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.