Periodic Reporting for period 1 - PIM PROTEOMICS (Unravelling PIM kinase signal integration in the T cell response)
Berichtszeitraum: 2017-07-01 bis 2019-06-30
The immune response provides powerful protection against harmful infections. An important component of this response involves a cell type called a “T cell” which provides protection by killing virus infected cells or cancerous cells. However, in patients with autoimmune disease T cells can incorrectly target parts of the human body and cause disease. During organ transplantation T cells will also attack and lead to rejection of the newly transplanted organ unless their function is suppressed. Therefore, understanding how T cell responses can be reduced or switched off is of critical importance for designing drug therapies for treating autoimmune diseases or during organ transplantation.
In order for T cells to respond to a threat they must receive multiple external signals that indicate danger. These signals and transmitted internally by multiple pathways and cause the T cells to change their protein composition so that they can survive, multiply and develop the killing capability needed to form a strong immune response. One way that a T cell response can be switched off is to inhibit the internal transmission pathways from these signals so that T cells become unable to survive, multiply and kill. Therefore, understanding what these internal transmission pathways are and how they work together is of critical importance for designing immune suppressive therapies.
In this project I investigated how two types of signalling pathway proteins called the PIM kinases, and mTORC1 acted individually and together to control the strength and function of the T cell immune response.
PIM kinases have also been shown to play a role in supporting the T cell immune response, promoting T cell multiplication and survival. How the PIM kinases have this effect in T cells in not well understood. Drugs that inhibit the PIM kinases are in clinical development and testing. mTORC1 is known to promote cell growth and killing function in T cells. A drug inhibiting mTORC1 function is used clinically in order to suppress the T cell immune response during organ transplantation. Inhibition of mTORC1 alone is not always effective at suppressing the T cell immune response. Therefore, combining mTORC1 inhibition with other drugs, such as inhibitors of PIM kinases, to increase therapeutic effectiveness is of clinical interest. Previously, it was shown that removal of PIM kinases and inhibition of mTORC1 acts synergistically to prevent T cells from switching on and multiplying. How these two proteins work together to control T cell growth is not known.
The overarching goal of this project was to better understand what PIM kinases are doing in T cells and to explore how PIM kinases were synergising with mTORC1 to control the T cell response. In this context, the objectives of this project were:
1) To measure how removal of PIM kinases changed the protein composition of T cells
2) To look for overlaps in changes to T cell protein composition when PIM kinases have been removed or mTORC1 inhibited
3) Test the effect PIM kinase removal and inhibition of mTORC1 on the ability of T cells to multiply, survive and develop the killing capability necessary to form a strong and effective immune response.
In order for T cells to respond to a threat they must receive multiple external signals that indicate danger. These signals and transmitted internally by multiple pathways and cause the T cells to change their protein composition so that they can survive, multiply and develop the killing capability needed to form a strong immune response. One way that a T cell response can be switched off is to inhibit the internal transmission pathways from these signals so that T cells become unable to survive, multiply and kill. Therefore, understanding what these internal transmission pathways are and how they work together is of critical importance for designing immune suppressive therapies.
In this project I investigated how two types of signalling pathway proteins called the PIM kinases, and mTORC1 acted individually and together to control the strength and function of the T cell immune response.
PIM kinases have also been shown to play a role in supporting the T cell immune response, promoting T cell multiplication and survival. How the PIM kinases have this effect in T cells in not well understood. Drugs that inhibit the PIM kinases are in clinical development and testing. mTORC1 is known to promote cell growth and killing function in T cells. A drug inhibiting mTORC1 function is used clinically in order to suppress the T cell immune response during organ transplantation. Inhibition of mTORC1 alone is not always effective at suppressing the T cell immune response. Therefore, combining mTORC1 inhibition with other drugs, such as inhibitors of PIM kinases, to increase therapeutic effectiveness is of clinical interest. Previously, it was shown that removal of PIM kinases and inhibition of mTORC1 acts synergistically to prevent T cells from switching on and multiplying. How these two proteins work together to control T cell growth is not known.
The overarching goal of this project was to better understand what PIM kinases are doing in T cells and to explore how PIM kinases were synergising with mTORC1 to control the T cell response. In this context, the objectives of this project were:
1) To measure how removal of PIM kinases changed the protein composition of T cells
2) To look for overlaps in changes to T cell protein composition when PIM kinases have been removed or mTORC1 inhibited
3) Test the effect PIM kinase removal and inhibition of mTORC1 on the ability of T cells to multiply, survive and develop the killing capability necessary to form a strong and effective immune response.
Objective 1: To test how removal of PIM kinases changes the protein composition of T cells.
Normal T cells and T cells in which the PIM kinases had been removed were grown in plastic dishes under a series of conditions that mimicked the environment T cells encounter during an immune response. Protein composition of the T cells was measured using a powerful technique call mass spectrometry. With this technology we measured the amounts of each of the 4000-7000 main proteins that make up a T cell. We compared whether the quantity of each protein was the same in normal T cells versus PIM kinase-deficient T cells. Surprisingly, removal of PIM kinases did not change the amounts of many proteins. Only 0.5-3% of the proteins changed in quantity when PIM kinases were removed. We examined if the proteins that changed in quantity were the same in each of the different growth conditions tested and found that there was almost no overlap.
Collectively, these results indicated that PIM kinases only have a small, context-specific effect the protein composition of T cells i.e. there is no universal function the PIM kinases perform in all the conditions T cells encounter during an immune response.
Objective 2: To look for overlaps in proteins controlled by PIM kinases and mTORC1.
From the work in objective 1 we had a list of the proteins changed due to PIM removal. We also had a list of the protein changed due to mTORC1 inhibition - this data was generated previously within the Cantrell laboratory by Dr Andy Howden. We compared the list of PIM kinase-regulated and mTORC1-regulated proteins to look at how much they overlapped for each of the growth conditions tested. It had previously been reported that PIM kinases and mTORC1 work together to control T cell growth and survival therefore we expected that there would be a large overlap between the PIM kinase-controlled and mTORC1-controlled proteins. Surprisingly, we saw the opposite. There was almost no overlap between the proteins lists in all the environmental conditions tested.
This suggested that the PIM kinases and mTORC1 were not functioning similarly in T cells and were not working together synergistically to control the strength of the T cell immune response.
Objective 3: To test the effect of PIM kinases and mTORC1 on the ability of T cells to multiply and survive.
Normal T cells or T cells in which PIM kinases were removed were labelled with a dye that enabled us to measure how many times the cells multiplied. We grew normal T cells and PIM kinase-deficient T cells in plastic dishes in the presence or absence of mTORC1 inhibitor. We measured the number of cells and how many times they had divided in each condition daily for a week. Contrary to previous reports, we found there was no difference in the amount of cell division or cell survival when normal versus PIM kinase-deficient T cells were treated with an mTORC1 inhibitor.
This supported the idea that PIM kinases and mTORC1 did not synergise to control the strength of the T cell response.
Exploitation and Dissemination:
At present the results from these studies have been presented at scientific conferences both locally and internationally. We are in the process of assembling these results into a manuscript that we will submit for publication in a scientific journal.
Normal T cells and T cells in which the PIM kinases had been removed were grown in plastic dishes under a series of conditions that mimicked the environment T cells encounter during an immune response. Protein composition of the T cells was measured using a powerful technique call mass spectrometry. With this technology we measured the amounts of each of the 4000-7000 main proteins that make up a T cell. We compared whether the quantity of each protein was the same in normal T cells versus PIM kinase-deficient T cells. Surprisingly, removal of PIM kinases did not change the amounts of many proteins. Only 0.5-3% of the proteins changed in quantity when PIM kinases were removed. We examined if the proteins that changed in quantity were the same in each of the different growth conditions tested and found that there was almost no overlap.
Collectively, these results indicated that PIM kinases only have a small, context-specific effect the protein composition of T cells i.e. there is no universal function the PIM kinases perform in all the conditions T cells encounter during an immune response.
Objective 2: To look for overlaps in proteins controlled by PIM kinases and mTORC1.
From the work in objective 1 we had a list of the proteins changed due to PIM removal. We also had a list of the protein changed due to mTORC1 inhibition - this data was generated previously within the Cantrell laboratory by Dr Andy Howden. We compared the list of PIM kinase-regulated and mTORC1-regulated proteins to look at how much they overlapped for each of the growth conditions tested. It had previously been reported that PIM kinases and mTORC1 work together to control T cell growth and survival therefore we expected that there would be a large overlap between the PIM kinase-controlled and mTORC1-controlled proteins. Surprisingly, we saw the opposite. There was almost no overlap between the proteins lists in all the environmental conditions tested.
This suggested that the PIM kinases and mTORC1 were not functioning similarly in T cells and were not working together synergistically to control the strength of the T cell immune response.
Objective 3: To test the effect of PIM kinases and mTORC1 on the ability of T cells to multiply and survive.
Normal T cells or T cells in which PIM kinases were removed were labelled with a dye that enabled us to measure how many times the cells multiplied. We grew normal T cells and PIM kinase-deficient T cells in plastic dishes in the presence or absence of mTORC1 inhibitor. We measured the number of cells and how many times they had divided in each condition daily for a week. Contrary to previous reports, we found there was no difference in the amount of cell division or cell survival when normal versus PIM kinase-deficient T cells were treated with an mTORC1 inhibitor.
This supported the idea that PIM kinases and mTORC1 did not synergise to control the strength of the T cell response.
Exploitation and Dissemination:
At present the results from these studies have been presented at scientific conferences both locally and internationally. We are in the process of assembling these results into a manuscript that we will submit for publication in a scientific journal.
Collectively, this project has demonstrated that the previously reported synergy between PIM kinases and mTORC1 in T cells is not reproducible. In complete contrast to what had previously been reported, we did not observe evidence of PIM kinases and mTORC1 acting together to control T cell function. They in fact appeared to have distinct effects on the T cell response. This is an important step forward in our understanding of the PIM kinases, as it calls into question the previously accepted idea that the function of PIM kinases was to work together with mTORC1. This has an important wider impact for clinical drug development as it suggests that trialling a combination of mTORC1 and PIM kinase inhibition as a therapy would not be effective. This has the potential to save considerable time, money and effort in the development of effective immune suppressive therapies.