Final Report Summary - PEPTIDE DIVERSITY (Diversity of MHC/peptide complexes in thymic compartments and their role in shaping T cell repertoire)
In the Peptide Diversity project we are assessing the role of antigen processing pathways that are utilized by distinct thymic antigen presenting cell (APC)-types that mediate positive selection and tolerance induction. Specifically, there is increasing evidence that cortical thymic epithelial cells (cTECs), which mediate the positive selection of T cells, use different pathways of antigen processing and loading of both MHC class I and MHC class II molecules as compared to APCs that reside in the medulla: medullary Thymic Epithelial Cells (mTECs) and Dendritic Cells (DCs). However, the biological relevance of this observation has not been resolved.
There are several proteolytic enzymes in the MHC class II loading pathway that are differentially expressed in APCs. The first class of proteases that was shown to have a differential expression pattern in cTECs in comparison to other APCs are cathepsins. Cathepsins are lysosomal proteases involved in invariant chain (li) processing and also in the processing of protein antigens that are to be displayed on the cell surface in the context of MHC class II molecules. While thymic DCs and mTECs make use of Cathepsin S, cTECs rely on the activity of Cathepsin L protease. Cathepsin L knock out animals display a severe loss of CD4 T cells (60-80% reduction of CD4 T cells in comparison to WT animals), which has been suggested to result from inefficient positive selection due to a reduced diversity and/or different nature of MHC-peptide complexes on cTECs (Nakagawa et al., 1998). However, this hypothesis has not been experimentally tested due to technical limitations of available peptide-sequencing technologies. Furthermore, the functional quality of the T cell repertoire that is selected in the absence of Cathepsin L enzymatic activity remained uncharacterized. Here, we set out to address these open questions, which may have a substantial impact on our understanding of TCR-MHC/peptide interactions and their influence on T cell biology.
In order to gain insight into the specific role of Cathepsin L in T cell “education” we proposed to:
i) Analyze in detail Cathepsin L knock out and Cathepsin L conditional knock out animals (whereby the gene is specifically deleted only in thymic epithelium due to expression of Cre recombinase under the Foxn1 promoter - CatLΔTEC animals). Particular emphasis has been put on understanding the arm of the selection process that is impaired in the system (positive or negative selection of developing T cell repertoire);
ii) Analyze in detail the functionality of T cells that develop on Cathepsin L deficient thymic epithelium in a polyclonal settings – assessment of immune-competence;
iii) Establish a new mouse model where the selected T cell repertoire has a limited diversity- ‘oligo-clonal’ TCR transgenic mouse; The new animal model will allow for monitoring the "fate" of every single cell that is "born" in the thymus in different experimental settings (e.g. WT versus Cathepsin L knock out animals), a task that remains impossible to accomplish in WT TCR repertoire, as any given mouse has around hundred million different T cell specificities. By taking advantage of single cell TCR gene sequencing technology this model will enable us to generate an "index" of all possible T cell specificities that can be selected in WT animals. Thus, comparative analyses of T cell receptor sequences of cells selected on Cathepsin L sufficient and deficient thymic epithelium will for the first time allow us to qualitatively asses the influence of a cTEC-specific protease on the nature of the selected T cell repertoire in high resolution.
To this end, we have characterized in detail the phenotype of Cathepsin L knock out and CatLΔTEC animals. CatLΔTEC animals phenocopy Cathepsin L deficient animals, thus the impairment in development of CD4 T cell repertoire is due to the absence of this cysteine protease in thymic epithelium. Specifically, the analyses have confirmed published findings that in the absence of a functional Cathepsin L protease, a specific impairment in development of CD4 cells is observed (around 70% reduction in CD4 T cell numbers). Detailed analyses of thymocyte development have revealed that thymocytes accumulate at the immature stage of CD4 development (as defined by expression of CD24, CD69 and CD62L molecular markers). Analyses of naïve CD4 T cells in peripheral lymphoid organs (lymph nodes and spleen) have revealed that CatL deficient mice exhibit a loss of T cells bearing receptors with low affinity for self (as assessed by molecular markers Nur77, CD5 and Ly6C). Furthermore, we documented that the peripheral repertoire of T cells educated on Cathepsin L knock out epithelium displays a significantly increased ratio of regulatory to effector T cells in lymph nodes and spleen. We have also observed that the T cell repertoire selected on CatL knock out epithelium shows significant differences in usage of a set of variable genes both in TCR alpha and TCR beta chain of T cell receptor. The differences in the repertoire of CD4 T cells are evident in both primary and peripheral lymphoid organs, and are not observed in the CD8 T cell repertoire. Thus, Cathepsin L protease molds the T cell repertoire both quantitatively and qualitatively.
Our analyses of thymic antigen presenting cells (cortical thymic epithelial cells, medullary thymic epithelial cells, dendritic cells and B cells) have documented that there are no major perturbations in the APCs composition in Cathepsin L knock out animals. Specifically, the number of antigen presenting cells as well as expression level of both MHC class II and MHC class I are not impaired.
Furthermore, we have been able to show that deficiency of CatL in TECs specifically affects positive selection of developing T cells, while we did not observe any alterations in negative selection. Briefly, we have established a model where all antigen presenting cells involved in negative selection (mTECs, DCs and B cells) have impaired expression of MHC class II, and thus cannot efficiently present peptides to developing T cells. Therefore, if the observed reduction in the CD4 T cell compartment results from excessive negative selection in the medulla one would observe a rescue of the developmental defect. However, even when we rendered negative selection inefficient in the CatL deficient mouse model, we did no observe the rescue of CD4 T cell development. Thus, we conclude that negative selection does not contribute to the observed deficiency in CD4 T cell selection.
We have assessed the positive selection of T cells with a known specificity for foreign- antigens by taking advantage of TCR transgenic animals, where virtually all CD4 T cells express the receptor of defined specificity. We analyzed the positive selection of Dep, OT II and AD10 TCR transgenic thymocytes which express T cell receptors specific for human C- reactive protein, ovalbumin and moth cytochrome c, respectively. The selection of all tested TCR transgenic cells is impaired in Cathepsin L deficient animals. Furthermore, we analyzed the efficacy of positive selection at the CD4+CD8+ DP stage of development in polyclonal, WT settings. In order to monitor interactions between the developing T cells and MHC class II –peptide complexes only, we have crossed CatL deficient animals with MHC class I null mouse model. By doing so, we were able to monitor only the interaction of T cell receptor and MHC class II –complexes displayed on the surface of CatL sufficient and deficient TECs. We were able to document that positive selection is indeed impaired in a polyclonal system as well, as assessed by decreased expression of molecular markers CD69, CD5 and TCR complex (TCRβ and CD3ε chain) on the surface of CD4+CD8+ DP thymocytes. Thus, these findings in a polyclonal system go in line with our observations of inefficient positive selection of T cells with a known specificity for an antigen.
In conclusion, we have revealed that Cat L deficiency specifically affects positive selection of the developing T cell repertoire, while tolerance induction remains intact in the studied system.
Subsequent to our detailed characterization of T cell development, we have started to assess the functionality of selected cells in in vivo settings. Specifically, we have monitored the ability of CD4 T cells to provide help to B cells during the immune response to foreign pathogens by monitoring the germinal center reaction upon sheep red blood cell immunization. This set of experiments revealed that the T cell repertoire educated on CatL deficient thymic epithelium is unable to provide efficient help to B cells during the immune response, as evidenced by a reduction in the number of germinal center B cells. We are currently investigating the efficacy of immune response to a set of pathogen - derived antigens (Listeriolyisin O protein of Listeria monocytogenes, Cholera Toxin B subunit of Vibrio cholerae and Glycoprotein 1 of Lymphocytic choriomeningitis virus) by taking advantage of MHC II- tetramers.
Lastly, we have successfully established the conventional TCR transgenic model with a limited diversity of a T cell repertoire, TCRmini PLP500 mini mouse model – ‘oligoclonal’ mouse. We have chosen to work with TCR genes that encode a receptor that is specific for a self-peptide/MHC Class II complex. Specifically, we have cloned in a TCR alpha expression cassette a TCR mini locus that contains variable and joint genes that have been identified to be a part of a T cell receptor, which is specific for myelin proteolipid protein (PLP), a self-protein expressed in the central nervous system and in both cortical and medullary thymic epithelium. Importantly, the gene segments in the engineered TCR mini locus are flanked with recombination signal sequences (RSS) that allow for generation of junctional diversity. As PLP knock out animals are available, we can use this oligo-clonal mouse model to study positive selection and anti-foreign immune - responses upon breeding TCRmini PLP500 mini mouse model onto PLP deficient background. This mouse model will be also used to study how the presence or absence of an antigen, and processing of antigen in question, influence and shape the development of T cell repertoire (studies of tolerance induction).
Taken together, the results of the ‘Peptide Diversity’ project will precisely characterize Cathepsin L’s role in the generation of an immuno-competent and self- tolerant T cell compartment. Combined, the described model systems will lead to understanding of the causal links between the positive selection of ‘altered’ T cell repertoire and (dys)functionality of T cell responses. By understanding this phenomenon, we can pave the way for development of new pharmacological therapies that would modulate activities of antigen processing enzymes in pre-determined ways in individuals who are suffering from auto-immune related disorders on one hand and immune-deficiencies, on the other.
There are several proteolytic enzymes in the MHC class II loading pathway that are differentially expressed in APCs. The first class of proteases that was shown to have a differential expression pattern in cTECs in comparison to other APCs are cathepsins. Cathepsins are lysosomal proteases involved in invariant chain (li) processing and also in the processing of protein antigens that are to be displayed on the cell surface in the context of MHC class II molecules. While thymic DCs and mTECs make use of Cathepsin S, cTECs rely on the activity of Cathepsin L protease. Cathepsin L knock out animals display a severe loss of CD4 T cells (60-80% reduction of CD4 T cells in comparison to WT animals), which has been suggested to result from inefficient positive selection due to a reduced diversity and/or different nature of MHC-peptide complexes on cTECs (Nakagawa et al., 1998). However, this hypothesis has not been experimentally tested due to technical limitations of available peptide-sequencing technologies. Furthermore, the functional quality of the T cell repertoire that is selected in the absence of Cathepsin L enzymatic activity remained uncharacterized. Here, we set out to address these open questions, which may have a substantial impact on our understanding of TCR-MHC/peptide interactions and their influence on T cell biology.
In order to gain insight into the specific role of Cathepsin L in T cell “education” we proposed to:
i) Analyze in detail Cathepsin L knock out and Cathepsin L conditional knock out animals (whereby the gene is specifically deleted only in thymic epithelium due to expression of Cre recombinase under the Foxn1 promoter - CatLΔTEC animals). Particular emphasis has been put on understanding the arm of the selection process that is impaired in the system (positive or negative selection of developing T cell repertoire);
ii) Analyze in detail the functionality of T cells that develop on Cathepsin L deficient thymic epithelium in a polyclonal settings – assessment of immune-competence;
iii) Establish a new mouse model where the selected T cell repertoire has a limited diversity- ‘oligo-clonal’ TCR transgenic mouse; The new animal model will allow for monitoring the "fate" of every single cell that is "born" in the thymus in different experimental settings (e.g. WT versus Cathepsin L knock out animals), a task that remains impossible to accomplish in WT TCR repertoire, as any given mouse has around hundred million different T cell specificities. By taking advantage of single cell TCR gene sequencing technology this model will enable us to generate an "index" of all possible T cell specificities that can be selected in WT animals. Thus, comparative analyses of T cell receptor sequences of cells selected on Cathepsin L sufficient and deficient thymic epithelium will for the first time allow us to qualitatively asses the influence of a cTEC-specific protease on the nature of the selected T cell repertoire in high resolution.
To this end, we have characterized in detail the phenotype of Cathepsin L knock out and CatLΔTEC animals. CatLΔTEC animals phenocopy Cathepsin L deficient animals, thus the impairment in development of CD4 T cell repertoire is due to the absence of this cysteine protease in thymic epithelium. Specifically, the analyses have confirmed published findings that in the absence of a functional Cathepsin L protease, a specific impairment in development of CD4 cells is observed (around 70% reduction in CD4 T cell numbers). Detailed analyses of thymocyte development have revealed that thymocytes accumulate at the immature stage of CD4 development (as defined by expression of CD24, CD69 and CD62L molecular markers). Analyses of naïve CD4 T cells in peripheral lymphoid organs (lymph nodes and spleen) have revealed that CatL deficient mice exhibit a loss of T cells bearing receptors with low affinity for self (as assessed by molecular markers Nur77, CD5 and Ly6C). Furthermore, we documented that the peripheral repertoire of T cells educated on Cathepsin L knock out epithelium displays a significantly increased ratio of regulatory to effector T cells in lymph nodes and spleen. We have also observed that the T cell repertoire selected on CatL knock out epithelium shows significant differences in usage of a set of variable genes both in TCR alpha and TCR beta chain of T cell receptor. The differences in the repertoire of CD4 T cells are evident in both primary and peripheral lymphoid organs, and are not observed in the CD8 T cell repertoire. Thus, Cathepsin L protease molds the T cell repertoire both quantitatively and qualitatively.
Our analyses of thymic antigen presenting cells (cortical thymic epithelial cells, medullary thymic epithelial cells, dendritic cells and B cells) have documented that there are no major perturbations in the APCs composition in Cathepsin L knock out animals. Specifically, the number of antigen presenting cells as well as expression level of both MHC class II and MHC class I are not impaired.
Furthermore, we have been able to show that deficiency of CatL in TECs specifically affects positive selection of developing T cells, while we did not observe any alterations in negative selection. Briefly, we have established a model where all antigen presenting cells involved in negative selection (mTECs, DCs and B cells) have impaired expression of MHC class II, and thus cannot efficiently present peptides to developing T cells. Therefore, if the observed reduction in the CD4 T cell compartment results from excessive negative selection in the medulla one would observe a rescue of the developmental defect. However, even when we rendered negative selection inefficient in the CatL deficient mouse model, we did no observe the rescue of CD4 T cell development. Thus, we conclude that negative selection does not contribute to the observed deficiency in CD4 T cell selection.
We have assessed the positive selection of T cells with a known specificity for foreign- antigens by taking advantage of TCR transgenic animals, where virtually all CD4 T cells express the receptor of defined specificity. We analyzed the positive selection of Dep, OT II and AD10 TCR transgenic thymocytes which express T cell receptors specific for human C- reactive protein, ovalbumin and moth cytochrome c, respectively. The selection of all tested TCR transgenic cells is impaired in Cathepsin L deficient animals. Furthermore, we analyzed the efficacy of positive selection at the CD4+CD8+ DP stage of development in polyclonal, WT settings. In order to monitor interactions between the developing T cells and MHC class II –peptide complexes only, we have crossed CatL deficient animals with MHC class I null mouse model. By doing so, we were able to monitor only the interaction of T cell receptor and MHC class II –complexes displayed on the surface of CatL sufficient and deficient TECs. We were able to document that positive selection is indeed impaired in a polyclonal system as well, as assessed by decreased expression of molecular markers CD69, CD5 and TCR complex (TCRβ and CD3ε chain) on the surface of CD4+CD8+ DP thymocytes. Thus, these findings in a polyclonal system go in line with our observations of inefficient positive selection of T cells with a known specificity for an antigen.
In conclusion, we have revealed that Cat L deficiency specifically affects positive selection of the developing T cell repertoire, while tolerance induction remains intact in the studied system.
Subsequent to our detailed characterization of T cell development, we have started to assess the functionality of selected cells in in vivo settings. Specifically, we have monitored the ability of CD4 T cells to provide help to B cells during the immune response to foreign pathogens by monitoring the germinal center reaction upon sheep red blood cell immunization. This set of experiments revealed that the T cell repertoire educated on CatL deficient thymic epithelium is unable to provide efficient help to B cells during the immune response, as evidenced by a reduction in the number of germinal center B cells. We are currently investigating the efficacy of immune response to a set of pathogen - derived antigens (Listeriolyisin O protein of Listeria monocytogenes, Cholera Toxin B subunit of Vibrio cholerae and Glycoprotein 1 of Lymphocytic choriomeningitis virus) by taking advantage of MHC II- tetramers.
Lastly, we have successfully established the conventional TCR transgenic model with a limited diversity of a T cell repertoire, TCRmini PLP500 mini mouse model – ‘oligoclonal’ mouse. We have chosen to work with TCR genes that encode a receptor that is specific for a self-peptide/MHC Class II complex. Specifically, we have cloned in a TCR alpha expression cassette a TCR mini locus that contains variable and joint genes that have been identified to be a part of a T cell receptor, which is specific for myelin proteolipid protein (PLP), a self-protein expressed in the central nervous system and in both cortical and medullary thymic epithelium. Importantly, the gene segments in the engineered TCR mini locus are flanked with recombination signal sequences (RSS) that allow for generation of junctional diversity. As PLP knock out animals are available, we can use this oligo-clonal mouse model to study positive selection and anti-foreign immune - responses upon breeding TCRmini PLP500 mini mouse model onto PLP deficient background. This mouse model will be also used to study how the presence or absence of an antigen, and processing of antigen in question, influence and shape the development of T cell repertoire (studies of tolerance induction).
Taken together, the results of the ‘Peptide Diversity’ project will precisely characterize Cathepsin L’s role in the generation of an immuno-competent and self- tolerant T cell compartment. Combined, the described model systems will lead to understanding of the causal links between the positive selection of ‘altered’ T cell repertoire and (dys)functionality of T cell responses. By understanding this phenomenon, we can pave the way for development of new pharmacological therapies that would modulate activities of antigen processing enzymes in pre-determined ways in individuals who are suffering from auto-immune related disorders on one hand and immune-deficiencies, on the other.