IMMUNOLOGICAL TOLERANCE
Autoimmune disease | thymus | negative selection | Tregs | neonatal tolerance | cell differentiation | gene regulation | chromatin organization/human
Autoimmunity results from a breakdown in central or peripheral mechanisms of lymphocyte tolerance. Central T-cell tolerance is imposed within the thymus, key orchestrators being medullary thymic epithelial cells (mTECs), which play critical roles in both negative selection of T effector cells and positive selection of Tregs. A unique feature of mTECs is their expression of thousands of loci encoding antigens typically associated with peripheral parenchymal cells. Most of this “misplaced” gene expression is driven by the transcriptional regulator Aire (1M. S. Anderson, E. S. Venanzi et al. Projection of an immunological self shadow within the thymus by the aire protein. Science (2002) 298: 1395). Loss of Aire results in multi-organ autoimmune disease. We have studied both the molecular mechanisms of Aire’s control of gene expression (an unusual long-range scenario) (2M. S. Anderson, E. S. Venanzi et al. The cellular mechanism of Aire control of T cell tolerance. Immunity (2005) 23: 227) (3M. Guerau-de-Arellano, M. Martinic et al. Neonatal tolerance revisited: a perinatal window for Aire control of autoimmunity. J Exp. Med. (2009) 206: 1245) and how Aire drives tolerance at the cellular level (clonal deletion and diversion) (4S. Yang, N. Fujikado et al. Regulatory T cells generated early in life play a distinct role in maintaining self-tolerance. Science (2015) 348: 589) (5J. Tuncel, C. Benoist et al. T cell anergy in perinatal mice is promoted by T reg cells and prevented by IL-33. J Exp. Med (2019) 216: 1328.) (6J. Abramson, M. Giraud et al. Aire's partners in the molecular control of immunological tolerance. Cell (2010) 140: 123) (7K. Bansal, H. Yoshida et al. The transcriptional regulator Aire binds to and activates super-enhancers. Nat Immunol (2017) 18: 263) (8D. A. Michelson, K. Hase et al. Thymic epithelial cells co-opt lineage-defining transcription factors to eliminate autoreactive T cells. Cell (2022) 185: 2542) .
Recently, we discovered a second arm of thymocyte tolerization: thymic mimetic cells (8D. A. Michelson, K. Hase et al. Thymic epithelial cells co-opt lineage-defining transcription factors to eliminate autoreactive T cells. Cell (2022) 185: 2542) (9D. A. Michelson and D. Mathis. Thymic mimetic cells: tolerogenic masqueraders. Trends Immunol (2022) 43: 782) . These “misplaced” cells are a heterogeneous set of (mostly) Aire-dependent but Aire-nonexpressing mTECs that have co-opted the lineage-defining transcription factors, chromatin-accessibility landscapes, and gene-expression profiles of particular extra-thymic cell-types, while maintaining their mTEC identity. To date, 15 mimetic-cell subtypes have been identified, including tuft, muscle, ciliated, microfold and neuroendocrine mTECs. Accrual of mimetic cells depends critically on the LDTF(s) they express. Directing expression of a neoantigen to them tolerizes cognate thymocytes. Current interests include: human analogs, differentiation mechanisms and pathways, modulation to promote tolerance.
It’s been known for over half a century that just after birth is a critical window for self-tolerization, but the special mechanisms involved remain largely unknown. We’ve found: key participation by Aire (3M. Guerau-de-Arellano, M. Martinic et al. Neonatal tolerance revisited: a perinatal window for Aire control of autoimmunity. J Exp. Med. (2009) 206: 1245) , and Treg-promoted effector-cell anergy in nonlymphoid tissues (5J. Tuncel, C. Benoist et al. T cell anergy in perinatal mice is promoted by T reg cells and prevented by IL-33. J Exp. Med (2019) 216: 1328.) .
Autoimmune disease | thymus | negative selection | Tregs | neonatal tolerance | cell differentiation | gene regulation | chromatin organization/human
Autoimmunity results from a breakdown in central or peripheral mechanisms of lymphocyte tolerance. Central T-cell tolerance is imposed within the thymus, key orchestrators being medullary thymic epithelial cells (mTECs), which play critical roles in both negative selection of T effector cells and positive selection of Tregs. A unique feature of mTECs is their expression of thousands of loci encoding antigens typically associated with peripheral parenchymal cells. Most of this “misplaced” gene expression is driven by the transcriptional regulator Aire (1M. S. Anderson, E. S. Venanzi et al. Projection of an immunological self shadow within the thymus by the aire protein. Science (2002) 298: 1395). Loss of Aire results in multi-organ autoimmune disease. We have studied both the molecular mechanisms of Aire’s control of gene expression (an unusual long-range scenario) (2M. S. Anderson, E. S. Venanzi et al. The cellular mechanism of Aire control of T cell tolerance. Immunity (2005) 23: 227) (3M. Guerau-de-Arellano, M. Martinic et al. Neonatal tolerance revisited: a perinatal window for Aire control of autoimmunity. J Exp. Med. (2009) 206: 1245) and how Aire drives tolerance at the cellular level (clonal deletion and diversion) (4S. Yang, N. Fujikado et al. Regulatory T cells generated early in life play a distinct role in maintaining self-tolerance. Science (2015) 348: 589) (5J. Tuncel, C. Benoist et al. T cell anergy in perinatal mice is promoted by T reg cells and prevented by IL-33. J Exp. Med (2019) 216: 1328.) (6J. Abramson, M. Giraud et al. Aire's partners in the molecular control of immunological tolerance. Cell (2010) 140: 123) (7K. Bansal, H. Yoshida et al. The transcriptional regulator Aire binds to and activates super-enhancers. Nat Immunol (2017) 18: 263) (8D. A. Michelson, K. Hase et al. Thymic epithelial cells co-opt lineage-defining transcription factors to eliminate autoreactive T cells. Cell (2022) 185: 2542) .
Recently, we discovered a second arm of thymocyte tolerization: thymic mimetic cells (8D. A. Michelson, K. Hase et al. Thymic epithelial cells co-opt lineage-defining transcription factors to eliminate autoreactive T cells. Cell (2022) 185: 2542) (9D. A. Michelson and D. Mathis. Thymic mimetic cells: tolerogenic masqueraders. Trends Immunol (2022) 43: 782) . These “misplaced” cells are a heterogeneous set of (mostly) Aire-dependent but Aire-nonexpressing mTECs that have co-opted the lineage-defining transcription factors, chromatin-accessibility landscapes, and gene-expression profiles of particular extra-thymic cell-types, while maintaining their mTEC identity. To date, 15 mimetic-cell subtypes have been identified, including tuft, muscle, ciliated, microfold and neuroendocrine mTECs. Accrual of mimetic cells depends critically on the LDTF(s) they express. Directing expression of a neoantigen to them tolerizes cognate thymocytes. Current interests include: human analogs, differentiation mechanisms and pathways, modulation to promote tolerance.
It’s been known for over half a century that just after birth is a critical window for self-tolerization, but the special mechanisms involved remain largely unknown. We’ve found: key participation by Aire (3M. Guerau-de-Arellano, M. Martinic et al. Neonatal tolerance revisited: a perinatal window for Aire control of autoimmunity. J Exp. Med. (2009) 206: 1245) , and Treg-promoted effector-cell anergy in nonlymphoid tissues (5J. Tuncel, C. Benoist et al. T cell anergy in perinatal mice is promoted by T reg cells and prevented by IL-33. J Exp. Med (2019) 216: 1328.) .
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INTERPLAY BETWEEN THE GUT MICROBIOME AND THE IMMUNE SYSTEM
Host/microbe | Treg differentiation | Neuroimmune | Maternal transmission | Teff phenotypes
From early studies demonstrating an essential influence of gut microbes on arthritis and diabetes in the KRN and NOD models (1H. J. Wu, I. I. Ivanov et al. Gut-residing segmented filamentous bacteria drive autoimmune arthritis via T helper 17 cells. Immunity. (2010) 32: 815) (2M. Silverman, L. Kua et al. Protective major histocompatibility complex allele prevents type 1 diabetes by shaping the intestinal microbiota early in ontogeny. Proc Natl Acad Sci U S A (2017) 114: 9671) , we have tackled more generally the question of how gut microbes influence the immune system. We demonstrated a widespread migration of immunocytes from the gut, in particular Treg cells (3B. S. Hanna, G. Wang et al. The gut microbiota promotes distal tissue regeneration via RORg+ regulatory T cell emissaries. Immunity (2023) 56: 829) , to systemic immunologic organs and sites of inflammation or tumors (4S. Galván-Peña, Y. Zhu, B. S. Hanna, D. Mathis, C. Benoist, A dynamic atlas of immunocyte migration from the gut. bioRxiv. https://doi.org/10.1101/2022.11.16.516757 (2022).) . Large screens in gnotobiotic mice, in collaboration with the Kasper lab, revealed a wealth of influences, individual microbes affecting different immunocyte populations. Paradoxically, microbes sold as health-food probiotics induce pro-inflammatory T cells (5T. G. Tan, E. Sefik et al. Identifying species of symbiont bacteria from the human gut that, alone, can induce intestinal Th17 cells in mice. Proc Natl Acad Sci U S A (2016) 113: E8141-E8150) . We discovered the induction, by microbes of diverse phyla but with much species-level variability, of a distinct subset of Treg cells that paradoxically expresses the transcription factor RORγ (6E. Sefik, N. Geva-Zatorsky et al. Individual intestinal symbionts induce a distinct population of RORg+ regulatory T cells. Science (2015) 349: 993) . The capacity to induce RORg Tregs is tied, at least in part, to the microbes’ ability to trigger sensory neurons, and we described a triangular interaction between gut microbes, gut-innervating neurons, and Treg cells (7N. Yissachar, Y. Zhou et al. An intestinal organ culture system uncovers a role for the nervous system in microbe-immune crosstalk. Cell (2017) 168: 1135) (8Y. Yan, D. Ramanan et al. Interleukin-6 produced by enteric neurons regulates the number and phenotype of microbe-responsive regulatory T cells in the gut. Immunity. (2021) 54: 499) . We are further exploring, with the Chiu lab, the fascinating connections between neuronal subtypes and of immunocytes in the gut. Intriguingly, the homeostatic setpoint of RORγ+ Tregs, and of the coating of gut microbes by IgA, is transmitted by mothers to their offspring during a narrow postnatal window, in a manner that supersedes genetic control, and can be transmitted across generations (9D. Ramanan, E. Sefik et al. An immunologic mode of multigenerational transmission governs a gut Treg setpoint. Cell (2020) 181: 1276) . Single-cell transcriptomics and TCR sequencing have revealed unexpected relationships between RORγ+ and other Treg cells; we are using TCR engineering to investigate the locales and determinants of peripheral Treg differentiation and, conversely, try to understand how microbe-specific T cells affect the gut microbiota.
Host/microbe | Treg differentiation | Neuroimmune | Maternal transmission | Teff phenotypes
From early studies demonstrating an essential influence of gut microbes on arthritis and diabetes in the KRN and NOD models (1H. J. Wu, I. I. Ivanov et al. Gut-residing segmented filamentous bacteria drive autoimmune arthritis via T helper 17 cells. Immunity. (2010) 32: 815) (2M. Silverman, L. Kua et al. Protective major histocompatibility complex allele prevents type 1 diabetes by shaping the intestinal microbiota early in ontogeny. Proc Natl Acad Sci U S A (2017) 114: 9671) , we have tackled more generally the question of how gut microbes influence the immune system. We demonstrated a widespread migration of immunocytes from the gut, in particular Treg cells (3B. S. Hanna, G. Wang et al. The gut microbiota promotes distal tissue regeneration via RORg+ regulatory T cell emissaries. Immunity (2023) 56: 829) , to systemic immunologic organs and sites of inflammation or tumors (4S. Galván-Peña, Y. Zhu, B. S. Hanna, D. Mathis, C. Benoist, A dynamic atlas of immunocyte migration from the gut. bioRxiv. https://doi.org/10.1101/2022.11.16.516757 (2022).) . Large screens in gnotobiotic mice, in collaboration with the Kasper lab, revealed a wealth of influences, individual microbes affecting different immunocyte populations. Paradoxically, microbes sold as health-food probiotics induce pro-inflammatory T cells (5T. G. Tan, E. Sefik et al. Identifying species of symbiont bacteria from the human gut that, alone, can induce intestinal Th17 cells in mice. Proc Natl Acad Sci U S A (2016) 113: E8141-E8150) . We discovered the induction, by microbes of diverse phyla but with much species-level variability, of a distinct subset of Treg cells that paradoxically expresses the transcription factor RORγ (6E. Sefik, N. Geva-Zatorsky et al. Individual intestinal symbionts induce a distinct population of RORg+ regulatory T cells. Science (2015) 349: 993) . The capacity to induce RORg Tregs is tied, at least in part, to the microbes’ ability to trigger sensory neurons, and we described a triangular interaction between gut microbes, gut-innervating neurons, and Treg cells (7N. Yissachar, Y. Zhou et al. An intestinal organ culture system uncovers a role for the nervous system in microbe-immune crosstalk. Cell (2017) 168: 1135) (8Y. Yan, D. Ramanan et al. Interleukin-6 produced by enteric neurons regulates the number and phenotype of microbe-responsive regulatory T cells in the gut. Immunity. (2021) 54: 499) . We are further exploring, with the Chiu lab, the fascinating connections between neuronal subtypes and of immunocytes in the gut. Intriguingly, the homeostatic setpoint of RORγ+ Tregs, and of the coating of gut microbes by IgA, is transmitted by mothers to their offspring during a narrow postnatal window, in a manner that supersedes genetic control, and can be transmitted across generations (9D. Ramanan, E. Sefik et al. An immunologic mode of multigenerational transmission governs a gut Treg setpoint. Cell (2020) 181: 1276) . Single-cell transcriptomics and TCR sequencing have revealed unexpected relationships between RORγ+ and other Treg cells; we are using TCR engineering to investigate the locales and determinants of peripheral Treg differentiation and, conversely, try to understand how microbe-specific T cells affect the gut microbiota.
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TISSUE-Tregs: IMMUNOMETABOLISM
Diabetes | obesity | stromal cells | adipocyte differentiation | temperature regulation
Beyond its role in combatting microbial challenges, the immune system has a second major function: safeguarding tissue homeostasis (1A. R. Muñoz-Rojas and D. Mathis. Tissue regulatory T cells: regulatory chameleons. Nat Rev Immunol (2021) 21: 597) . Our group has pioneered the exploration of Treg control of non-immunological processes, especially organismal metabolism. Our society is currently facing an epidemic of obesity and, hand-in-hand, an increase in insulin resistance, type-2 diabetes and cardiovascular disease. Obesity-induced chronic, low-grade inflammation is a major factor promoting this “metabolic syndrome.” We discovered a unique Treg compartment in visceral-adipose tissue (VAT) of lean, but not obese, individuals that regulates T cells, macrophages and even adipocytes to control insulin sensitivity (2M. Feuerer, L. Herrero et al. Lean, but not obese, fat is enriched for a unique population of regulatory T cells that affect metabolic parameters. Nat Med (2009) 15: 930) , the first parenchymal-tissue-Treg population identified. Interestingly, the supreme regulator of adipocyte differentiation, PPARγ, controls the unique VAT-Treg phenotype (3D. Cipolletta, M. Feuerer et al. PPAR-g is a major driver of the accumulation and phenotype of adipose tissue Treg cells. Nature (2012) 486: 549) . By constructing a VAT-Treg TCR-transgenic mouse line, wherein VAT Tregs are over-represented, we could establish their dependencies (TCR specificity, Foxp3, IL-33) as well as follow their generation by a two-step, two-site scenario (4C. Li, J. R. Dispirito et al. TCR transgenic mice reveal stepwise, multi-site acquisition of the distinctive fat-Treg phenotype. Cell (2018) 174: 285) . This TCR-transgenic line also permitted us to determine that in obese mice VAT Tregs die off due to directly toxic effects of type-I interferon produced by a locally expanded plasmacytoid dendritic cell population (5C. Li, G. Wang et al. Interferon-a-producing plasmacytoid dendritic cells drive the loss of adipose tissue regulatory T cells during obesity. Cell Metab (2021) S1550(21)00277-1) . We have also studied several other interesting aspects of adipose-tissue Tregs: cross-talk with local stromal cells (6R. G. Spallanzani, D. Zemmour et al. Distinct immunocyte-promoting and adipocyte-generating stromal components coordinate adipose tissue immune and metabolic tenors. Sci Immunol (2019) 4: eaaw3658) , their transcription factor network (7J. R. Dispirito, D. Zemmour et al. Molecular diversification of regulatory T cells in nonlymphoid tissues. Sci Immunol (2018) 3: eaat5861) , mimotope TCR ligands, circadian influences (8T. Xiao, P. K. Langston et al. Tregs in visceral adipose tissue up-regulate circadian-clock expression to promote fitness and enforce a diurnal rhythm of lipolysis. Sci Immunol (2022) 7: eabl7641) , their control of adipocyte differentiation, implication in temperature regulation.
More broadly, we are exploring the tissue-Treg compartments of several other metabolically important organs: muscle, liver, the gut, the brain. Each compartment is unique – phenotypically and functionally – although common themes are emerging.
Diabetes | obesity | stromal cells | adipocyte differentiation | temperature regulation
Beyond its role in combatting microbial challenges, the immune system has a second major function: safeguarding tissue homeostasis (1A. R. Muñoz-Rojas and D. Mathis. Tissue regulatory T cells: regulatory chameleons. Nat Rev Immunol (2021) 21: 597) . Our group has pioneered the exploration of Treg control of non-immunological processes, especially organismal metabolism. Our society is currently facing an epidemic of obesity and, hand-in-hand, an increase in insulin resistance, type-2 diabetes and cardiovascular disease. Obesity-induced chronic, low-grade inflammation is a major factor promoting this “metabolic syndrome.” We discovered a unique Treg compartment in visceral-adipose tissue (VAT) of lean, but not obese, individuals that regulates T cells, macrophages and even adipocytes to control insulin sensitivity (2M. Feuerer, L. Herrero et al. Lean, but not obese, fat is enriched for a unique population of regulatory T cells that affect metabolic parameters. Nat Med (2009) 15: 930) , the first parenchymal-tissue-Treg population identified. Interestingly, the supreme regulator of adipocyte differentiation, PPARγ, controls the unique VAT-Treg phenotype (3D. Cipolletta, M. Feuerer et al. PPAR-g is a major driver of the accumulation and phenotype of adipose tissue Treg cells. Nature (2012) 486: 549) . By constructing a VAT-Treg TCR-transgenic mouse line, wherein VAT Tregs are over-represented, we could establish their dependencies (TCR specificity, Foxp3, IL-33) as well as follow their generation by a two-step, two-site scenario (4C. Li, J. R. Dispirito et al. TCR transgenic mice reveal stepwise, multi-site acquisition of the distinctive fat-Treg phenotype. Cell (2018) 174: 285) . This TCR-transgenic line also permitted us to determine that in obese mice VAT Tregs die off due to directly toxic effects of type-I interferon produced by a locally expanded plasmacytoid dendritic cell population (5C. Li, G. Wang et al. Interferon-a-producing plasmacytoid dendritic cells drive the loss of adipose tissue regulatory T cells during obesity. Cell Metab (2021) S1550(21)00277-1) . We have also studied several other interesting aspects of adipose-tissue Tregs: cross-talk with local stromal cells (6R. G. Spallanzani, D. Zemmour et al. Distinct immunocyte-promoting and adipocyte-generating stromal components coordinate adipose tissue immune and metabolic tenors. Sci Immunol (2019) 4: eaaw3658) , their transcription factor network (7J. R. Dispirito, D. Zemmour et al. Molecular diversification of regulatory T cells in nonlymphoid tissues. Sci Immunol (2018) 3: eaat5861) , mimotope TCR ligands, circadian influences (8T. Xiao, P. K. Langston et al. Tregs in visceral adipose tissue up-regulate circadian-clock expression to promote fitness and enforce a diurnal rhythm of lipolysis. Sci Immunol (2022) 7: eabl7641) , their control of adipocyte differentiation, implication in temperature regulation.
More broadly, we are exploring the tissue-Treg compartments of several other metabolically important organs: muscle, liver, the gut, the brain. Each compartment is unique – phenotypically and functionally – although common themes are emerging.
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TISSUE-Tregs: DRIVERS OF TISSUE REPAIR / REGENERATION
Muscular dystrophy | aging | stromal cells | stem cells | immunometabolism | neuroimmunology | exercise
Subsequent to our discovery of a unique population of Tregs in visceral adipose tissue (1A. R. Muñoz-Rojas and D. Mathis. Tissue regulatory T cells: regulatory chameleons. Nat Rev Immunol (2021) 21: 597) , we found an analogous tissue-Treg population in skeletal muscle (2D. Burzyn, W. Kuswanto et al. A special population of regulatory T cells potentiates muscle repair. Cell (2013) 155: 1282) . These cells proved critical for muscle repair/regeneration in models of acute and chronic injury (eg, a muscular dystrophy model). Old mice have fewer muscle Tregs due to a dearth of stroma-produced IL-33; injection of IL-33 corrects the Treg deficit and improves muscle repair (3W. Kuswanto, D. Burzyn et al. Poor repair of skeletal muscle in aging mice reflects a defect in local, interleukin-33-dependent accumulation of regulatory T cells. Immunity (2016) 44: 355) . scRNA-seq revealed muscle Tregs to be a consortium of distinct subtypes that wax and wane during the injury response, performing distinct functions (4B. S. Hanna, G. Wang et al. The gut microbiota promotes distal tissue regeneration via RORg+ regulatory T cell emissaries. Immunity (2023) 56: 829) . For example, we uncovered a gut:injured-muscle axis involving RORγ+ Treg control of IL-17A production, thereby reining in inflammation and promoting stem-cell differentiation. Highlights from our current efforts include: definition of a gut-derived antigen and a cross-reactive muscle antigen recognized by muscle Tregs; molecular dissection of Treg control of the benefits of exercise (5P. K. Langston, Y. Sun et al. Regulatory T cells shield muscle mitochondria from interferon-mediated damage to promote the beneficial effects of exercise. Science Imm (2023) In press) ; exploration of how muscle-mimetic thymic cells (6D. A. Michelson, K. Hase et al. Thymic epithelial cells co-opt lineage-defining transcription factors to eliminate autoreactive T cells. Cell (2022) 185: 2542) prevent muscle autoimmunity.
More broadly, we are actively pursuing an emerging general theme: Treg control of local stem/progenitor cells. Tregs regulate the proliferation, differentiation and/or fate of neighboring parenchymal precursor cells in several tissues. That this might be a primordial function is suggested by the fact that Tregs control precursor cell activities in several tissue-injury models in zebrafish. Multiple direct (2D. Burzyn, W. Kuswanto et al. A special population of regulatory T cells potentiates muscle repair. Cell (2013) 155: 1282) and indirect (4B. S. Hanna, G. Wang et al. The gut microbiota promotes distal tissue regeneration via RORg+ regulatory T cell emissaries. Immunity (2023) 56: 829) mechanisms come into play in the various mouse and fish models. We are currently exploring Treg:stem/progenitor cell cross-talk in muscle, visceral adipose tissue and the meninges of the brain.
Muscular dystrophy | aging | stromal cells | stem cells | immunometabolism | neuroimmunology | exercise
Subsequent to our discovery of a unique population of Tregs in visceral adipose tissue (1A. R. Muñoz-Rojas and D. Mathis. Tissue regulatory T cells: regulatory chameleons. Nat Rev Immunol (2021) 21: 597) , we found an analogous tissue-Treg population in skeletal muscle (2D. Burzyn, W. Kuswanto et al. A special population of regulatory T cells potentiates muscle repair. Cell (2013) 155: 1282) . These cells proved critical for muscle repair/regeneration in models of acute and chronic injury (eg, a muscular dystrophy model). Old mice have fewer muscle Tregs due to a dearth of stroma-produced IL-33; injection of IL-33 corrects the Treg deficit and improves muscle repair (3W. Kuswanto, D. Burzyn et al. Poor repair of skeletal muscle in aging mice reflects a defect in local, interleukin-33-dependent accumulation of regulatory T cells. Immunity (2016) 44: 355) . scRNA-seq revealed muscle Tregs to be a consortium of distinct subtypes that wax and wane during the injury response, performing distinct functions (4B. S. Hanna, G. Wang et al. The gut microbiota promotes distal tissue regeneration via RORg+ regulatory T cell emissaries. Immunity (2023) 56: 829) . For example, we uncovered a gut:injured-muscle axis involving RORγ+ Treg control of IL-17A production, thereby reining in inflammation and promoting stem-cell differentiation. Highlights from our current efforts include: definition of a gut-derived antigen and a cross-reactive muscle antigen recognized by muscle Tregs; molecular dissection of Treg control of the benefits of exercise (5P. K. Langston, Y. Sun et al. Regulatory T cells shield muscle mitochondria from interferon-mediated damage to promote the beneficial effects of exercise. Science Imm (2023) In press) ; exploration of how muscle-mimetic thymic cells (6D. A. Michelson, K. Hase et al. Thymic epithelial cells co-opt lineage-defining transcription factors to eliminate autoreactive T cells. Cell (2022) 185: 2542) prevent muscle autoimmunity.
More broadly, we are actively pursuing an emerging general theme: Treg control of local stem/progenitor cells. Tregs regulate the proliferation, differentiation and/or fate of neighboring parenchymal precursor cells in several tissues. That this might be a primordial function is suggested by the fact that Tregs control precursor cell activities in several tissue-injury models in zebrafish. Multiple direct (2D. Burzyn, W. Kuswanto et al. A special population of regulatory T cells potentiates muscle repair. Cell (2013) 155: 1282) and indirect (4B. S. Hanna, G. Wang et al. The gut microbiota promotes distal tissue regeneration via RORg+ regulatory T cell emissaries. Immunity (2023) 56: 829) mechanisms come into play in the various mouse and fish models. We are currently exploring Treg:stem/progenitor cell cross-talk in muscle, visceral adipose tissue and the meninges of the brain.
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Treg IDENTITY
Transcription factor networks | Chromatin structure | Enhancers | CRISPR engineering | IPEX
T regulatory cells are a fascinating cell population, given their diversity of functions, and an overarching goal is to work out, in a holistic manner, the molecular biology of their differentiation and identity(ies). We pioneered the use of single-cell transcriptomics to chart the landscape of Treg states, and define how signals from the TCR influence it (1D. Zemmour, R. Zilionis et al. Single-cell gene expression reveals a landscape of regulatory T cell phenotypes shaped by the TCR. Nat Immunol (2018) 19: 291) (2J. R. Dispirito, D. Zemmour et al. Molecular diversification of regulatory T cells in nonlymphoid tissues. Sci Immunol (2018) 3: eaat5861) (3C. Li, J. R. Dispirito et al. TCR transgenic mice reveal stepwise, multi-site acquisition of the distinctive fat-Treg phenotype. Cell (2018) 174: 285) . The transcription factor FoxP3 is quasi-uniquely expressed in Treg cells, and controls a good part, but not all, of their identity and function. We have long been interested in the relationships between FoxP3’s structural domains and its transcriptional activity, collaborating with structural biologists (4H. S. Bandukwala, Y. Wu et al. Structure of a domain-swapped FOXP3 dimer on DNA and its function in regulatory T cells. Immunity. (2011) 34: 479) (5F. Leng, W. Zhang et al. The transcription factor FoxP3 can fold into two dimerization states with divergent implications for regulatory T cell function and immune homeostasis. Immunity (2022) 55: 1354) (6J. Leon, K. Chowdhary et al. Mutations from patients with IPEX ported to mice reveal different patterns of FoxP3 and Treg dysfunction. Cell Rep (2023) 42: 113018) , or performing a full scanning mutagenesis of FoxP3 (7H. K. Kwon, H. M. Chen et al. Different molecular complexes that mediate transcriptional induction and repression by FoxP3. Nat Immunol (2017) 18: 1238) that revealed that it partakes in multiple molecular complexes, cooperating with alternative partners to either activate or suppress transcription. These complexes may be related to the DNA loops that FoxP3 anchors (8R. N. Ramirez, K. Chowdhary, J. Leon, D. Mathis, C. Benoist, FoxP3 associates with enhancer-promoter loops to regulate Treg-specific gene expression. Sci Immunol. 7, eabj9836 (2022)) . Single-cell chromatin profiling, combined with high-density genetic variation, is bringing a higher-level understanding the Treg GRN, by connecting fine variation in transcription factor binding motifs with chromatin patterns across the space of Treg variegation (9K. Chowdhary, J. Léon et al. An interwoven network of transcription factors, with divergent influences from FoxP3, underlies Treg diversity. bioRxiv (2023)) , which machine learning tools are helping to organize into consistent programs. Single-cell cytometric and genomic analyses of mice or IPEX patients with FOXP3 missense mutations, or their heterozygous relatives, uncovered Treg-like cells whose altered characteristics reflect both cell-intrinsic effects, linked to the mutation, and extrinsic effects that can be reverted by the presence of normal Tregs. We are determining how WT and FoxP3-deficient Tregs compete with each other in mixed environments. Porting into mice IPEX mutations in several domains of FoxP3 revealed specific effects on autoimmune diseases, with impacts revealed only by inflammatory or genetic perturbations, explaining the disease constellation in IPEX patients.
Treg cells can differentiate either in the thymus (tTreg) or in the periphery (pTreg). We are investigating how cells of different origins contribute to various Treg pools, and whether the same molecular signals are involved in tTreg and pTreg differentiation, and how TCR/antigen specificity affects pTreg propensity.
Transcription factor networks | Chromatin structure | Enhancers | CRISPR engineering | IPEX
T regulatory cells are a fascinating cell population, given their diversity of functions, and an overarching goal is to work out, in a holistic manner, the molecular biology of their differentiation and identity(ies). We pioneered the use of single-cell transcriptomics to chart the landscape of Treg states, and define how signals from the TCR influence it (1D. Zemmour, R. Zilionis et al. Single-cell gene expression reveals a landscape of regulatory T cell phenotypes shaped by the TCR. Nat Immunol (2018) 19: 291) (2J. R. Dispirito, D. Zemmour et al. Molecular diversification of regulatory T cells in nonlymphoid tissues. Sci Immunol (2018) 3: eaat5861) (3C. Li, J. R. Dispirito et al. TCR transgenic mice reveal stepwise, multi-site acquisition of the distinctive fat-Treg phenotype. Cell (2018) 174: 285) . The transcription factor FoxP3 is quasi-uniquely expressed in Treg cells, and controls a good part, but not all, of their identity and function. We have long been interested in the relationships between FoxP3’s structural domains and its transcriptional activity, collaborating with structural biologists (4H. S. Bandukwala, Y. Wu et al. Structure of a domain-swapped FOXP3 dimer on DNA and its function in regulatory T cells. Immunity. (2011) 34: 479) (5F. Leng, W. Zhang et al. The transcription factor FoxP3 can fold into two dimerization states with divergent implications for regulatory T cell function and immune homeostasis. Immunity (2022) 55: 1354) (6J. Leon, K. Chowdhary et al. Mutations from patients with IPEX ported to mice reveal different patterns of FoxP3 and Treg dysfunction. Cell Rep (2023) 42: 113018) , or performing a full scanning mutagenesis of FoxP3 (7H. K. Kwon, H. M. Chen et al. Different molecular complexes that mediate transcriptional induction and repression by FoxP3. Nat Immunol (2017) 18: 1238) that revealed that it partakes in multiple molecular complexes, cooperating with alternative partners to either activate or suppress transcription. These complexes may be related to the DNA loops that FoxP3 anchors (8R. N. Ramirez, K. Chowdhary, J. Leon, D. Mathis, C. Benoist, FoxP3 associates with enhancer-promoter loops to regulate Treg-specific gene expression. Sci Immunol. 7, eabj9836 (2022)) . Single-cell chromatin profiling, combined with high-density genetic variation, is bringing a higher-level understanding the Treg GRN, by connecting fine variation in transcription factor binding motifs with chromatin patterns across the space of Treg variegation (9K. Chowdhary, J. Léon et al. An interwoven network of transcription factors, with divergent influences from FoxP3, underlies Treg diversity. bioRxiv (2023)) , which machine learning tools are helping to organize into consistent programs. Single-cell cytometric and genomic analyses of mice or IPEX patients with FOXP3 missense mutations, or their heterozygous relatives, uncovered Treg-like cells whose altered characteristics reflect both cell-intrinsic effects, linked to the mutation, and extrinsic effects that can be reverted by the presence of normal Tregs. We are determining how WT and FoxP3-deficient Tregs compete with each other in mixed environments. Porting into mice IPEX mutations in several domains of FoxP3 revealed specific effects on autoimmune diseases, with impacts revealed only by inflammatory or genetic perturbations, explaining the disease constellation in IPEX patients.
Treg cells can differentiate either in the thymus (tTreg) or in the periphery (pTreg). We are investigating how cells of different origins contribute to various Treg pools, and whether the same molecular signals are involved in tTreg and pTreg differentiation, and how TCR/antigen specificity affects pTreg propensity.
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SYSTEM IMMUNOLOGY AND AI
Multiomics | Neural Networks | TCR repertoire | Deep learning
Many of the lab’s explorations have a Systems Immunology flavor, meaning that we try to approach immunological questions in a global manner, genome-wide or organism-wide. For instance, we aim to understand the genetic regulatory network that determines Treg cell identity as a whole, not just one transcription factor at a time. Single-cell genomics revealed that, unlike simple textbook pictures, Teff cells (and Tregs) belong to complex continua of phenotypes (1E. Kiner, E. Willie et al. Gut CD4+ T cell phenotypes are a continuum molded by microbes, not by TH archetypes. Nat Immunol (2021) 22: 216) (2K. Chowdhary, J. Léon et al. An interwoven network of transcription factors, with divergent influences from FoxP3, underlies Treg diversity. bioRxiv (2023)) (3J. Leon, K. Chowdhary et al. Mutations from patients with IPEX ported to mice reveal different patterns of FoxP3 and Treg dysfunction. Cell Rep (2023) 42: 113018) , stem cells or mature T cells and other immunocytes. For instance, a system-wide screen of the role of Nuclear Receptor family members is revealing unexpected roles of particular receptors.
The complexity of the components and interactions in biological systems dwarf the possibilities of the human brain. We are fascinated by the potential of Artificial Intelligence to help us make sense of questions like transcriptional regulation (1E. Kiner, E. Willie et al. Gut CD4+ T cell phenotypes are a continuum molded by microbes, not by TH archetypes. Nat Immunol (2021) 22: 216) (2K. Chowdhary, J. Léon et al. An interwoven network of transcription factors, with divergent influences from FoxP3, underlies Treg diversity. bioRxiv (2023)) (4A. Maslova, R. N. Ramirez et al. Deep learning of immune cell differentiation. Proc Natl Acad Sci U S A (2020) 117: 25655) , the rules of TCR specificity, or cell interactions in the immune system.
The lab is an active participant in ImmGen, a collaborative group of immunology and computational biology laboratories aiming to completely chart the patterns and regulation of gene expression in the immune system. ImmGen has become a widely used community resource. This participation allows us to tackle questions on a scale beyond what a single lab could do, and to design experiments and analyze complex datasets with an array of collaborative expertise (5H. Yoshida, C. A. Lareau et al. The cis-Regulatory Atlas of the Mouse Immune System. Cell (2019) 176: 897) (6A. Baysoy, K. Seddu et al. The interweaved signatures of common-gamma-chain cytokines across immunologic lineages. J Exp Med (2023) 220:) .
Multiomics | Neural Networks | TCR repertoire | Deep learning
Many of the lab’s explorations have a Systems Immunology flavor, meaning that we try to approach immunological questions in a global manner, genome-wide or organism-wide. For instance, we aim to understand the genetic regulatory network that determines Treg cell identity as a whole, not just one transcription factor at a time. Single-cell genomics revealed that, unlike simple textbook pictures, Teff cells (and Tregs) belong to complex continua of phenotypes (1E. Kiner, E. Willie et al. Gut CD4+ T cell phenotypes are a continuum molded by microbes, not by TH archetypes. Nat Immunol (2021) 22: 216) (2K. Chowdhary, J. Léon et al. An interwoven network of transcription factors, with divergent influences from FoxP3, underlies Treg diversity. bioRxiv (2023)) (3J. Leon, K. Chowdhary et al. Mutations from patients with IPEX ported to mice reveal different patterns of FoxP3 and Treg dysfunction. Cell Rep (2023) 42: 113018) , stem cells or mature T cells and other immunocytes. For instance, a system-wide screen of the role of Nuclear Receptor family members is revealing unexpected roles of particular receptors.
The complexity of the components and interactions in biological systems dwarf the possibilities of the human brain. We are fascinated by the potential of Artificial Intelligence to help us make sense of questions like transcriptional regulation (1E. Kiner, E. Willie et al. Gut CD4+ T cell phenotypes are a continuum molded by microbes, not by TH archetypes. Nat Immunol (2021) 22: 216) (2K. Chowdhary, J. Léon et al. An interwoven network of transcription factors, with divergent influences from FoxP3, underlies Treg diversity. bioRxiv (2023)) (4A. Maslova, R. N. Ramirez et al. Deep learning of immune cell differentiation. Proc Natl Acad Sci U S A (2020) 117: 25655) , the rules of TCR specificity, or cell interactions in the immune system.
The lab is an active participant in ImmGen, a collaborative group of immunology and computational biology laboratories aiming to completely chart the patterns and regulation of gene expression in the immune system. ImmGen has become a widely used community resource. This participation allows us to tackle questions on a scale beyond what a single lab could do, and to design experiments and analyze complex datasets with an array of collaborative expertise (5H. Yoshida, C. A. Lareau et al. The cis-Regulatory Atlas of the Mouse Immune System. Cell (2019) 176: 897) (6A. Baysoy, K. Seddu et al. The interweaved signatures of common-gamma-chain cytokines across immunologic lineages. J Exp Med (2023) 220:) .
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