Final Report Summary - MIELT1D (MHC Class I Expression Level in Type 1 Diabetes)
Abstract: To achieve the overall goal of MIELT1D I have distinguished 3 specific objectives to be met, i.e (1) Understanding the implication of the light chain of MHC class I, ß2M, on CD8T cell thymic selection in T1D susceptibility (2) Study the level of expression of the ß2M to determine if it can be used as a diagnostic marker for T1D risk, and (3) Study the polymorphisms in the human ß2M promoter that control differential ß2M expression level to determine if it can act as genetic markers for T1D risk. Each aim is developed below.
Aim 1: Understanding the implication of the light chain of MHC class I, ß2M on the thymic selection of CD8T cell that cause T1D. The methodological approach consisted of extensively phenotyping CD8T cells: I should have determined the molecular markers that distinguish diabetogenic CD8 T cells from non-diabetogenic CD8 T cells with the same T cell receptor (TcR) specificity. I should have worked on a uniquely generated genetically engineered model of T1D to compare the influence of ß2M/MHC class I expression on disease. Unfortunately the transgenic animals required for these experiments could not be imported to the host institution without a 9 months delay. A requirement of the host facility was that the animals undergo a rederivation procedure to create specific pathogen free (SPF) mice. Because of the time limitation on the Marie Curie funded research this 9 month delay meant that the proposed animal housing conditions were not feasible. Alternative arrangements for housing the transgenic mice directly from Australia in a quarantine environment (obviating the need for time consuming re-derivation) were sought at Sciences Facilty. Unfortunately there was insufficient quarantine space available at Sciences Facilty to house the transgenic mice required for these experiments. None of the animal experiments could therefore be performed. Aim 2: Identification of diagnostic markers for T1D.The methodological approach consisted in studying the expression level of ß2M, in clinical samples. To achieve this part, I should have worked on a french serum collection (Prof. B. Viallettes) comprising 200 samples collected prospectively for the project (within Prof Viallettes Unit) and 200 control samples ( from Bank Transfusion Center, provided through Prof. B. Vialettes). Unfortunately these studies could not be performed due to a 6 month delay in the administrative process of reviewing the human ethics application necessary for collecting human blood. The ethics application was submitted to the hospital in March 2009, however there were repeated delays in the review process, and the application was still not approved in September 2009.
While waiting for the approval of the ethics application collaborations were established with Dr. Terese Letreut at Hopital Nord for the preparation of a methodology to quantify the surface expression of ß2M on peripheral blood cells from normal human blood. Reagents were ordered for primary and secondary antibodies that would allow quantitative detection of beta2microglobulin and HLA. Antibody reagents to identify cell population, including antibodies to CD4, CD8, CD14, CD20 and isotype controls were also ordered. Reagents for quantification of the surface level of HLA and ß2M arrived and were titrated on human blood with collaborator Prof Terese LeTreut (collaboration ongoing). Titrations of ß2M and HLA on cells defined by the markers CD4, CD8, CD14, and CD20 were performed for optimal working dilutions of antibodies. Numerous experiments were done to work out the best conditions for the protocol, using the minimum amount of human blood. The latter allowed us to use high throughput 96 well plates for analyses. We also sampled blood that had been stored at different temperatures so that we could determine the simplest mode of transport for the blood samples from T1D patients to arrive from Hopital Latimone to Hopital Nord. These experiments were successful.
Aim 1: Understanding the implication of the light chain of MHC class I, ß2M on the thymic selection of CD8T cell that cause T1D. The methodological approach consisted of extensively phenotyping CD8T cells: I should have determined the molecular markers that distinguish diabetogenic CD8 T cells from non-diabetogenic CD8 T cells with the same T cell receptor (TcR) specificity. I should have worked on a uniquely generated genetically engineered model of T1D to compare the influence of ß2M/MHC class I expression on disease. Unfortunately the transgenic animals required for these experiments could not be imported to the host institution without a 9 months delay. A requirement of the host facility was that the animals undergo a rederivation procedure to create specific pathogen free (SPF) mice. Because of the time limitation on the Marie Curie funded research this 9 month delay meant that the proposed animal housing conditions were not feasible. Alternative arrangements for housing the transgenic mice directly from Australia in a quarantine environment (obviating the need for time consuming re-derivation) were sought at Sciences Facilty. Unfortunately there was insufficient quarantine space available at Sciences Facilty to house the transgenic mice required for these experiments. None of the animal experiments could therefore be performed. Aim 2: Identification of diagnostic markers for T1D.The methodological approach consisted in studying the expression level of ß2M, in clinical samples. To achieve this part, I should have worked on a french serum collection (Prof. B. Viallettes) comprising 200 samples collected prospectively for the project (within Prof Viallettes Unit) and 200 control samples ( from Bank Transfusion Center, provided through Prof. B. Vialettes). Unfortunately these studies could not be performed due to a 6 month delay in the administrative process of reviewing the human ethics application necessary for collecting human blood. The ethics application was submitted to the hospital in March 2009, however there were repeated delays in the review process, and the application was still not approved in September 2009.
While waiting for the approval of the ethics application collaborations were established with Dr. Terese Letreut at Hopital Nord for the preparation of a methodology to quantify the surface expression of ß2M on peripheral blood cells from normal human blood. Reagents were ordered for primary and secondary antibodies that would allow quantitative detection of beta2microglobulin and HLA. Antibody reagents to identify cell population, including antibodies to CD4, CD8, CD14, CD20 and isotype controls were also ordered. Reagents for quantification of the surface level of HLA and ß2M arrived and were titrated on human blood with collaborator Prof Terese LeTreut (collaboration ongoing). Titrations of ß2M and HLA on cells defined by the markers CD4, CD8, CD14, and CD20 were performed for optimal working dilutions of antibodies. Numerous experiments were done to work out the best conditions for the protocol, using the minimum amount of human blood. The latter allowed us to use high throughput 96 well plates for analyses. We also sampled blood that had been stored at different temperatures so that we could determine the simplest mode of transport for the blood samples from T1D patients to arrive from Hopital Latimone to Hopital Nord. These experiments were successful.