RORγt-expressing dendritic cells are functionally versatile and evolutionarily conserved antigen-presenting cells

Abstract

Conventional dendritic cells (cDCs) are potent antigen-presenting cells (APCs) that integrate signals from their environment allowing them to direct situation-adapted immunity. Thereby they harbor great potential for being targeted in vaccination, autoimmunity, and cancer. Here, we use fate mapping, functional analyses, and comparative cross-species transcriptomics to show that RORγt+ DCs are a conserved, functionally versatile, and transcriptionally distinct type of DCs. RORγt+ DCs entail various populations described in different contexts including Janus cells/RORγt-expressing extrathymic Aire-expressing cells (eTACs), subtypes of Thetis cells, RORγt+-DC (R-DC) like cells, cDC2C and ACY3+ DCs. We show that in response to inflammatory triggers, RORγt+ DCs can migrate to lymph nodes and in the spleen can activate naïve CD4+ T cells. These findings expand the functional repertoire of RORγt+ DCs beyond the known role of eTACs and Thetis cells in inducing T cell tolerance to self-antigens and intestinal microbes in mice. We further show that RORγt+ DCs with proinflammatory features accumulate in autoimmune neuroinflammation in mice and men. Thus, our work establishes RORγt+ DCs as immune sentinel cells that exhibit a broad functional spectrum ranging from inducing peripheral T cell tolerance to T cell activation depending on signals they integrate from their environment.

Reprogramming Stars #20:Attenuating Cancer Cell Memory and Discovering Cancer Biomarkers with Cellular Reprogramming—An Interview with Dr. Jungsun Kim

REPROGRAMMING STAR: Dr. Jungsun Kim is a member of the Early Detection Advanced Research Center and Cancer Biology Program at Knight Cancer Institute, Oregan, USA. Her research group is interested in the reversibility of regulatory mechanisms in cancer development and progression, with the long-term goal of applying the generated knowledge in early cancer therapy. Defined combinations of transcription factors (TFs) can reprogram somatic cells into induced pluripotent stem cells (iPSCs) through epigenetic rewiring of the landscape of “starting” somatic cells, established by cell fate decisions during normal development. Dr. Kim’s lab explores TF-mediated reprogramming to rewire and reverse aberrant epigenetic alterations in cancer cells. They have demonstrated proof of principle of a pancreatic cancer reprogramming model, providing a unique platform to study different stages of human pancreatic cancer. Essentially, Dr. Kim’s research group addresses the following questions: To what extent and reproducibility aberrant cancer transcriptional networks can be destabilized through TF-mediated reprogramming? How do cells that fail reprogramming regain an aggressive tumor phenotype? What chromatin regulators maintain a cancer cell state? Addressing these questions can provide understanding of how cancer cells establish and maintain cancer identity.

Transcription Factor–Mediated Reprogramming to Antigen-Presenting Cells

Abstract

Antigen-presenting cells (APCs) are a heterogenous group of immune cells composed by dendritic cells (DCs) and macrophages (Mϕ), which are critical for orchestrating immunity against cancer or infections. Several strategies have been explored to generate APC subsets, including enrichment from peripheral blood and differentiation from pluripotent or multipotent cells. During development, the generation of APC subsets is instructed by transcription factors (TFs). Direct cell reprogramming, also known as transdifferentiation, offers an approach to harness combinations of TFs to generate APCs from unrelated somatic cells, including cancer cells. In this review, we summarize the transcriptional specification of DC subsets, highlight transcriptional networks for their generation, and discuss future applications of DC reprogramming in cancer immunotherapy.

Reprogramming Stars #17:Breaking Down the Barriers of Direct Reprogramming Using a Model Organism—An Interview with Dr. Baris Tursun

REPROGRAMMING STAR: Prof. Dr. Baris Tursun leads the Molecular Cell Biology unit at the University of Hamburg’s Institute of Cell and Systems Biology of Animals. His group uses the nematode Caenorhabditis elegans (C. elegans) to study direct reprogramming of cells in vivo. Their aim is to identify and better understand cellular processes that limit the conversion of cell identities. The genetic factors identified by the Tursun group contribute to safeguard cell fates and thereby act as reprogramming barriers. Using C. elegans facilitates unbiased genetic screening for such factors. By interrogating all 20,000 genes of the worm, the Tursun group identifies unanticipated molecular mechanisms that counteract cell conversion, ensuring the maintenance of cell function and health. Tursun’s research focuses on epigenetic and physiological mechanisms in cellular reprogramming and aging

Reprogramming Stars #19:Upgrading Cell Fate Conversions with Engineered Reprogramming Factors—An Interview with Dr. Ralf Jauch

REPROGRAMMING STAR: Professor Ralf Jauch leads the protein and cell engineering laboratory at the School of Biomedical Sciences in the Faculty of Medicine of the University of Hong Kong (HKUMed). His group aims to boost cellular reprogramming with the help of unconventional factors from exotic species and reengineered biomolecules. To achieve this, they look at the natural evolution of pioneer transcription factors, perform direct molecular evolution in mammalian cells, and use structural information for protein design. With this toolkit, they aim to make new types of stem cells that can help to model and revert age linked disease, enhance the developmental potential of stem cells, and direct stem cell differentiation. The team aims to translate their technologies within the Centre for Translational Stem Cell Biology (CTSCB).

Reprogramming Stars #18:Engineering Cell Fates and Preventing Diseaseby Repressing Unwanted Plasticity—An Interview with Dr. Moritz Mall

REPROGRAMMING STAR: Dr. Moritz Mall is a research group leader at the Hector Institute for Translational Brain Research (HITBR) and the German Cancer Research Center (DKFZ). The Mall lab employs mouse models, pluripotent stem cells, organoids, and cell fate engineering to reconstruct and investigate human development and disease. Their mission is to understand the mechanisms that determine and maintain cell fate, with the goal of treating diseases associated with increased cellular plasticity. Their immediate research goal is to understand the role of cell plasticity in brain disorders and cancer, with a focus on autism spectrum disorders and the emerging field of phenotypic plasticity in cancer.

HMGA1 stimulates cancer stem-like features and sensitivity to monensin in gastric cancer

Abstract

Gastric cancer represents a serious health problem worldwide, with insufficient molecular biomarkers and therapeutic options. Consequently, several efforts have been directed towards finding specific disease markers in order to develop new therapies capable of defeating gastric cancer. Attention has been pointed to cancer stem cells (CSCs) as they are primarily responsible for tumor initiation and recurrence, making them essential therapeutic targets. Using the SORE6-GFP reporter system, based on the expression of SOX2 and/or OCT4 to drive GFP expression, we isolated gastric cancer stem-like cells (SORE6+ cells) enriched in several molecules, including SOX2, C-MYC, KLF4, HIF-1α, NOTCH1 and HMGA1. Here, we explored the previously undisclosed link of HMGA1 with gastric CSCs. Our results indicated that HMGA1 can activate a transcriptional program that includes SOX2, C-MYC, and KLF4 and endows cells with CSC features. We further showed that chemical induction of gastric CSCs using ciclopirox (CPX) can be mediated by HMGA1. Finally, we showed that HMGA1 GFP+ cells were sensitive to monensin confirming the selective activity of this drug over CSCs. Thus, HMGA1 is a key player in the cellular reprogramming of gastric non-CSCs to cancer stem-like cells.

In Vivo Dendritic Cell Reprogramming for Cancer Immunotherapy

Abstract

Immunotherapy can lead to long-term survival for some cancer patients, yet generalized success has been hampered by insufficient antigen presentation and exclusion of immunogenic cells from the tumor microenvironment. Here, we developed an approach to reprogram tumor cells in vivo by adenoviral delivery of the transcription factors PU.1, IRF8, and BATF3, which enabled them to present antigens as type 1 conventional dendritic cells. Reprogrammed tumor cells remodeled their tumor microenvironment, recruited, and expanded polyclonal cytotoxic T cells, induced tumor regressions, and established long-term systemic immunity in multiple mouse melanoma models. In human tumor spheroids and xenografts, reprogramming to immunogenic dendritic-like cells progressed independently of immunosuppression, which usually limits immunotherapy. Our study paves the way for human clinical trials of in vivo immune cell reprogramming for cancer immunotherapy.

Image credit: Joana Carvalho


Graphical Abstract

In vivo reprogramming of tumor cells to dendritic cells. (1) Adenoviral delivery of PIB to tumors generates cDC1-like cells, marked by XCR1, CLEC9A, MHC-I/II and CD40 expression. (2) Reprogrammed tumor cells promote the formation of tertiary lymphoid structures, infiltration of CD8+ T cells and polyclonal cytotoxic CD4+ T cells, (3) leading to tumor regression, immunological memory and the control of abscopal tumors and lung metastasis.

Perspective Published on Science

Reprogramming Tumor Cells to Fight Cancer by Haibo Zhou and Li Wu

Web-Based Application for Data

https://cellreprolab.shinyapps.io/inVivo_DC1_atlas

Reprogramming Is Key to Unlock Antitumor Immunity – About the Cover

This cover depicts a cancer immunotherapy modality by reprogramming tumor cells within the tumor microenvironment into dendritic cells that glow as a light bulb in the dark symbolizing the dawn of a new class of cancer treatments. The key, an adenoviral vector, delivers the reprogramming factors PU.1, IRF8 and BATF3 to the tumor cells in vivo and unlocks an immunogenic program in the tumor cells to present antigens as type 1 dendritic cells. Reprogrammed cells ultimately illuminate the way for the immune system in cold and “dark” tumors to generate robust, long-lasting, and systemic antitumor responses.
Image credit: Joana Carvalho

Summary and Interview by ACIR

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Uncovering Novel Regulators of Dendritic Cell Reprogramming Through Crispr/Cas9 Screening

Cellular identity generally remains stable and is regulated by specific transcription factors. However, perturbation of gene expression can alter cellular identity. Direct cell reprogramming induces a desired cell fate without transitioning through a pluripotent stage, making it a powerful approach for therapeutic development and mechanistic studies of cell fate conversion. Using PU.1, IRF8 and BATF3, mouse and human fibroblasts can be converted into type one conventional dendritic cell-like cells. However, the regulatory mechanisms of chromatin regulators or RNA modifiers in iDC1 reprogramming are still poorly understood. CRISPR/Cas9 screenings are a powerful approach to identify regulatory networks in biological processes. Therefore, we used CRISPR/Cas9 screening to target epigenetic regulators and characterize barriers and facilitators of DC reprogramming. Furthermore, 18 barriers and 13 facilitators were top candidates for individual validation. Therefore, multiplexed guide RNA vectors were cloned to induce gene knockouts, confirmed by sequencing and validated for most selected barriers. The expression levels of CD45 and HLA-DR were determined by flow cytometry on day 9. Out of the 13 candidate facilitators, 5 slightly increased the non-reprogrammed population. Moreover, 7 of the 18 candidate barriers increased the CD45 and HLA-DR positive reprogrammed population. Furthermore, those hits were stained for both reprogramming markers at various time points to study the kinetics and efficiency of DC reprogramming. CD40, CD226, XCR1, crucial for cDC1 cells, were stained for the assessment of further characteristics. For the selected facilitators, no change of the non-reprogrammed population could be characterized over time. Moreover, the identified candidate barriers increased the reprogrammed population over time, with SND1 being the most significant and HIRA showing interesting mechanisms. For the facilitators and barriers, CD40 and CD226 remain unaltered, and XCR1 could not be determined throughout the time course as XCR1 gene expression had not been identified and the alternative staining approach did not work. In the past, the histone chaperone HIRA has already been identified as a barrier in neuronal reprogramming. The oncogene SND1 could act as a barrier by having an important role in the influence of the NF-κB pathway and miR-221, which has been previously identified as targets by the characterized barrier c-Jun. Overall, our investigation into the effects of selected barriers and facilitators has revealed some interesting insights into the regulatory mechanisms in DC reprogramming. To gain deeper understanding of the mechanisms mediated through HIRA and SND1 silencing, further experiments, like ATAC-Seq analysis or RNA- sequencing, need to be conducted.

Reprogramming Stars #16:Reprogramming, from Cells to Embryos—An Interviewwith Dr. José Silva

REPROGRAMMING STAR: Dr. José Silva is a full time Principal Investigator at the Guangzhou National Laboratory in Guangzhou, China. He leads a team that is developing new mouse and human embryo models to generate relevant cell types and tissues that are molecularly and functionally identical to their in vivo counterparts. Their approach combines reprogramming with developmental biology principles and an array of advanced tools to generate high-quality, relevant cell types for potential medical applications

CD4+ T Cells Drive Anti-Tumor Immunity Elicited by In Vivo Dendritic Cell Reprogramming

Immunotherapies have revolutionized the field of cancer treatment and rely on the activation of anti-tumor T cell responses. Downregulation of antigen presentation in tumor cells, the immunosuppressive microenvironment and dysfunction of dendritic cells limit the efficacy of current treatments. Conventional type-1 dendritic cells (cDC1s) play a crucial role in orchestrating anti-tumor T cell responses and their presence correlates with better survival in cancer patients. However, there is no efficient method to generate cDC1s for immunotherapy. In vivo cell fate reprogramming enables the conversion of cells within tissues at the disease location into another cell identity with therapeutic potential. Previously, our lab has shown that direct cell reprogramming can be used to convert fibroblasts or tumor cells into cDC1-like cells by overexpression of the transcription factors PU.1, IRF8 and BATF3, called PIB, in vitro. In this master thesis, we hypothesize that PIB mediate in vivo reprogramming of cancer cells into tumor antigen-presenting cDC1-like cells (tumor-APCs). PIB overexpression generates tumor-APCs that persist in the tumor for 9 days, acquire a CCR7- resident profile and elicit robust and durable anti-tumor immunity. CD4+ T cells are recruited and interact with reprogrammed cells in the tumor and are required for complete tumor regression. In vivo reprogrammed cells employed complementary immune mechanisms to induce tumor growth control, demonstrated by the use of single knock-outs for MHC-II, MHC-I, CD40 and XCR1. Lastly, we identified a model that escaped immunological memory and showed resistance to anti-tumor immunity elicited by in vivo reprogramming which was not mediated by downregulation of cDC1-related immune mechanisms. Ultimately, we show that in vivo reprogrammed cells remain in the tumor and drive a CD4+ T cell response by employing multifaceted immune mechanisms. This study paves the way for the generation of a gene therapy based on in vivo cDC1 reprogramming and identification of resistance mechanisms.

Induction Of Alveolar Epithelial Type II-Like Cells By Direct Reprogramming

Alveolar type II cells (AT2s) are essential for the lung’s regenerative capacity following injury, and AT2 disorders are associated with multiple lung diseases. These cells are complex to maintain in culture, and the lack of a model to study AT2s remains a major roadblock in understanding their self-renewal and identity. Direct reprogramming by transcription factor (TF) overexpression allows for the fast
generation of specific cell types without transitioning through a pluripotent state. There are, however, no reports of pulmonary cell induction by cell reprogramming in the respiratory system, keeping this lineage largely unexplored despite its therapeutic potential. Therefore, we hypothesized that overexpression of AT2-specific TFs would allow for the direct conversion of fibroblasts to AT2-like cells.
In this study, we validated a direct reprogramming approach to screen TFs on mouse embryonic fibroblasts harboring the AT2-specific surfactant protein C (SPC) reporter. We identified 28 TFs based on gene enrichment, loss-of-function studies, and bioinformatic analysis as candidates to induce AT2 cell fate. To mimic the distal lung environment, we isolated AT2s from murine lungs and established alveolospheres capable of proliferating and maintaining of surface marker expression. In both 3D spheroids and 2D in vitro cultures, combined overexpression of the 28 TFs was sufficient to activate the SPC reporter. We then refined the combination to 5 AT2-restricted TFs, FOXA1, FOXA2, CEBPA, NKX2.1, and ETV5, and confirmed their capacity to activate the SPC reporter. Removal of FOXA1,
FOXA2 or CEBPA from the combination reduced reporter activation suggesting their requirement to induce AT2s. We suggest a network where FOXA1 and FOXA2 open the chromatin for CEBPA which recruits NKX2.1, promoting AT2 identity. Generating AT2-like cells by direct reprogramming not only gives insight into the transcriptional regulators of their identity, but also the constant supply of AT2s is an attractive potential strategy for clinical therapies.

Harnessing Diverse Dendritic Cell Reprogramming for Immunotherapy

Cancer immunotherapy re-establishes the function of the immune system of recognizing tumour-associated neoantigens. Although many patients benefited from these forms of therapy, others have still proven resistant.
Dendritic cells (DCs), subdivided in type 1 (cDC1), 2 (cDC2) or plasmacytoid (pDC), are immune cells with important role in antigen-presentation, proven to be implicated in cancer immunosurveillance. Through cellular reprogramming, induced DCs have been previously generated from fibroblasts with the overexpression of different combinations of transcription factors.
Here, we evaluated the reprogramming of different melanoma and breast cancers (B16, Yumm1.7, EO771. PymT and BRAF) into multiple DC subsets in vitro and in vivo and investigated their antitumor effect. We found that Yumm1.7 exclusively responded to induced cDC1 while EO771 responded to induced cDC2 and pDC. By combining DC reprogramming we observed tumor-induced cDC2s in Yumm1.7 negatively affected the anti-tumorigenicity of tumor-induced cDC1s, while combined tumor-induced cDC2s and pDCs inhibited EO771 tumor growth.

Genomic deletion of Bcl6 differentially affects conventional dendritic cell subsets and compromises Tfh/Tfr/Th17 cell responses

Abstract

Conventional dendritic cells (cDC) play key roles in immune induction, but what drives their heterogeneity and functional specialization is still ill-defined. Here we show that cDC-specific deletion of the transcriptional repressor Bcl6 in mice alters the phenotype and transcriptome of cDC1 and cDC2, while their lineage identity is preserved. Bcl6-deficient cDC1 are diminished in the periphery but maintain their ability to cross-present antigen to CD8+ T cells, confirming general maintenance of this subset. Surprisingly, the absence of Bcl6 in cDC causes a complete loss of Notch2-dependent cDC2 in the spleen and intestinal lamina propria. DC-targeted Bcl6-deficient mice induced fewer T follicular helper cells despite a profound impact on T follicular regulatory cells in response to immunization and mounted diminished Th17 immunity to Citrobacter rodentium in the colon. Our findings establish Bcl6 as an essential transcription factor for subsets of cDC and add to our understanding of the transcriptional landscape underlying cDC heterogeneity.

Reprogramming Stars #15:Colliding Cellular Reprogramming Paths—An Interview with Dr. Stefan Stricker

ReprogrammingStar: Prof. Dr. Stefan H. Stricker leads the Epigenetic Engineering group at the Institute of Stem Cell Research, Helmholtz Munich, and concurrently holds the position of Professor of Reprogramming and Regeneration at the Biomedical Center of LMU Munich. Their primary objective is to uncover and manipulate mediators and barriers of cell identity conversion, leveraging these insights to enhance both established and innovative reprogramming strategies. Their multifaceted approach integrates computational analyses, epigenome editing techniques, transcriptional engineering, and advanced single-cell methodologies to propel their research toward achieving transformative outcomes in regenerative medicine.

Reprogramming Stars #14:Fast-Forwarding Cellular Reprogramming—An Interview with Dr. Mark Kotter

Reprogramming Star: Dr. Mark Kotter is a stem cell biologist and neurosurgeon at the University of Cambridge and a serial entrepreneur. As a neurosurgeon, he treats patients with spinal cord injury. He is CEO and founder of bit.bio, the cell coding company generating human cells for research, drug discovery, and cell therapy, cofounder of Meatable and clock.bio and cofounder and trustee of myelopathy.org, the first charity dedicated to a common yet often overseen condition causing a ‘‘slow motion spinal cord injury.’’ Mark set up bit.bio to democratize access to human cells for research, drug discovery, and cell therapies.

Orchestrating an Immune Response to Cancer with Cellular Reprogramming

In recent years, immunotherapy has transformed cancer treatment by harnessing the patient’s own immune system. However, cancer cells develop mechanisms to evade immune surveillance, compromising the body’s natural defences against cancer. The lack of immune control and immunotherapy failure can be attributed to both tumour intrinsic and extrinsic factors favouring the survival of tumour cells. These encompass primarily the downregulation of antigen presentation and major immunohistocompatibility complex (MHC) molecules on the cell surface, increasing tumour heterogeneity and exclusion or functional impairment of immune effectors within the tumour microenvironment (TME).

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Reprogramming Cancer Cells to Antigen-presenting Cells

Abstract

Cancer cells evade the immune system by downregulating antigen presentation. Although immune checkpoint inhibitors (ICI) and adoptive T-cell therapies revolutionized cancer treatment, their efficacy relies on the intrinsic immunogenicity of tumor cells and antigen presentation by dendritic cells. Here, we describe a protocol to directly reprogram murine and human cancer cells into tumor-antigen-presenting cells (tumor-APCs), using the type 1 conventional dendritic cell (cDC1) transcription factors PU.1, IRF8, and BATF3 delivered by a lentiviral vector. Tumor-APCs acquire a cDC1 cell-like phenotype, transcriptional and epigenetic programs, and function within nine days (Zimmermannova et al., 2023). Tumor-APCs express the hematopoietic marker CD45 and acquire the antigen presentation complexes MHC class I and II as well as co-stimulatory molecules required for antigen presentation to T cells, but do not express high levels of negative immune checkpoint regulators. Enriched tumor-APCs present antigens to Naïve CD8+ and CD4+ T cells, are targeted by activated cytotoxic T lymphocytes, and elicit anti-tumor responses in vivo. The tumor-APC reprogramming protocol described here provides a simple and robust method to revert tumor evasion mechanisms by increasing antigen presentation in cancer cells. This platform has the potential to prime antigen-specific T-cell expansion, which can be leveraged for developing new cancer vaccines, neoantigen discovery, and expansion of tumor-infiltrating lymphocytes. Key features • This protocol describes the generation of antigen-presenting cells from cancer cells by direct reprogramming using lineage-instructive transcription factors of conventional dendritic cells type I. • Verification of reprogramming efficiency by flow cytometry and functional assessment of tumor-APCs by antigen presentation assays.

High-Throughput Drug Screening Revealed That Ciclopirox Olamine Can Engender Gastric Cancer Stem-like Cells

Abstract

Cancer stem cells (CSCs) are relevant therapeutic targets for cancer treatment. Still, the molecular circuits behind CSC characteristics are not fully understood. The low number of CSCs can sometimes be an obstacle to carrying out assays that explore their properties. Thus, increasing CSC numbers via small molecule-mediated cellular reprogramming appears to be a valid alternative tool. Using the SORE6-GFP reporter system embedded in gastric non-CSCs (SORE6−), we performed a high-throughput image-based drug screen with 1200 small molecules to identify compounds capable of converting SORE6− to SORE6+ (CSCs). Here, we report that the antifungal agent ciclopirox olamine (CPX), a potential candidate for drug repurposing in cancer treatment, is able to reprogram gastric non-CSCs into cancer stem-like cells via activation of SOX2 expression and increased expression of C-MYC, HIF-1α, KLF4, and HMGA1. This reprogramming depends on the CPX concentration and treatment duration. CPX can also induce cellular senescence and the metabolic shift from oxidative phosphorylation (OXPHOS) to glycolysis. We also disclose that the mechanism underlying the cellular reprogramming is similar to that of cobalt chloride (CoCl2), a hypoxia-mimetic agent.

Reprogramming Stars #13:Establishing Connections with Cellular Reprogramming–An Interview with Dr. Daniella Rylander Ottosson

Reprogramming Star: Dr. Rylander Ottosson is an Associate Professor at the Lund University and Wallenberg Academy Fellow. The Ottosson laboratory studies cell fate specification toward GABAergic interneurons from human neuronal progenitor cells and somatic cells using neuronal differentiation and reprogramming approaches. The long-term goal is in brain repair and rewiring of altered brain plasticity in disorders such as Schizophrenia and Alzheimer’s disease. Her laboratory includes cell-based models, mouse in vivo model, and human organotypic cultures with a clear focus on the functional and integrational aspects of in vivo reprogrammed cells. In parallel the laboratory applies in vitro reprogramming of patient skin fibroblast to develop interneuron disease models for pathologies related to these neurons.

GATA2 Mitotic Bookmarking Is Required for Definitive Haematopoiesis

Abstract

In mitosis, most transcription factors detach from chromatin, but some are retained and bookmark genomic sites. Mitotic bookmarking has been implicated in lineage inheritance, pluripotency and reprogramming. However, the biological significance of this mechanism in vivo remains unclear.
Here, we address mitotic retention of the hemogenic factors GATA2, GFI1B and FOS during hematopoietic specification. We show that GATA2 remains bound to chromatin throughout mitosis, in contrast to GFI1B and FOS, via C-terminal zinc finger-mediated DNA binding. GATA2 bookmarks a subset of its interphase targets that are co-enriched for RUNX1 and other regulators of definitive haematopoiesis. Remarkably, homozygous mice harbouring the cyclin B1 mitosis degradation domain upstream Gata2 partially phenocopy knockout mice. Degradation of GATA2 at mitotic exit abolishes definitive haematopoiesis at aorta-gonad-mesonephros, placenta and foetal liver, but does not impair yolk sac haematopoiesis.
Our findings implicate GATA2-mediated mitotic bookmarking as critical for definitive haematopoiesis and highlights a dependency on bookmarkers for lineage commitment.


An Animated Video on the Mitotic Bookmarking by GATA2

Genome Browser for ChIP-Seq

https://genome-euro.ucsc.edu/s/ilyak/GATA2-ChipSeq-hg38

Editor’s Summary

Most transcription factors detach from chromatin during mitosis, but some are retained and bookmark genomic sites. Here, the authors show that GATA2-mediated mitotic bookmarking is critical for definitive hematopoiesis.

Restoring Tumor Immunogenicity with Dendritic Cell Reprogramming

Abstract

Decreased antigen presentation contributes to the ability of cancer cells to evade the immune system. We used the minimal gene regulatory network of type 1 conventional dendritic cells (cDC1) to reprogram cancer cells into professional antigen-presenting cells (tumor-APCs). Enforced expression of the transcription factors PU.1, IRF8, and BATF3 (PIB) was sufficient to induce cDC1 phenotype in 36 cell lines derived from human and mouse hematological and solid tumors. Within 9 days of reprogramming, tumor-APCs acquired transcriptional and epigenetic programs associated with cDC1 cells. Reprogramming restored the expression of antigen presentation complexes and costimulatory molecules on the surface of tumor cells, allowing the presentation of endogenous tumor antigens on MHC-I, and facilitating targeted killing by CD8 T cells.
Functionally, tumor-APCs engulfed and processed proteins and dead cells, secreted inflammatory cytokines, and cross-presented antigens to naïve CD8 T cells. Human primary tumor cells could also be reprogrammed to increase their capability to present antigens and to activate patient-specific tumor-infiltrating lymphocytes. In addition to acquiring improved antigen presentation, tumor-APCs had impaired tumorigenicity in vitro and in vivo. Injection of in vitro-generated melanoma-derived tumor-APCs into subcutaneous melanoma tumors delayed tumor growth and increased survival in mice. Antitumor immunity elicited by tumor-APCs was synergistic with immune checkpoint inhibitors.
Our approach serves as a platform for the development of immunotherapies that endow cancer cells with the capability to process and present endogenous tumor antigens.


Web-Based Application for Processed RNA-seq and ATAC-seq Data

https://cellreprolab.shinyapps.io/tumorAPC_atlas/

An Animated Video on the TrojanDC Technology

About the Illustration

Using the minimal regulatory network of type 1 conventional dendritic cells (connections and cells inside the horse), Zimmermannova & Ferreira et al. reprogrammed human and mouse cancer cells into dendritic cells. This strategy bypassed tumor evasion mechanisms and endowed tumor cells with professional antigen presentation leading to activation of specific CD8+ T cells (soldiers), and anti-tumor immunity in vivo. This study paves the way for a new class of cancer immunotherapies based on cell fate reprogramming. The illustration depicts a novel Trojan horse approach to cancer immunotherapy by reprogramming cancer cells to become traitors to their kind.

CREDIT: Sandeep Menon.

Summary and Interview by ACIR

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Compositions For Reprogramming Cells Into Hemogenic And/Or Hematopoietic Stem Cell-Like Cells, Methods And Uses Thereof

The present disclosure relates to a construct or a vector for reprogramming stem cells, differentiated cells, or mixtures thereof into hemogenic and/or hematopoietic stem cell-like cells, wherein the construct or the vector encodes a peptide comprising a combination of two isolated or synthetic transcription factors, preferably at least three isolated or synthetic transcription factors. The disclosure also relates to a composition comprising said construct or vector, to a method for reprogramming or inducing a stem cell or a differentiated cell into hemogenic and/or hematopoietic stem cell-like cells comprising a step of transducing a cell with at least one of said vectors; to a induced hemogenic and/or hematopoietic stem cell-like cell obtained by said method; to a composition comprising said induced hemogenic and/or hematopoietic stem cell-like cell; and to a kit comprising at least one of the following components: the induced hemogenic and/or hematopoietic stem cell-like cell; the composition as described in any of the previous claims; the vector or the construct as disclosed; or mixtures thereof.

Identifying Epigenetic and Mutational Barriers Limiting Cancer Cell Reprogramming Into Dendritic Cells

Cell reprogramming underscored a remarkable plasticity of somatic cells, opening avenues for regenerative medicine as well as cell replacement therapies. Through enforced expression of the three transcription factors (TFs) PU.1, IRF8 and BATF3, our group demonstrated that fibroblasts can be reprogrammed into conventional type 1 dendritic cells (cDC1) fate. Furthermore, we showed that the same combination of TFs can be applied to reprogram cancer cells, generating tumor-antigen presenting cells (tumor-APCs) with highly immunogenic features and functional capacity to restore anti-cancer immune response. However, the efficiency of reprogramming varied significantly among different cancer cell lines. We hypothesized that the efficiency of cancer cell reprogramming into cDC1 might be limited by preexisting epigenetic and genetic barriers. Here, we investigated the impact of chromatin-based barriers on cDC1 reprogramming by removing repressive marks using histone deacetylase inhibitor valproic acid (VPA) and inhibitor of DNA demethylases, 5-azacytidine (AZA) during reprogramming in 26 various human cancer cell lines. Overall, VPA had a superior effect on increasing reprogramming efficiency compared to treatment with AZA, suggesting that histone deacetylation represents a more frequent barrier in reprogramming. Although AZA showed a generally lower increase in reprogramming efficiency, it revealed that DNA methylation is also a limiting barrier in reprogramming, particularly in certain cancer cell lines. Mechanistically, we showed that treatment with VPA accelerates the PIB-mediated reprogramming process, leading to faster acquisition of a mature phenotype in reprogrammed tumor-APCs. Nevertheless, the majority of tested cell lines only showed partial improvement in reprogramming efficiency in response to treatment with epigenetic modifiers, suggesting preexisting genetic barriers (mutations) hindering reprogramming. By integrating mutational profiles of cell lines identified by whole exome sequencing with the epigenetic analysis, we predicted mutations that negatively affect reprogramming efficiencies and cannot be reverted by epigenetic modulation. We identified mutations in 6 genes (BPIFB6, BPIFB3, ITGA4, OR5H15, KRTAP10-6, and SPATA22) that correlated with impaired reprogramming. Although the identified mutations do not affect cDC1 signature genes, they may play a role in cellular plasticity and maintenance of identity in cells. Validating these mutations and assessing their impact on reprogramming efficiency and functionality of reprogrammed cells is crucial for further understanding of their role. To this end, understanding the factors limiting cell reprogramming is of key importance to improve the efficacy of cDC1 reprogramming and advancing its translation into cancer immunotherapy, as well as improving the efficiency of other reprogramming-based strategies.

Reprogramming Stars #12:At the Heart of In Vivo Reprogramming—An Interview with Dr. Li Qian

Reprogramming Star: Dr. Li Qian is a Professor at the University of North Carolina. Dr. Qian’s laboratory is interested in developing innovative approaches to regenerate or repair an injured heart. Their goal is to understand the molecular basis of cardiomyocyte specification and maturation and apply this knowledge to improve efficiency and clinical applicability of cellular reprogramming in heart disease. To achieve these goals, they utilize in vivo modeling of cardiac disease in the mouse, including myocardial infarction, cardiac hypertrophy, chronic heart failure, and congenital heart disease. In addition, they take advantage of traditional mouse genetics, cell and molecular biology, biochemistry, reprogramming technologies, and the latest single-cell genomics approaches in combination with mathematical modeling to investigate the fundamental events underlying the progression of various cardiovascular diseases as well as to discover the basic mechanisms of cell reprogramming.

Reprogramming Stars #11: Teaming Up to Uncoverthe Epitranscriptomics of Reprogramming—An Interview with Dr. Miguel Fidalgo and Dr. Diana Guallar

Reprogramming Stars: Dr. Diana Guallar is an Assistant Professor at the Department of Biochemistry and Molecular Biology in the University of Santiago de Compostela (USC) and group leader at the Center for Research in Molecular Medicine and Chronic Diseases (CIMUS). The Guallar laboratory studies how epigenetic and epitranscriptomic cross talk is involved in the regulation of cell identity and plasticity in cellular rejuvenation and pluripotency. Her ultimate aim is to dissect critical pathways accompanying the loss of molecular fidelity observed during aging and in aging-related disorders that could be very valuable for clinical application. Dr. Miguel Fidalgo is an Associate Professor at the Department of Physiology (USC) and group leader at the CIMUS. The Fidalgo laboratory is focused on understanding how regulatory information encoded by the genome is integrated with the metabolic, epigenetic, and epitranscriptomic machineries to control cellular plasticity in the context of cell reprogramming and pluripotency, and how perturbations of these mechanisms could be associated with development and disease.

Reprogramming Stars #10:Modeling Cancer with Cellular Reprogramming—An Interview with Dr. Dung-Fang Lee

Reprogramming Star: Dr. Dung-Fang Lee is an assistant professor and CPRIT scholar in cancer research at the University of Texas Health Science Center at Houston. The Lee research laboratory is dedicated to understanding the pathological mechanisms behind cancer by applying patient-specific induced pluripotent stem cells (iPSCs) and/or engineered embryonic stem cells (ESCs) as models. The central directions of the Lee laboratory include (1) systems-level analyses and characterization of mutant p53 in Li–Fraumeni syndrome-associated tumor initiation, (2) systematic analyses of genome alterations during Li–Fraumeni syndrome-associated osteosarcoma development, and (3) modeling of familial cancer syndromes with a predisposition to osteosarcoma through patient-specific iPSC methods.

Reprogramming Stars #9:Spacing Out Cellular Reprogramming—An Interview with Dr. Valentina Fossati

Reprogramming Star: Dr. Valentina Fossati is a senior investigator at the New York Stem Cell Foundation Research Institute where she focuses on building human induced pluripotent stem cell (iPSC)-based models for studying progressive multiple sclerosis (MS) and other neurodegenerative diseases to investigate the role of glia in neuroinflammation and neurodegeneration. She has established protocols to generate human iPSCderived oligodendrocytes, astrocytes, microglia, and neuronal cell types and is developing organoids and coculture systems to study the crosstalk between neurons and glial cells.Her ultimate goal is to identify and target key glia-driven pathogenic mechanisms leading to neurodegeneration in progressive MS, Alzheimer’s disease, and other disorders of the central nervous system (CNS).

Reprogramming Stars #8:A Synthetic Biology Approach to Cellular Reprogramming—An Interview with Dr. Katie Galloway

Reprogramming star: Dr. Katie Galloway is the W.M. Keck Career Development Professor in biomedical engineering and chemical engineering at MIT. As a chemical engineer working in stem cell and molecular systems biology, her research focuses on elucidating the fundamental principles of constructing and integrating synthetic circuitry to drive cellular reprogramming. Her laboratory leverages synthetic biology to understand cell fate transitions with the goal of building genetic controllers that support disease modeling and cell-based therapies.

Direct Reprogramming of Fibroblasts to NK Cells

Abstract

Natural killer (NK) cells are innate lymphocytes with remarkable cytotoxic abilities that control cancer and viral infections independent of antigen specificity. Indeed, NK cells are the first induced pluripotent stem cell (iPSCs)-derived hematopoietic cells to be tested in clinical trials against hematological tumors. However, limited persistence in vivo and complexity of differentiation protocols, pose significant obstacles to widespread NK-based therapeutics. We hypothesize that direct cell reprogramming mediated by cell type-specific transcription factors (TFs) can be employed to generate NK lymphocytes from somatic cells. To define combinations of TF that induce NK cell identity we tested a list of 19 candidate TFs for their ability to activate a NK-specific reporter in mouse embryonic fibroblasts (MEFs). We identified Ets1, Nfil3, T-bet, Eomes (TENE) that activated a NCR1-driven reporter and induce NK progenitor and mature NK global gene expression programs as assessed by single-cell mRNA seq. To evaluate species conservation, we transduced human fibroblasts with 4 TFs and assessed activation of NK-specific markers by flow cytometry. We show that CD34 and CD56 expression is induced by enforced expression of TENE, suggesting that this combination of TFs to induce NK cell identity are conserved in human. CD34 expression increased with time in culture and was enhanced by the addition of cytokines important for lymphocyte development, suggesting cell expansion. Finally, we employed a barcoded TF approach coupled with single-cell mRNA-seq to inform the specification of progenitor versus mature programs. We screened 48 TFs and first confirmed the involvement of Ets1 in immature NK development, T-bet and Eomes in mature NK, and Nfil3 in both stages. We also suggest Runx family TFs and Ikzf1 as novel
regulators of NK development.

Taken together, our results contribute to the understanding of the transcriptional network driving NK lineage commitment and pave the way for the generation of patient- specific NK cells by direct cell reprogramming approaches.