Advances in Experimental Medicine and Biology, 2016
Interleukin (IL)-10 is an essential anti-inflammatory cytokine that plays important roles as a ne... more Interleukin (IL)-10 is an essential anti-inflammatory cytokine that plays important roles as a negative regulator of immune responses to microbial antigens. Loss of IL-10 results in the spontaneous development of inflammatory bowel disease as a consequence of an excessive immune response to the gut microbiota. IL-10 also functions to prevent excessive inflammation during the course of infection. IL-10 can be produced in response to pro-inflammatory signals by virtually all immune cells, including T cells, B cells, macrophages, and dendritic cells. Given its function in maintaining the delicate balance between effective immunity and tissue protection, it is evident that IL-10 expression is highly dynamic and needs to be tightly regulated. The transcriptional regulation of IL-10 production in myeloid cells and T cells is the topic of this review. Drivers of IL-10 expression as well as their downstream signaling pathways and transcription factors will be discussed. We will examine in more detail how various signals in CD4+ T cells converge on common transcriptional circuits, which fine-tune IL-10 expression in a context-dependent manner.
CRISPR (clustered, regularly interspaced, short palindromic repeats)/Cas9 (CRISPR-associated prot... more CRISPR (clustered, regularly interspaced, short palindromic repeats)/Cas9 (CRISPR-associated protein 9) has become the tool of choice for generating gene knockouts across a variety of species. The ability for efficient gene editing in primary T cells not only represents a valuable research tool to study gene function but also holds great promise for T cell–based immunotherapies, such as next-generation chimeric antigen receptor (CAR) T cells. Previous attempts to apply CRIPSR/Cas9 for gene editing in primary T cells have resulted in highly variable knockout efficiency and required T cell receptor (TCR) stimulation, thus largely precluding the study of genes involved in T cell activation or differentiation. Here, we describe an optimized approach for Cas9/RNP transfection of primary mouse and human T cells without TCR stimulation that results in near complete loss of target gene expression at the population level, mitigating the need for selection. We believe that this method will gr...
CRISPR (clustered, regularly interspaced, short palindromic repeats)/Cas9 (CRISPR-associated prot... more CRISPR (clustered, regularly interspaced, short palindromic repeats)/Cas9 (CRISPR-associated protein 9) has become the tool of choice for generating gene knockouts across a variety of species. The ability for efficient gene editing in primary T cells not only represents a valuable research tool to study gene function but also holds great promise for T cell–based immunotherapies, such as next-generation chimeric antigen receptor (CAR) T cells. Previous attempts to apply CRIPSR/Cas9 for gene editing in primary T cells have resulted in highly variable knockout efficiency and required T cell receptor (TCR) stimulation, thus largely precluding the study of genes involved in T cell activation or differentiation. Here, we describe an optimized approach for Cas9/RNP transfection of primary mouse and human T cells without TCR stimulation that results in near complete loss of target gene expression at the population level, mitigating the need for selection. We believe that this method will gr...
Loss of function of the nuclear deubiquitinating enzyme BRCA1-associated protein-1 (BAP1) is asso... more Loss of function of the nuclear deubiquitinating enzyme BRCA1-associated protein-1 (BAP1) is associated with a wide spectrum of cancers. We report that tamoxifen-induced BAP1 deletion in adult mice resulted in severe thymic atrophy. BAP1 was critical for T cell development at several stages. In the thymus, BAP1 was required for progression through the pre-T cell receptor checkpoint. Peripheral T cells lacking BAP1 demonstrated a defect in homeostatic and antigen-driven expansion. Deletion of BAP1 resulted in suppression of E2F target genes and defects in cell cycle progression, which was dependent on the catalytic activity of BAP1, but did not require its interaction with host cell factor-1 (HCF-1). Loss of BAP1 led to increased monoubiquitination of histone H2A at Lys (H2AK119ub) throughout the T cell lineage, in particular in immature thymocytes, but did not alter trimethylation of histone H3 at Lys (H3K27me3). Deletion of BAP1 also abrogated B cell development in the bone marrow....
The E3 ubiquitin ligase Itch limits interleukin 17A (IL-17A)-mediated intestinal inflammation by ... more The E3 ubiquitin ligase Itch limits interleukin 17A (IL-17A)-mediated intestinal inflammation by targeting the transcription factor ROR-γt for proteasomal degradation.
The interleukin (IL)-20 subfamily of cytokines is comprised of IL-19, IL-20, IL-22, IL-24, and IL... more The interleukin (IL)-20 subfamily of cytokines is comprised of IL-19, IL-20, IL-22, IL-24, and IL-26. These cytokines are part of the larger IL-10 cytokine family, but share a common biology based on structural homology and common receptor usage. Their heterodimeric class II receptors are preferentially expressed on epithelial tissues, which uniquely positions IL-20 subfamily cytokines as a means of communication between leukocytes and epithelial cells. IL-20 subfamily cytokines generally function to enhance innate defense mechanisms, wound healing, and tissue repair at epithelial surfaces. This biology has been extensively studied in the skin where IL-20 subfamily cytokines have critical roles during the wound healing response but are also key drivers of psoriasis. These functions are mediated through their ability to stimulate epithelial cell proliferation and differentiation, to induce antimicrobial responses in epithelial cells, and to promote inflammation through the recruitment and activation of neutrophils and other leukocytes. IL-22, the best-studied member of the group, has important host-protective roles during bacterial infections of the intestine and the lung, consistent with the high expression of the IL-22 receptor on mucosal epithelial cells.
The basic helix-loop-helix transcriptional repressor twist1, as an antagonist of nuclear factor κ... more The basic helix-loop-helix transcriptional repressor twist1, as an antagonist of nuclear factor κB (NF-κB)–dependent cytokine expression, is involved in the regulation of inflammation-induced immunopathology. We show that twist1 is expressed by activated T helper (Th) 1 effector memory (EM) cells. Induction of twist1 in Th cells depended on NF-κB, nuclear factor of activated T cells (NFAT), and interleukin (IL)-12 signaling via signal transducer and activator of transcription (STAT) 4. Expression of twist1 was transient after T cell receptor engagement, and increased upon repeated stimulation of Th1 cells. Imprinting for enhanced twist1 expression was characteristic of repeatedly restimulated EM Th cells, and thus of the pathogenic memory Th cells characteristic of chronic inflammation. Th lymphocytes from the inflamed joint or gut tissue of patients with rheumatic diseases, Crohn's disease or ulcerative colitis expressed high levels of twist1. Expression of twist1 in Th1 lympho...
Genome engineering of T lymphocytes, the main effectors of antitumor adaptive immune responses, h... more Genome engineering of T lymphocytes, the main effectors of antitumor adaptive immune responses, has the potential to uncover unique insights into their functions and enable the development of next-generation adoptive T cell therapies. Viral gene delivery into T cells, which is currently used to generate CAR T cells, has limitations in regard to targeting precision, cargo flexibility, and reagent production. Nonviral methods for effective CRISPR/Cas9-mediated gene knock-out in primary human T cells have been developed, but complementary techniques for nonviral gene knock-in can be cumbersome and inefficient. Here, we report a convenient and scalable nonviral method that allows precise gene edits and transgene integration in primary human T cells, using plasmid donor DNA template and Cas9-RNP. This method is highly efficient for single and multiplex gene manipulation, without compromising T cell function, and is thus valuable for use in basic and translational research.
Advances in Experimental Medicine and Biology, 2016
Interleukin (IL)-10 is an essential anti-inflammatory cytokine that plays important roles as a ne... more Interleukin (IL)-10 is an essential anti-inflammatory cytokine that plays important roles as a negative regulator of immune responses to microbial antigens. Loss of IL-10 results in the spontaneous development of inflammatory bowel disease as a consequence of an excessive immune response to the gut microbiota. IL-10 also functions to prevent excessive inflammation during the course of infection. IL-10 can be produced in response to pro-inflammatory signals by virtually all immune cells, including T cells, B cells, macrophages, and dendritic cells. Given its function in maintaining the delicate balance between effective immunity and tissue protection, it is evident that IL-10 expression is highly dynamic and needs to be tightly regulated. The transcriptional regulation of IL-10 production in myeloid cells and T cells is the topic of this review. Drivers of IL-10 expression as well as their downstream signaling pathways and transcription factors will be discussed. We will examine in more detail how various signals in CD4+ T cells converge on common transcriptional circuits, which fine-tune IL-10 expression in a context-dependent manner.
CRISPR (clustered, regularly interspaced, short palindromic repeats)/Cas9 (CRISPR-associated prot... more CRISPR (clustered, regularly interspaced, short palindromic repeats)/Cas9 (CRISPR-associated protein 9) has become the tool of choice for generating gene knockouts across a variety of species. The ability for efficient gene editing in primary T cells not only represents a valuable research tool to study gene function but also holds great promise for T cell–based immunotherapies, such as next-generation chimeric antigen receptor (CAR) T cells. Previous attempts to apply CRIPSR/Cas9 for gene editing in primary T cells have resulted in highly variable knockout efficiency and required T cell receptor (TCR) stimulation, thus largely precluding the study of genes involved in T cell activation or differentiation. Here, we describe an optimized approach for Cas9/RNP transfection of primary mouse and human T cells without TCR stimulation that results in near complete loss of target gene expression at the population level, mitigating the need for selection. We believe that this method will gr...
CRISPR (clustered, regularly interspaced, short palindromic repeats)/Cas9 (CRISPR-associated prot... more CRISPR (clustered, regularly interspaced, short palindromic repeats)/Cas9 (CRISPR-associated protein 9) has become the tool of choice for generating gene knockouts across a variety of species. The ability for efficient gene editing in primary T cells not only represents a valuable research tool to study gene function but also holds great promise for T cell–based immunotherapies, such as next-generation chimeric antigen receptor (CAR) T cells. Previous attempts to apply CRIPSR/Cas9 for gene editing in primary T cells have resulted in highly variable knockout efficiency and required T cell receptor (TCR) stimulation, thus largely precluding the study of genes involved in T cell activation or differentiation. Here, we describe an optimized approach for Cas9/RNP transfection of primary mouse and human T cells without TCR stimulation that results in near complete loss of target gene expression at the population level, mitigating the need for selection. We believe that this method will gr...
Loss of function of the nuclear deubiquitinating enzyme BRCA1-associated protein-1 (BAP1) is asso... more Loss of function of the nuclear deubiquitinating enzyme BRCA1-associated protein-1 (BAP1) is associated with a wide spectrum of cancers. We report that tamoxifen-induced BAP1 deletion in adult mice resulted in severe thymic atrophy. BAP1 was critical for T cell development at several stages. In the thymus, BAP1 was required for progression through the pre-T cell receptor checkpoint. Peripheral T cells lacking BAP1 demonstrated a defect in homeostatic and antigen-driven expansion. Deletion of BAP1 resulted in suppression of E2F target genes and defects in cell cycle progression, which was dependent on the catalytic activity of BAP1, but did not require its interaction with host cell factor-1 (HCF-1). Loss of BAP1 led to increased monoubiquitination of histone H2A at Lys (H2AK119ub) throughout the T cell lineage, in particular in immature thymocytes, but did not alter trimethylation of histone H3 at Lys (H3K27me3). Deletion of BAP1 also abrogated B cell development in the bone marrow....
The E3 ubiquitin ligase Itch limits interleukin 17A (IL-17A)-mediated intestinal inflammation by ... more The E3 ubiquitin ligase Itch limits interleukin 17A (IL-17A)-mediated intestinal inflammation by targeting the transcription factor ROR-γt for proteasomal degradation.
The interleukin (IL)-20 subfamily of cytokines is comprised of IL-19, IL-20, IL-22, IL-24, and IL... more The interleukin (IL)-20 subfamily of cytokines is comprised of IL-19, IL-20, IL-22, IL-24, and IL-26. These cytokines are part of the larger IL-10 cytokine family, but share a common biology based on structural homology and common receptor usage. Their heterodimeric class II receptors are preferentially expressed on epithelial tissues, which uniquely positions IL-20 subfamily cytokines as a means of communication between leukocytes and epithelial cells. IL-20 subfamily cytokines generally function to enhance innate defense mechanisms, wound healing, and tissue repair at epithelial surfaces. This biology has been extensively studied in the skin where IL-20 subfamily cytokines have critical roles during the wound healing response but are also key drivers of psoriasis. These functions are mediated through their ability to stimulate epithelial cell proliferation and differentiation, to induce antimicrobial responses in epithelial cells, and to promote inflammation through the recruitment and activation of neutrophils and other leukocytes. IL-22, the best-studied member of the group, has important host-protective roles during bacterial infections of the intestine and the lung, consistent with the high expression of the IL-22 receptor on mucosal epithelial cells.
The basic helix-loop-helix transcriptional repressor twist1, as an antagonist of nuclear factor κ... more The basic helix-loop-helix transcriptional repressor twist1, as an antagonist of nuclear factor κB (NF-κB)–dependent cytokine expression, is involved in the regulation of inflammation-induced immunopathology. We show that twist1 is expressed by activated T helper (Th) 1 effector memory (EM) cells. Induction of twist1 in Th cells depended on NF-κB, nuclear factor of activated T cells (NFAT), and interleukin (IL)-12 signaling via signal transducer and activator of transcription (STAT) 4. Expression of twist1 was transient after T cell receptor engagement, and increased upon repeated stimulation of Th1 cells. Imprinting for enhanced twist1 expression was characteristic of repeatedly restimulated EM Th cells, and thus of the pathogenic memory Th cells characteristic of chronic inflammation. Th lymphocytes from the inflamed joint or gut tissue of patients with rheumatic diseases, Crohn's disease or ulcerative colitis expressed high levels of twist1. Expression of twist1 in Th1 lympho...
Genome engineering of T lymphocytes, the main effectors of antitumor adaptive immune responses, h... more Genome engineering of T lymphocytes, the main effectors of antitumor adaptive immune responses, has the potential to uncover unique insights into their functions and enable the development of next-generation adoptive T cell therapies. Viral gene delivery into T cells, which is currently used to generate CAR T cells, has limitations in regard to targeting precision, cargo flexibility, and reagent production. Nonviral methods for effective CRISPR/Cas9-mediated gene knock-out in primary human T cells have been developed, but complementary techniques for nonviral gene knock-in can be cumbersome and inefficient. Here, we report a convenient and scalable nonviral method that allows precise gene edits and transgene integration in primary human T cells, using plasmid donor DNA template and Cas9-RNP. This method is highly efficient for single and multiplex gene manipulation, without compromising T cell function, and is thus valuable for use in basic and translational research.
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Papers by Sascha Rutz