Periodic Reporting for period 1 - GD TCR LIGAND (Identification of the ligand of a human public anti-HCMV/cancer γδ T cell receptor)
Reporting period: 2017-03-01 to 2019-02-28
γδ T cells, an important compartment of adaptive immune responses against infections and malignant transformation, have been proposed as a promising target for cancer immunotherapy. However, progress in this field is blocked by limited knowledge regarding the ligands of γδ T cell receptors (TCR). Our project was driven by the idea that the acquisition of basic knowledge on γδ T cell biological functions could lead to new strategies for the elaboration of anti-viral and anti-tumor immunotherapies. So, our project aimed to boost research effort on this topic, on the basis of a work published by David Vermijlen (the promoter of the project). His group discovered a human γδ TCR which was highly enriched in every individual (therefore ‘public’) infected in utero with human cytomegalovirus (HCMV), and that reacts towards adult cancer cells, suggesting that it can detect signs of ‘cellular stress’ induced in both cell transformation and infection. Thus, the objective of this research project was the identification of the ligand of the public (cancer and infection) cross-reactive γδ TCR. Thanks to the work performed, it’s possible to conclude that: i) the ligand of the public γ TCR is not a soluble molecule; ii) about 150 genes that can represent candidate ligands were identified starting from previous gene expression profiling data; iii) a candidate ligand we selected from literature is not involved in the activation of the public TCR, thus it is not its ligand; iv) the immunoprecipitation protocols applied so far by using blocking antibodies were not able to provide us the expected results and support us in the identification of the ligand.
- Public γTCR reactivity. Working with ‘public-cells’ that express the public TCR, we checked the upregulation of an activation marker expressed by activated cells upon co-incubation with stimulator cells. As controls we used cells that express a non-public γδ TCR as well as not-transduced cells. By using this system a screening of a series of human cell lines has been previously performed (and now confirmed) in order to check their ability to activate the public γδ TCR, leading to group these cell lines in a ‘non-stimulatory’ (NS) or ‘stimulatory’ (S) class.
- Transwell experiments. The transwell insert is porous, allowing for dynamic interactions between the cells via secreted factors. Our results from transwell co-culture (S cells and public-cells) experiments demonstrated that the ligand of the public TCR is not a soluble molecule. Therefore, we hypothesized that the ligand could be a plasma membrane protein.
- Generation of a list of candidate ligands starting from previous microarray data. Assuming that the ligand is expressed by stimulator cells and not (or less) by non-stimulator cell lines, and starting from previous microarray data, we were able to generate a list of 150 genes to be tested as candidate ligands.
- Study of the role of a Butyrophilin protein in public-TCR activation and during HCMV infection. We selected this candidate ligand (we will call it BTNx) starting from literature, in particular because it is member of the butyrophilin (BTN) family and recent data indicates that this family of proteins could play an important role in γδ T cells activation. In order to analyse BTNx-counterreceptor expression, we worked with a recombinant BTNx-Fc fusion protein (and the relative control-Fc fusion protein). By incubating the biotinylated recombinant proteins with peripheral blood mononuclear cells derived both from HCMV- and HCMV+ new borns, we observed the binding of BTNx on γδ T cells derived from HCMV+ samples. At this point we checked the expression of BTNx protein in our different cell lines. We performed several runs of the experiments but finally we were not able to set up the protocols because of the results obtained were neither clear nor reproducible. The next step was to evaluate BTNx gene expression in our strongest stimulator cell line via RT-PCR. Surprisingly, several experiments demonstrated that this cell line does not express BTNx, thus our candidate protein can’t be the ligand of the public TCR.
- Screening of antibodies produced by hybridoma cells previously generated by the laboratory, in order to find a blocking antibody able to immunoprecipitate putative ligands of the public TCR. Upon the identification of blocking antibodies, able to block the activation of our public TCR, we performed immunoprecipitation experiments starting from protein extracts of stimulator cells, obtaining some nice and apparently specific bands. Unfortunately, the subsequent mass spectrometry analysis did not result in the identification of the putative ligand.
- Generation of γδbodies as an alternative tool to immunoprecipitate the ligand (collaboration with University of Hannover). A body is a chimeric structure which consist of the variable regions of the TCR and the constant regions of an antibody. In collaboration with the group of Immo Prinz we were in the process of the generation of such γδ bodies starting from the public TCR, as an alternative tool to immunoprecipitate the ligand. Unfortunately, our collaborators have encountered many difficulties in the production of these structures, and have failed to develop a protocol able to provide a sufficient amount of γδ bodies that could allow us to perform the planned experiments.
- Development of a surface proximity-dependent biotinylation assay (collaboration with University of South Dakota). The group of K. Roux developed a simply and quick technique for identifying interacting proteins. The difficulty with this method was that it has always been used as an intracellular system, and we needed to apply it in the extracellular interface. This is the reason why finally our collaborators have not been able to apply successfully this technique to our purpose, so we could not work with this key reagent.
The results obtained by working at this project has been presented and discussed during periodic meetings of the team, as well as during the annual meeting of the Institute for Medical Immunology (IMI) where the experiments were conducted. Taking into account that the prospects for the dissemination/exploitation of the results were based on the assumption of finding the ligand of the public γδ TCR, and finally we were not able to identify it, we didn’t have the opportunity to carry out any exploitation activity. For the same reason we decided not to share our data at scientific conferences, having been confident until now to get closer and closer to achieving our goal.
- Transwell experiments. The transwell insert is porous, allowing for dynamic interactions between the cells via secreted factors. Our results from transwell co-culture (S cells and public-cells) experiments demonstrated that the ligand of the public TCR is not a soluble molecule. Therefore, we hypothesized that the ligand could be a plasma membrane protein.
- Generation of a list of candidate ligands starting from previous microarray data. Assuming that the ligand is expressed by stimulator cells and not (or less) by non-stimulator cell lines, and starting from previous microarray data, we were able to generate a list of 150 genes to be tested as candidate ligands.
- Study of the role of a Butyrophilin protein in public-TCR activation and during HCMV infection. We selected this candidate ligand (we will call it BTNx) starting from literature, in particular because it is member of the butyrophilin (BTN) family and recent data indicates that this family of proteins could play an important role in γδ T cells activation. In order to analyse BTNx-counterreceptor expression, we worked with a recombinant BTNx-Fc fusion protein (and the relative control-Fc fusion protein). By incubating the biotinylated recombinant proteins with peripheral blood mononuclear cells derived both from HCMV- and HCMV+ new borns, we observed the binding of BTNx on γδ T cells derived from HCMV+ samples. At this point we checked the expression of BTNx protein in our different cell lines. We performed several runs of the experiments but finally we were not able to set up the protocols because of the results obtained were neither clear nor reproducible. The next step was to evaluate BTNx gene expression in our strongest stimulator cell line via RT-PCR. Surprisingly, several experiments demonstrated that this cell line does not express BTNx, thus our candidate protein can’t be the ligand of the public TCR.
- Screening of antibodies produced by hybridoma cells previously generated by the laboratory, in order to find a blocking antibody able to immunoprecipitate putative ligands of the public TCR. Upon the identification of blocking antibodies, able to block the activation of our public TCR, we performed immunoprecipitation experiments starting from protein extracts of stimulator cells, obtaining some nice and apparently specific bands. Unfortunately, the subsequent mass spectrometry analysis did not result in the identification of the putative ligand.
- Generation of γδbodies as an alternative tool to immunoprecipitate the ligand (collaboration with University of Hannover). A body is a chimeric structure which consist of the variable regions of the TCR and the constant regions of an antibody. In collaboration with the group of Immo Prinz we were in the process of the generation of such γδ bodies starting from the public TCR, as an alternative tool to immunoprecipitate the ligand. Unfortunately, our collaborators have encountered many difficulties in the production of these structures, and have failed to develop a protocol able to provide a sufficient amount of γδ bodies that could allow us to perform the planned experiments.
- Development of a surface proximity-dependent biotinylation assay (collaboration with University of South Dakota). The group of K. Roux developed a simply and quick technique for identifying interacting proteins. The difficulty with this method was that it has always been used as an intracellular system, and we needed to apply it in the extracellular interface. This is the reason why finally our collaborators have not been able to apply successfully this technique to our purpose, so we could not work with this key reagent.
The results obtained by working at this project has been presented and discussed during periodic meetings of the team, as well as during the annual meeting of the Institute for Medical Immunology (IMI) where the experiments were conducted. Taking into account that the prospects for the dissemination/exploitation of the results were based on the assumption of finding the ligand of the public γδ TCR, and finally we were not able to identify it, we didn’t have the opportunity to carry out any exploitation activity. For the same reason we decided not to share our data at scientific conferences, having been confident until now to get closer and closer to achieving our goal.
In collaboration with the group head by Matthias Eberl (Cardiff University) at the beginning of 2018 we published the review “γδ T cell responses: How many ligands will it take till we know?” on Seminars in Cell and Developmental Biology. This is a review journal dedicated to keeping scientists informed of developments in the field of molecular cell and developmental biology.
With this work we reviewed the latest progress in the identification and validation of putative γδ T cell ligands and discuss the implications of such findings for γδ T cell responses in health and disease.
With this work we reviewed the latest progress in the identification and validation of putative γδ T cell ligands and discuss the implications of such findings for γδ T cell responses in health and disease.