Ph.D. Projects


Investigating the Tolerogenic Properties of CD45RO/RB Antibody Therapy
Supervisor: Prof. Maria-Grazia Roncarolo
Host Institute: HSR-TIGET

A loss of immune tolerance is linked to the expansion of pathogenic effector T cells and a functional deficit in tolerogenic responses, particularly those mediated by regulatory T cells. Thus, an ideal therapy for treatment of autoimmune diseases would be one that depletes auto-reactive effector T cells, and concomitantly restores or induces regulatory T cell responses that keep autoimmunity in check. One successful approach to induce tolerance developed by our lab is the use of an antibody targeting the isoforms RO and RB of human CD45 (CD45RO/RB). We previously demonstrated that this antibody has potent immuno-modulatory properties capable of controlling robust allogeneic responses by inducing apoptosis of effector/memory T cells through the intrinsic pathway, and plays a role in the development of a subset of regulatory T cells called regulatory type 1 cells (Tr1). Based upon our preliminary results, we believe applying anti-CD45RO/RB antibody to induce tolerance in T cell-mediated autoimmune diseases is a promising approach. Therefore, a more in-depth analysis of the mechanisms underlying the tolerogenic properties of anti-CD45RO/RB therapy is not only important to explain how tolerance can be achieved, but will also enhance the development of biomarkers used in translation studies.

The aims of this project are directed toward, 1) defining the mode of action of anti-CD45RO/RB therapy in apoptosis, 2) to determine the immunomodulatory properties of CD45RO/RB antibody on regulatory T cells, 3) and to evaluate the effects of CD45RO/RB on cell subsets in peripheral lymph organs pertinent in autoimmunity. To answer many of the questions addressed in this project, we will use a variety of techniques ranging from the use of multi-parameter flow cytometeric analysis, cell culture of primary human cells, development of a humanized mouse model to study the human immune system, cytokine secretion assays, humanized transplantation models, suppressive assays, and gene arrays to determine the effect of anti-CD45RO/RB treatment.  In addition, we will focus on developing a pertinent antigen-specific assay to evaluate how effective anti-CD45RO/RB is in regulating antigen-specific T cell responses.

Importantly, the findings from this study will not only propel the functional knowledge of CD45 RO and RB on human effector and memory T lymphocytes, but will also elucidate their significance in the induction and function of regulatory T cells. The clinical ramifications of these findings will provide insight into how the immune system may respond to anti-CD45RO/RB therapy, and what potential obstacles we may face during such a therapy. In summary, the translational nature of this project may provide an exciting avenue in which we will be able to treat T cell-mediated autoimmune diseases by inducing tolerance with anti-CD45RO/RB.


Dissecting the Cellular and Molecular Mechanisms Responsible for Tr1 Cell Phenotype and Functions.
Supervisor: Prof. Maria Grazia Roncarolo
Host Institute: HSR-TIGET

CD4+ type 1 regulatory T (Tr1) cells are important players in modulating immune responses to foreign antigens (Ag), self-Ags, and allo-Ags. Tr1 cells are characterized by high production of IL-10, and by the ability to suppress T-cell mediated responses mainly via IL-10 and TGF-beta. In addition, Tr1 cells express and secrete high levels of granzyme B, and specifically lyse antigen-presenting cells.

Several protocols have been developed to generate Tr1 cells in vitro and in vivo. The use of tolerogenic DC to differentiate Tr1 cells represents one of the most efficient methods to induce anergic T cell populations that contain IL-10-producing Tr1 cells. Unfortunately, this population still includes a subset of contaminating effector T cells that represent an obstacle to both further investigate the function of Tr1 cells, and to develop therapeutic adoptive strategies with Tr1 cells generated in vitro. To overcome this limitaton, we recently developed a lentiviral vector (LV) encoding for human IL-10 (LV-hIL-10). We demonstrated that human peripheral blood CD4+ T cells transduced with LV-hIL-10 acquired Tr1-like phenotype and function.

The aim of this project is to improve our understanding of Tr1 cells and further determine their impact in the modulation of the immune responses. To this aim, we will continue to study the role of endogenous IL-10 in modulating Tr1 cell induction and to investigate their mode of action. We will also further characterize LV-hIL-10 transduced CD4+ T cells in terms of phenotype and function, and compare them to bone fide Tr1 cell lines and clones generated in vitro or in vivo. Moreover, LV-hIL-10 transduced CD4+ T cells will be characterized at the molecular level using microarray analyses. The molecular signature of LV-hIL-10 transduced CD4+ T cells will be compared to that of bona fide Tr1 cells in order to define the specific surface molecules and transcription factors driven by IL-10 that endgender the induction and function of Tr1 cells. To further dissect the role of endogenous IL-10 in maintaining Tr1 cell phenotype and function, we will knock-down IL-10 by transducing either naïve T cells or Tr1 cell clones with regulated LV encoding for short hairpin RNA (shRNA) specific for human IL-10. Phenotype and function of resulting cells will be extensively investigated.

A better understanding of the biology of IL-10 producing Tr1 cells and the identification of a specific molecular signature will be fundamental to define the mechanisms underlying the induction, activation, and function of Tr1 cells, and also to develop new therapeutic approaches aimed at modulating T-cell mediated disease through induction of Tr1 cells.