Periodic Reporting for period 4 - REGiREG (Regulating the immune regulators: targeting adaptive immune control)
Berichtszeitraum: 2019-08-01 bis 2021-06-30
The immune system with its complex interactions of cells and molecules needs a very tight and specific interplay of control elements to ensure the establishment and re-establishment of immune homeostasis after challenges. Regulatory T cells are key-players in this regulatory network. It is now well accepted that deficiency or dysfunction of regulatory T cells causes various severe immune disorders due to immune hyperactivation. Conversely, an increased number of regulatory T cells in tumor-bearing individuals suppresses efficient anti-tumor immunity and, thereby, is often associated with poor prognosis. Cancer immunology is now one of the most exciting and promising frontiers in cancer research, and recent clinical trials have proven that immunotherapies driving to activate T cells can induce durable responses. In this sense, harnessing the potential of regulatory T cells is one of the most promising new approaches to control immune function and to treat cancer. This proposal has the objectives: identification and characterization of tissue-resident regulatory T cells to principally understand the unique features of regulatory T cell specialization in tissues and their function in organ-homeostasis, a phenomenon that is hardly understood, but holds great promise for local, tissue-specific immune intervention. And to interfere with regulatory T cells. We expect from these studies new basic insights into a fascinating and still arcane aspect of organ-homeostasis as maintained by regulatory T cells, as well as novel candidate molecules that target regulatory T cells.
Within this project, we were able to establish several novel tools and technologies to study regulatory T cells within tissues. We established new in-vivo model systems and in-vitro test systems. We characterized regulatory T cells on a molecular level from different organs and tissues.
For examples, we performed epigenetic analysis with a tagmentation-based whole-genome bisulfite sequencing technology, which allowed us to analyze the DNA methylation status of very few Treg cells isolated from tissues. With this technology, we were able to study the epigenetic changes of DNA methylation with the highest possible resolution (single nucleotide and genome-wide). Similarities of the epigenetic landscape led to the identification of a common tissue Treg population, present in many organs and characterized by gain and loss of DNA methylation, including many TH2-specific sites. Thus, we could show that tissue Treg cells integrate different waves of epigenetic reprogramming which define their tissue-restricted specializations. The corresponding manuscript has been published in Nature Immunology (Delacher M et al. Genome-wide DNA-methylation landscape defines specialization of regulatory T cells in tissues. Nature Immunology, 2017;18(10):1160-1172).
We wanted to understand the development of these tissue-resident Treg cells. By using our novel Nfil3-Cre-eGFP reporter mouse line, single-cell RNA-sequencing and bulk ATAC-sequencing, we identified two precursor stages of these IL-33 receptor ST2-expressing nonlymphoid tissue Treg population in spleen and lymph nodes. Molecular profiling of these precursors revealed their sequence of differentiation. Global chromatin profiling of nonlymphoid tissue Treg cells and the two precursor stages revealed a stepwise acquirement of open chromatin accessibility and reprogramming towards the nonlymphoid-tissue Treg cell phenotype. Mechanistically, we identified and validated the basic leucine zipper transcription factor, ATF-like (Batf) as the driver of the molecular tissue program in the precursors. We were able to publish these findings in the journal Immunity (Delacher et al., Precursors for Nonlymphoid-Tissue Treg Cells Reside in Secondary Lymphoid Organs and Are Programmed by the Transcription Factor BATF. Immunity, 2020; 18;52(2):295-312).
We were also able to shed light on human tissue-resident Tregs cells. We identified features that characterize human Treg cells with tissue-repair function. Single-cell chromatin accessibility profiles of murine and human tissue Treg cells defined a conserved, microbiota-independent tissue-repair Treg signature with a prevailing footprint of the transcription factor BATF. This signature, combined with gene expression profiling and TCR fate mapping, identified a population of tissue-like Treg cells in human peripheral blood that expressed BATF, chemokine receptor CCR8 and HLA-DR. Human BATF+CCR8+ Treg cells from normal skin and adipose tissue shared features with nonlymphoid T follicular helper-like (Tfh-like) cells, and induction of a Tfh-like differentiation program in naive human Treg cells partially recapitulated tissue Treg regenerative characteristics, including wound healing potential. Human BATF+CCR8+ Treg cells from healthy tissue share features with tumor-resident Treg cells, highlighting the importance of understanding the context-specific functions of these cells. The corresponding manuscript has been published in the journal Immunity (Delacher et al., Single-cell chromatin accessibility landscape identifies tissue repair program in human regulatory T cells. Immunity, 2021; 13;54(4):702-720).
We were also able to find an inhibitor of tissue-Treg cell differentiation. We described the function of the transcription factor Rbpj in Treg cells. Rbpj-deficient Treg cells adopt open chromatin landscapes and gene expression profiles similar to tissue Treg cells, with a prevailing signature of the transcription factor Gata-3. This study suggests that Treg cells require Rbpj to specifically restrain TH2 responses, including their own excessive TH2-like differentiation potential, thereby, inhibiting the tissue Treg pathway. Published in the journal Nature Communications (Delacher et al., Rbpj expression in regulatory T cells is critical for restraining TH2 responses. Nature Communications, 2019; 8;10(1):1621).
Regulatory T cells express the forkhead box transcription factor Foxp3 as a lineage-defining protein. Negative regulators of Foxp3 expression are not well understood. In our study, we generated double-stranded DNA probes complementary to the Foxp3 promoter sequence and performed a pull-down with nuclear protein in vitro, followed by elution of bound proteins and quantitative mass spectrometry. Of the Foxp3-promoter-binding transcription factors identified with this approach, one was T cell factor 1 (TCF1). Our data implicate a role of TCF1 in suppressing Foxp3 expression in activated T cells (Delacher et al., Quantitative Proteomics Identifies TCF1 as a Negative Regulator of Foxp3 Expression in Conventional T Cells. iScience, 2020; 22;23(5):101127).
For examples, we performed epigenetic analysis with a tagmentation-based whole-genome bisulfite sequencing technology, which allowed us to analyze the DNA methylation status of very few Treg cells isolated from tissues. With this technology, we were able to study the epigenetic changes of DNA methylation with the highest possible resolution (single nucleotide and genome-wide). Similarities of the epigenetic landscape led to the identification of a common tissue Treg population, present in many organs and characterized by gain and loss of DNA methylation, including many TH2-specific sites. Thus, we could show that tissue Treg cells integrate different waves of epigenetic reprogramming which define their tissue-restricted specializations. The corresponding manuscript has been published in Nature Immunology (Delacher M et al. Genome-wide DNA-methylation landscape defines specialization of regulatory T cells in tissues. Nature Immunology, 2017;18(10):1160-1172).
We wanted to understand the development of these tissue-resident Treg cells. By using our novel Nfil3-Cre-eGFP reporter mouse line, single-cell RNA-sequencing and bulk ATAC-sequencing, we identified two precursor stages of these IL-33 receptor ST2-expressing nonlymphoid tissue Treg population in spleen and lymph nodes. Molecular profiling of these precursors revealed their sequence of differentiation. Global chromatin profiling of nonlymphoid tissue Treg cells and the two precursor stages revealed a stepwise acquirement of open chromatin accessibility and reprogramming towards the nonlymphoid-tissue Treg cell phenotype. Mechanistically, we identified and validated the basic leucine zipper transcription factor, ATF-like (Batf) as the driver of the molecular tissue program in the precursors. We were able to publish these findings in the journal Immunity (Delacher et al., Precursors for Nonlymphoid-Tissue Treg Cells Reside in Secondary Lymphoid Organs and Are Programmed by the Transcription Factor BATF. Immunity, 2020; 18;52(2):295-312).
We were also able to shed light on human tissue-resident Tregs cells. We identified features that characterize human Treg cells with tissue-repair function. Single-cell chromatin accessibility profiles of murine and human tissue Treg cells defined a conserved, microbiota-independent tissue-repair Treg signature with a prevailing footprint of the transcription factor BATF. This signature, combined with gene expression profiling and TCR fate mapping, identified a population of tissue-like Treg cells in human peripheral blood that expressed BATF, chemokine receptor CCR8 and HLA-DR. Human BATF+CCR8+ Treg cells from normal skin and adipose tissue shared features with nonlymphoid T follicular helper-like (Tfh-like) cells, and induction of a Tfh-like differentiation program in naive human Treg cells partially recapitulated tissue Treg regenerative characteristics, including wound healing potential. Human BATF+CCR8+ Treg cells from healthy tissue share features with tumor-resident Treg cells, highlighting the importance of understanding the context-specific functions of these cells. The corresponding manuscript has been published in the journal Immunity (Delacher et al., Single-cell chromatin accessibility landscape identifies tissue repair program in human regulatory T cells. Immunity, 2021; 13;54(4):702-720).
We were also able to find an inhibitor of tissue-Treg cell differentiation. We described the function of the transcription factor Rbpj in Treg cells. Rbpj-deficient Treg cells adopt open chromatin landscapes and gene expression profiles similar to tissue Treg cells, with a prevailing signature of the transcription factor Gata-3. This study suggests that Treg cells require Rbpj to specifically restrain TH2 responses, including their own excessive TH2-like differentiation potential, thereby, inhibiting the tissue Treg pathway. Published in the journal Nature Communications (Delacher et al., Rbpj expression in regulatory T cells is critical for restraining TH2 responses. Nature Communications, 2019; 8;10(1):1621).
Regulatory T cells express the forkhead box transcription factor Foxp3 as a lineage-defining protein. Negative regulators of Foxp3 expression are not well understood. In our study, we generated double-stranded DNA probes complementary to the Foxp3 promoter sequence and performed a pull-down with nuclear protein in vitro, followed by elution of bound proteins and quantitative mass spectrometry. Of the Foxp3-promoter-binding transcription factors identified with this approach, one was T cell factor 1 (TCF1). Our data implicate a role of TCF1 in suppressing Foxp3 expression in activated T cells (Delacher et al., Quantitative Proteomics Identifies TCF1 as a Negative Regulator of Foxp3 Expression in Conventional T Cells. iScience, 2020; 22;23(5):101127).
We implemented novel technologies to analyze Treg and conventional T cells from peripheral organs and lymphoid tissues. Those included whole genome-wide DNA-methylation landscape analysis, single-cell chromatin accessibility profiles (sc-ATAC), single-cell RNA sequencing analysis (sc-RNA-seq), including single-cell TCR-sequencing and others. The assay for transposase-accessible chromatin (ATAC) is a technique to study chromatin accessibility. With these whole-genome sequencing technologies, we were able to study the epigenetic landscape and to recognize the permanent underlying molecular programs. Furthermore, we identified additional tissue-resident Treg cell targets for future therapeutic interventions.