological properties of dendritic cells

Scientific report prepared by: Regiane Cavinato


Dendritic cells (DCs) are specialized cells of the immune system. These cells migrate into lymphoid organs, particularly the thymus. The thymus is a lymphoid organ crucial for the development of the immune system that defends the organism against infections, but also drives the rejection of allograft.
A particular molecule denominated CCR9 has been shown to be a receptor for the CC chemokine TECK, a chemokine expressed by thymic cells. CCR9/TECK pairing has been shown to be important for cell homing. Hence, in this work we planned to enhance the migration of DCs into the host thymus to favor the development of donor-specific transplantation tolerance.

Aims
Transfect dendritic cells with gene encoding the CCR9 molecule and evaluate the effect of DS transduction with the CCR9 gene on their homing to the thymus and their ability to induce allograft tolerance.

METHODS

  • Growth of dendritic cells, Flow cytometry analysis (FACS) and Mixed Lymphocyte Reaction (MLR).
  • Cloning of CCR9
  • Analysis of CCR9 expression
  • Generation of adenoviral vector encoding for CCR9.

  • Final results

    1. CCR9 gene preparation
    CCR9 gene was cloned in E. coli. The obtained sequence was submitted to GenBank. After cloning, adenoviral vector containing the cDNA for CCR9 was generated.

    2. Dendritic cells preparation and transduction
    Dendritic cells were obtained at the end of the 11-day culture (figure 1). The adenoviral vector containing CCR9 gene was added to DCs culture to transfer CCR9 gene into donor DCs.
    Transduced DCs were collected and analyzed by FACS using OX-6 (anti-MHC II) and B7-2 antibodies (figure 2). FACS analysis with OX-6 antibody showed a lower fluorescence intensity of MHC II in CCR9-DCs than control DCs-AdV0. B7-2 expression was lower in transduced CCR9-DCs compared to DC-AdV0.
    In standard MLR, CCR9-DCs stimulated the proliferation of allogeneic lymph node cells less efficiently than control DCs-AdV0 (table 1).

    CONCLUSION
    Trasfection of DCs with CCR9 gene keeps them in a less immature state than transfection with a null adenovirus. Specifically, they express lower levels of MHCII and B7-2 and have a reduced capacity to stimulate allogeneic T lymphocytes.

    PERSPECTIVES
    We envisage to infuse donor CCR9 gene transduced -DCs into the recipient to induce tolerance to a subsequent immune incompatible kidney allograft .

    FIGURES

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