Mechanisms Leading to Rescue of Thymic Function in Immuno-Compromised Individuals

Rafick-Pierre Sékaly, Ph.D.

University of Montreal

Funded in December, 2005: $580000 for 2 years
LAY SUMMARY . ABSTRACT . BIOGRAPHY . SELECTED PUBLICATIONS .

LAY SUMMARY

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Determining how the Immune System Governs Graft-Versus-Host Disease in Transplantation Patients

This study will determine how the immune system governs the occurrence of graft-versus-host disease in some patients who receive donated bone marrow, and how this disease process might be prevented.

Patients with blood-related cancers, such as leukemia, lymphoma, and myeloma, can undergo transplantation to receive a donor's bone marrow. The transplant is designed to increase the patient's development of improved immunity through production of infection -fighting immune T cells. New T cells are generated within the patient's thymus (an organ located at the base of the neck), where the T cells learn the critical distinction between infectious invaders and the body's own tissues. This learning requires that the T cells be in close physical contact with thymic "epithelial" cells, including immune dendritic cells. In some instances of transplantation, however, the patient may develop "graft-versus-host" disease, in which the donor's T cells directly target and harm the patient's thymus, especially the epithelial cells, causing the thymus to atrophy. Patients then succumb to opportunistic infections.

The consortium researchers' preliminary studies have led them to hypothesize that a protein produced by immune cells, called "Type 1interferon-alpha" (INF), plays a harmful role in transplant patients who develop graft-versus-host disease in general, and specifically in immunodeficiency associated with this disease. The researchers will study an expanded number of patients, consisting of those who develop graft-versus-host disease, and those who do not, to identify which of 20 candidate genes that produce IFN-α are most likely to be involved. Then they will use a mouse model to determine how IFN-α produced by some of these candidate genes damages the thymus. This research could show how the thymus functions during bone marrow transplantation, explain why the reconstitution of immune T cells is so variable, and reveal the processes involved, such as whether too much IFN blocks T cell production in the thymus.

Significance: The results of this research could lead to markedly improved safety of bone-marrow transplantation in patients with blood-related cancers.

ABSTRACT

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Mechanisms Leading to Rescue of Thymic Function in Immuno-Compromised Individuals

Immune reconstitution has become a major component of the therapeutic arsenal in an increasing number of disease states where the immune system is immunocompromised, either as the result of an infectious agent (HIV) or following therapeutic manipulations (myeloablation, graft-versus-host disease [GVHD] after bone marrow transplantation). The central hypothesis developed here is that the activity of the thymus in producing new thymocytes can be used to reconstitute the immune system in immunocompromised individuals. Although critical for the successful outcome, thymic impairment is a characteristic feature of GVHD. Our recent data suggest that type I IFNs have a determinant role in the induction of thymic atrophy. IFN (i) contributes directly to the blockage of thymopoiesis, (ii) induces differentiation and apoptosis of cortical TEC, and (iii) increases the transcription and expression of MHC molecules, and thus affects the T cell selection processes in the thymus.

The consortium assembled herein includes four groups who have focused their work on 1) the mechanisms leading to immune dysfunction in GVHD following allogeneic haematopoietic cell transplant (Perreault) and HIV infection (Sekaly), 2) the importance of the thymic stroma in immune dysfunction and immune reconstitution (Hollander), 3) the impact of type I interferons (IFNs) on thymic dysfunction (Sekaly, Perreault) and, 4) developing tools that are aimed at understanding thymic function to develop strategies to correct thymic production defects (Zúñiga-Pflücker's, Sekaly, Hollander).

The major objectives of this proposal will be to understand the defects induced by type I IFNs on thymic development in order to provide the basis and rational for novel strategies of immune reconstitution in GVHD. The proposal includes three specific aims which are based in their development on a substantial amount of preliminary results obtained both on GVHD patients and in the murine system, where proof of concept experiments were carried out for obvious ethical reasons. We have access to an extremely well documented cohort of patients undergoing Allogenic Hematopoitic Cell Transplantation (AHCT) and which have different disease courses.

In Specific Aim 1 we will assess the influence of GVHD on Recent Thymic Emigrants (RTE) kinetics and function. We have defined a phenotype 10 fold enriched in TRECs, by quantifying both sjTREC and DJTRECs in various thymocytes subsets; we were also able to quantify the extent of proliferation undergone by thymocytes, which is a good surrogate marker for thymic function. We will monitor the frequency of RTEs and their functional properties, including their capacity to proliferate and to undergo apoptosis in three groups of patients all transplanted but with different disease courses. Multiparametric flow cytometry will be extensively used herein to determine the proliferative status of RTEs and their functional activities as determined by cytokine production in the different groups of patients. We will also investigate using the Phosflow technology that allows the analysis of signalling transduction pathways defects in the IL-2 and IL-7 downstream signalling that can result from exposure of RTEs to type I INFs. In preliminary experiments, we have shown that type I INF genes expressed in both donor and recipient PBMCs can predict the course of GVHD.

In Specific Aim 2, we will now validate our findings on larger number of individuals and investigate the expression and function of genes downstream of INF in T cells obtained from these patients. Our microarray-based gene profiling studies suggest that a set of 20 genes may have the accuracy and robustness required for clinical GVHD prediction. We will thus validate our hypothesis (based on analysis of 380 human microarrays) that expression profiling of type 1 IFNs–related genes can segregate donors that will induce or not GVHD following AHCT, and (ii) get a mechanistic understanding of how IFN-related genes may determine the occurrence of GVHD.

In Specific Aim 3, we will identify by functional criteria genes products that are directly involved in the loss of central T cell development following acute GVHD and demonstrate the impact of genes identified to be differentially up-regulated in TECs damaged by GVHD for their significance in thymopoiesis. Using an in vivo experimental transplantation model, expression profiling of TECs damaged by aGVHD has identified several candidate genes responsible for the complex impairment of thymopoiesis uniformly observed following allo-recognition of TECs by donor T cells. The specific aim of this set of experiments is to establish for a few of the candidate genes a function in the pathogenesis of thymic aGVHD. These include (i) signal transducer and activator of transcription-1 (STAT-1), which is involved in intracellular signal transduction and which is typically up-regulated in the course of IFN-ß-mediated signalling in target cells which typically occurs in the course of aGVHD; (ii) caspase-12, a gene associated with epithelial apoptosis in response to IFNs- ß and human CXCL9 (Mig), a chemokine that is specifically induced by IFNs. To assess the functional role of several target genes identified to be specifically up-regulated in TECs subjected to the damaging effect of aGVHD, we propose to transfect OP9-DLl stromal cells with the above listed genes and to analyse their capacity to support the differentiations of CD34+ cells derived from the different groups of GVHD patients.

Altogether, the experiments planned in this proposal will allow us to define the defects in thymic production resulting from exposure to type I INFs in patients undergoing AHCT and that result in GVHD and immunodeficiency. These results will direct us towards novel immune therapeutic strategies.

INVESTIGATOR BIOGRAPHIES

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Rafick-Pierre Sékaly, Ph.D.

Rafick-Pierre Sékaly, Ph.D., a full professor at Université de Montréal, is the Scientific Director and Program Leader of CANVAC. Dr. Sékaly obtained his Ph.D. in Biochemistry at the Université of Lausanne in 1984 and went on to perform a postdoctoral fellowship, from 1984 to 1987, on immunogenetics and molecular biology of major histocompatibility complex molecules. He received several honors and awards, including the Forgaty Fellowship of the National Institutes of Health in Bethesda, Maryland, and the fellowships named Chercheur-boursier «Senior 1» of the Fonds de la Recherche en Santé du Québec, Prix du jeune chercheur of the Club de recherches cliniques du Québec, and Senior Scientist Salary Support Awards from the Medical Research Council of Canada. He is now the Canada chair in Human Immunology.

He has been involved in the areas of AIDS and AIDS pathogenesis for the past fifteen years. Using several novel technologies developed in his lab, he was able to demonstrat,e in collaboration with the researchers Dr. Fauci and Dr. Pantaleo, the features of the primary cellular immune responses in HIV infected patients. His work on AIDS initially involved the characterization of the interactions between CD4, the envelope protein of HIV gp120, and class II MHC molecules of the major histocompatibility complex molecules and the natural ligand of CD4. He has also generated seminal observations on the interactions between class II molecules and several of their ligands including the T cell receptor and bacterial and retroviral superantigens. His group has pioneered several new modifications to assays that allow the characterization of the qualitative and quantitative features of the immune response using multiparametric flow cytometry at the single cell level, including class I and class II tetramers. His group was also amongst the first to demonstrate that the thymus, the major site of T cell production, was still active in adults and was the target of viruses such HIV.

As Founder and Scientific Director of the CANVAC Network of Centre of Excellence, Dr. Sékaly hopes to improve the quality of life of Canadians by using novel technologies and a multidisciplinary approach in order to develop vaccines for the prevention and treatment of chronic diseases such as AIDS, hepatitis C, and cancer.

Juan Carlos Zuniga-Pflucker, Ph.D., received a B.Sc. in Zoology in 1987 at the University of Maryland. From there, he studied at George Washington University, receiving his Ph.D. in Genetics-Immunology in 1991. He did his postdoctoral training at the NIH from 1990 to 1994. Currently, Dr. Zuniga-Pflucker is a senior scientist in Molecular and Cellular Biology at Sunnybrook and Women's College Health Sciences Centre and professor of Immunology at the University of Toronto. His research focus is Hematopoiesis, Lymphocyte differentiation, and T cell development.

SELECTED PUBLICATIONS

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Baron C., Somogyi R., Greller L.D., Rineau V., Wilkinson P., Cho C.R., Cameron M.J., Kelvin D.J., Chagnon P., Roy D.C., Busque L., Sékaly R.P., and Perreault C.   Prediction of graft-versus-host disease in humans by donor gene-expression profiling.   PLoS Med. 2007 Jan;4(1):e23.