Highlights

• Comprehensive protocol for producing arrayed lentiviral TF libraries
• Guidance for optimizing reprogramming and achieving uniform TF distribution
• Strategy for purifying cells and generating single-cell transcriptomes and barcodes
• Bioinformatics pipeline for detecting barcodes and assembling TF combinations

Summary

Direct reprogramming offers a powerful approach to generate therapeutic cell types, but progress is limited by an incomplete understanding of transcription factor (TF) cooperativity. Here, we present a protocol for performing combinatorial TF screening to resolve reprogramming factor networks that drive cell identity. We describe steps for arrayed lentiviral production, transduction, and reprogramming of human fibroblasts into distinct immune cells. We detail procedures for cell purification, library preparation, sequencing, and analysis to resolve TF combinations and dynamics.

Highlights

• REPROcode enables combinatorial single-cell screens of immune TFs
• Identifies TF stoichiometry, regulators of cDC1 cell state, and reprogramming fidelity
• Reveals dominant TFs that map hierarchical transcriptional networks for immune lineages
• Uncovers TF combinations for reprogramming to DC, monocyte, macrophage, and NK identities

Summary

Direct reprogramming of immune cells holds promise for immunotherapy but is constrained by limited knowledge of transcription factor (TF) networks. Here, we developed REPROcode, a combinatorial single-cell screening platform to identify TF combinations for immune cell reprogramming. We first validated REPROcode by inducing type-1 conventional dendritic cells (cDC1s) with multiplexed sets of 9, 22, and 42 factors. With cDC1-enriched TFs, REPROcode enabled identification of optimal TF stoichiometry, fidelity enhancers, and regulators of cDC1 states. We then constructed an arrayed lentiviral library of 408 barcoded immune TFs to explore broader reprogramming capacity. Screening 48 TFs enriched in dendritic cell subsets yielded myeloid and lymphoid phenotypes and enabled the construction of a TF hierarchy map to guide immune reprogramming. Finally, we validated REPROcode’s discovery power by inducing natural killer (NK)-like cells. This study deepens our understanding of immune transcriptional control and provides a versatile toolbox for engineering immune cells to advance immunotherapy.

Cover design credit: Lilith Lawrence.

Mapping the tulip field of immune cell identity – About the Cover

The artwork depicts a field of blooming tulips symbolizing the diversity of the immune system, with each flower representing a transcription factor and its stem rooted in unique combinations that define specific immune cell fates. Through combinatorial transcription factor screening, Kurochkin et al. map the landscape of immune identity, cultivating a framework for directing immune cell reprogramming. Illustration by Lilith Lawrence.

REPROGRAMMING STAR: Dr. Jerome Mertens is an Associate Professor at the Department of Neurosciences at the University of California San Diego (UCSD), USA. Dr. Mertens’s lab applies neuronal direct reprogramming and induced pluripotent stem cell (iPSC) generation to develop informative models of human biological aging and its intersection with age-related diseases, investigating the effects of epigenetic aging, rejuvenation, and stability of adult neuronal identity. With the goal of identifying key molecular players at the interface of human biological aging and disease to develop new treatments for age-related neurodegeneration, the lab relies on a patient-centric approach supported by cellular neuroscience and big data biology. By employing multi-omics strategies, functional genetics, and cell biology approaches to characterize human patient-based reprogramming models, Dr. Mertens’s work elucidates the interplay between non-genetic and age-related cellular changes and disease pathways.

REPROGRAMMING STAR: Dr. Fabrice Lavial is the team leader of the Cellular Reprogramming, Stem Cells, and Oncogenesis Laboratory at the Cancer Research Center of Lyon, France, and a research director at INSERM. Dr. Lavial’s lab joins the fields of developmental biology, cellular reprogramming, cancer biology, and bioinformatics to mechanistically understand embryonic development and the establishment of cellular identities. They explore the molecular mechanisms that control cell fate conversions in early development, reprogramming to pluripotency, transdifferentiation, and oncogenic transformation. Focusing on the regulation of cellular identity and plasticity, Dr. Lavial is interested in deciphering cellular and molecular networks that will lead to the development of nontumorigenic approaches for cell fate conversions in situ. Specifically, he and his group are trying to understand how we can harness the benefits of cellular reprogramming while avoiding cancer development.

Dr. Lavial’s lab is divided into two main themes: (1) control of cellular identity during reprogramming and oncogenic transformation and (2) pluripotent stem cell dynamics. The team’s first line of research aims to dissect the early molecular events of cellular reprogramming and transformation, using multi-omics approaches at the single-cell scale, 2D cellular models, 3D organoids, and murine genetic models. The second research subject focuses on understanding the molecular mechanisms controlling the self-renewal of pluripotent stem cells, as well as their ability to polarize and self-assemble. For that, the team uses models of embryonic or induced stem cells, cultured in 2D or induced to form 3D epiblast-like structures, as well as synthetic embryos.

Highlights

• Anchored screening identifies ETS-IRF pairs and a third factor specifying DC subsets
• PU.1, IRF4, and PRDM1 induce a pro-inflammatory cDC2B-like identity
• SPIB, IRF8, and IKZF2 drive an immature lymphoid pDC-like program
• Induced DC subsets trigger anti-tumor responses and durable immunological memory

Summary

Transcription factor cooperation is essential for specifying the heterogeneous dendritic cell (DC) lineages that orchestrate adaptive immunity, yet how it drives subset diversification remains poorly understood. Here, we employed a sequential anchored screen of 70 transcription factors using direct cellular reprogramming to identify regulators that specify type 2 conventional DCs (cDC2s) and plasmacytoid DCs (pDCs). We identified PU.1, IRF4, and PRDM1 as inducers of a pro-inflammatory cDC2B-like fate and SPIB, IRF8, and IKZF2 as mediators of an immature lymphoid DC program. Transcriptomic profiling linked these triads to lineage-specific signatures and demonstrated their requirement for subset identity. Mechanistically, lineage divergence was driven by chromatin co-engagement at subset-specific sites early in reprogramming. Functionally, reprogrammed DCs employed distinct immune mechanisms to elicit orthogonal anti-tumor responses in different tumor models. Collectively, our findings uncover transcriptional circuits that control DC diversification and pave the way to generate patient-tailored DC subsets for cancer immunotherapy.

Cover design credit: Avesta Rastan

Casting for Dendritic Cell Diversity – About the Cover

In this issue, Henriques-Oliveira et al. employ a direct cellular-repogramming-sequential anchored screen of 70 transcription factors to examine the transcription factor cooperativity that specifies distinct dendritic cell (DC) lineages. The authors identify transcriptional regulators of type 2 conventional DCs and plasmacytoid DCs and demonstrate distinct functions for these cells in anti-tumor immunity. The screen is depicted as a fishing net hauling distinct fish (DCs) from waters full of various immune cells as well as hidden tumor threats. Illustration by Avesta Rastan.

Web-Based Application for Data

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Initial Illustration for Press Releases

Mapping the Routes to DC Identity and anti-tumor immunity

In a barren immune desert, three reprogramming paths guided by transcription factor combinations PIB, PIP, and SII, converge on a lush oasis symbolizing effective anti-tumor immunity. Henriques-Oliveira, Altman, and Kurochkin et al. show how unique cell reprogramming routes give rise to functionally diverse dendritic cell subsets, trailing new paths for immunotherapy.
Illustration by Avesta Rastan.

REPROGRAMMING STAR: Dr. Effie Apostolou is an Associate Professor of Molecular Biology at Weill Cornell Medicine, New York, US, and the Group Leader of the Chromatin Organization & Cell Fate Decisions Lab. The main focus of the Apostolou lab is to dissect the critical interplay between TFs, 3D chromatin organization, and transcription during either (i) maintenance of cell fate (selfrenewal) or (ii) transition to a new fate. Their goal is to build 4D molecular roadmaps to address specific questions such as, How do cells maintain or change identity? What factors dictate cell fate decisions? What is the interplay between transcription, epigenetic factors, and 3D chromatin organization in development and disease?
Dr. Apostolou’s group uses several dynamic cellular systems, including induced pluripotent stem cell (iPSC) reprogramming, differentiation, immune response, tumorigenesis, and cell cycle. They use high throughput sequencing technologies such as 4C-Seq, HiC, HiChIP, ChIP-seq, andPRO-seq and integrative computational analysis to address regulatory mechanisms of cell type-specific gene expression programs. In addition, they employ advanced CRISPR/(d)Cas9-based genetic and epigenetic approaches for precise modulation and functional testing. Dr Apostolou’s lab is also tackling functional heterogeneity and rare stem-like populations in vivo and ex vivo by expanding into single-cell technologies.

Abstract

The efficacy of cancer immunotherapy relies on the recruitment and activation of cytotoxic T cell responses against solid tumors by type 1 conventional dendritic cells (cDC1s). However, the generation of cDC1s for cancer immunotherapy faces significant limitations, including poor cell yield, functional heterogeneity, and susceptibility to immunosuppression in the tumor microenvironment (TME). We recently developed an immunotherapy modality based on in vivo reprogramming of cancer cells into immunogenic cDC1-like cells, which enabled cancer cells to present tumor antigens as cDC1s and elicited polyclonal cytotoxic T cell responses and durable systemic anti-tumor immunity. Here, we describe a tractable protocol to generate cDC1-like cells within the TME by overexpressing the minimal cDC1-specific gene regulatory network-PU.1, IRF8, and BATF3 (collectively referred to as PIB)-in cancer cells, followed by subcutaneous implantation of a mixture of transduced and parental cells. PIB overexpression drives the gradual acquisition of the hematopoietic marker CD45 and the professional antigen presentation complex MHC class II on tumor cells, serving as cell surface readouts for in vivo cDC1 reprogramming. When compared to the reprogramming process in vitro, reprogramming of the YUMM1.7 mouse melanoma model in vivo demonstrated faster kinetics and higher efficiency. cDC1-like cells induced rapid remodeling of the TME by recruiting host immune cells within the first 3 days and leading to the formation of tertiary lymphoid structure by day 9. Reprogrammed cDC1-like cells persisted in tumors for at least 9 days but were undetected at day 15. The in vivo cDC1 reprogramming protocol described here provides a tractable and robust method to effectively transform “immune-cold” tumors into “immune-hot”. Overall, it offers a powerful platform to study the mechanisms underlying cDC1-mediated anti-tumor immunity and uncover synergistic combinations with other cancer immunotherapy modalities.

Immunotherapy has revolutionized cancer treatment, but the majority of patients do not benefit from current strategies. Antigen presentation and lymphoid-mediated cytotoxicity are essential immune properties to elicit antitumor immune responses and control tumor growth. However, high tumor heterogeneity and immune evasion mechanisms impair tumor immune surveillance. High frequencies of cytotoxic natural killer (NK) cells and antigen-presenting conventional dendritic cell type 1 (cDC1) in the tumor environment correlate with positive responses to immunotherapy and survival. However, current therapeutic strategies to enhance innate lymphoid cytotoxicity and antigen presentation function continue to fall short of their potential.

Direct cell reprogramming mediated by transcription factors (TFs) offers the possibility to induce immune functional properties in other cell types, allowing fast and efficient generation of effector cells for therapy. Ectopic expression of the TFs PU.1, IRF8 and BATF3 (PIB) induces cDC1 fate in fibroblasts, but instructor TFs for lymphoid programs have not been reported.

Here, I employed direct reprogramming strategies to impose antigen presentation function in cancer cells and to identify TF codes for innate cytotoxic function.Enforced expression of PIB imposed cDC1 morphology and immunophenotype in mouse and human tumor cells generating tumor antigen presenting cells (tumor-APCs) with reduced tumorigenicity. Tumor-APCs showed extensive transcriptional remodeling with upregulation of antigen-presenting machinery genes. cDC1 reprogramming activated surface expression of antigen presentation complexes and co-stimulatory molecules, enabling presentation of endogenous tumor antigens. Functionally, tumor-APCs acquired competence to engulf, process and cross-present antigens to CD8+ T cells. Adoptive transfer of tumor-APCs to melanoma tumors controlled tumor growth and increase survival, an effect that was synergistic with immune checkpoint blockade. To determine instructor factors for innate cytotoxic function, I overexpressed four canonical NK cell TFs- TBET, ETS1, NFIL3, EOMES (TENE) in human embryonic fibroblasts. TENE-transduced cells activated the expression of the critical NK marker CD56 and acquired morphological changes such as the defining intracellular granules. Furthermore, TENE induced the production and secretion of cytotoxic molecules, granzyme B and granulysin, and pro-inflammatory cytokines TNF-α and IL-2. Induced cytotoxic lymphocytes exhibit global transcriptional changes towards an NK cell program, with upregulation of immunomodulatory genes. Finaly, I showed that TENE-reprogramming was conserved across human dermal and mouse embryonic fibroblasts.

Overall, the findings described here lay the foundation for the development of immunotherapies centered on the induction of immune cell properties in non-immune cell types. In the future, conversion of tumor cells into cDC1 can be utilized in vivo to attenuate tumorigenesis and elicit antitumor immune responses in situ. Instructor TFs for innate cytotoxicity may be harnessed for reinvigorate exhausted NK cells or to confer killing capacity to tumor resident cells.

Abstract

Dendritic cells (DCs) are a heterogenous family of professional antigen presenting cells that specialize in the uptake, processing and presentation of antigens to T cells to promote downstream immune responses, therefore acting as orchestrators of immunity. Conventional type 1 DCs (cDC1) are cross-presenting cells, cDC type 2 (cDC2) cells govern type 1, 2 and 3 immunity, and plasmacytoid DCs (pDCs) are critical in type-I IFN (IFN-I) secretion for antiviral responses. However, the intricacy of DC diversity has become more apparent with recent studies highlighting the range and redundancy of some DC functions previously thought as restricted. In the context of cancer, the effectiveness of classical immunotherapy approaches, such as immune checkpoint blockade, has been associated with cDC1, but also with the presence of cDC2 and pDCs in the tumor microenvironment. Understanding how DC diversity is generated is crucial to predict and promote immune responses. However, the transcription factors (TFs) driving DC subset identity and functional divergence remain unclear.

By allowing the direct conversion of a somatic cell type into another, direct cell reprogramming offers not only a strategy to generate cells for regenerative medicine or immunotherapy, but also a platform to dissect the TF codes underlying heterogeneous cell lineages. The minimal network composed of PU.1, IRF8, and BATF3 was shown to be sufficient to induce cDC1s from fibroblasts, opening opportunities to understand DC diversity with reprogramming.

Here, I applied direct cell reprogramming to uncover the TF networks underlying the diversity of DCs, while also generating diverse DC subsets for cancer immunotherapy. Using an additive screening strategy, I identified two TF triads that induce cDC2 or pDC identities. PU.1, IRF4, and PRDM1 (PIP) induced cDC2s that acquired a pro-inflammatory cDC2B transcriptional program with an interferon gene signature and the ability to present antigens to T cells. SPIB, IRF8, and IKZF2 (SII) induced pDCs that displayed an immature pDC pro-inflammatory profile which could be primed to IFN-β secretion by the addition of IRF4. We further elucidated TF engagement at the onset of the reprogramming process, highlighting the need for cooperative action between an ETS and an IRF factor as the base program for DC induction, further aided by a third factor to establish subset-specific identities. The injection of all induced DCs into tumors promoted anti-tumor immunity in mice, solidifying their immunotherapeutic potential. In vivo reprogramming of two different tumor models revealed a differential response to cDC1 and cDC2 reprogramming, highlighting the specificity of each DC-reprogramming combination in driving tumor-protective responses. Additionally, I developed a combinatorial barcoded TF-based single-cell screening platform to further identify TF combinations for DC reprogramming. A set of 48 DC-specific barcoded TFs allowed the simultaneous induction of multiple DCs and other immune cell types and the construction of a TF hierarchy map to inform DC reprogramming.

Collectively, these findings contribute to better understanding TF dynamics in DC specification, heterogeneity, and function, paving the way for advancing precision cancer immunotherapies based on DC reprogramming. Additionally, I developed a multiplexed technology that opens opportunities to harness immune cell reprogramming for broader applications, ranging from cancer to autoimmunity and beyond.

REPROGRAMMING STAR: Dr. Janelle Drouin-Ouellet is an associate professor at the Université de Montréal, Canada, a neuroscientist, and a Canada Research Chair in Direct Neural Reprogramming. Her research group works with direct neural reprogramming to study neurodegenerative disorders and identify new therapeutic targets. Uncovering age-related drivers of neurodegeneration through cellular reprogramming, Dr. Drouin-Ouellet’slab uses specific approaches such as highcontent confocal microscopy, gene expression profiling, and proteomics. To study the interplay between cellular aging and Parkinson’s disease, in addition to using direct cellular reprogramming, her group investigates approaches to accelerate cellular aging in induced pluripotent stem cells (iPSCs)- derived immune cells such as microglia. Her group is working on understanding the mechanisms of neuronal reprogramming a means to improve current direct neuronal reprogramming methods and generate subtypespecific neurons resembling more closely the neuronal populations of the central nervous system to improve disease modeling and brain repair strategies.

Effective immune responses, within the tumor microenvironment (TME), including T and B cell activation, are key for successful cancer immunotherapies. Tertiary lymphoid structures(TLS), which generate persistent T and B cell responses, correlate with improved outcomes. However, tools to therapeutically induce TLS across cancers are lacking. We previously developed an approach to reprogram tumor cells in vivo into type 1 conventional dendritic-like (cDC1-like) cells, triggering T cell responses and TLS formation in melanoma. Here, we hypothesize that dendritic cell (DC) reprogramming can induce TLS and humoral responses across tumor types. In vivo cDC1 reprogramming led to consistent synthetic TLS (synTLS) formation and tumor-agnostic anti-tumor immunity. DC reprogramming induced complete responses (CR) and outperformed anti-PD-1 therapy, across subcutaneous and orthotopic models, including ICB-resistant YUMM1.7 (60% vs. 0% CR), MC38 (80% vs. 40% CR), and CT26 (100% vs. 0% CR). Orthotopic models such as 4T1 (100% vs. 25% CR), B16 (20% vs. 0%) and LLC, also responded, confirming location-independent efficacy. Strikingly, we even observed 50% CR in the orthotopic immunosuppressive SB28 glioblastoma model, which has so far not been shown treatable with immunotherapy modalities. SynTLS formed within 9 days, containing CD4+ and CD8+ T cells, CD19+ B cells and BCL6+ germinal centers and gradually disappeared with tumor regression. Formation occurred even in BATF3KO mice lacking endogenous cDC1s. B cells contributed to anti-tumor immunity, as 30% of B-cell-depleted mice showed impaired tumor growth control despite treatment with reprogramming. Serum binding assays and serum transfer between animals demonstrated tumor- specific antibody binding of tumor tissue and when transplanted tumor growth delay suggesting role in anti-tumor immunity. Furthermore, mice treated with DC reprogramming developed circulatory tumor-specific antibodies which could serve as blood-based biomarkers of therapeutic response. Overall, cDC1 reprogramming acts as a tumor-agnostic immunotherapy that drives TLS formation, elicits humoral immunity, and generates circulating biomarkers for treatment monitoring, supporting future clinical settings of DC reprogramming.

REPROGRAMMING STAR: Dr. Bruno Di Stefano is an Assistant Professor in the Department of Molecular and Cellular Biology at Baylor College of Medicine (Houston, Texas, USA). His research centers on the pivotal role of post-transcriptional regulation in mammalian cell fate decisions. Cell fate transitions are essential during embryonic development and for tissue homeostasis in adults. These transitions largely occur without changes in genomic content, highlighting the importance of RNAbased regulatory mechanisms in development and lineage specification. Despite recent advances, the contribution of post transcriptional regulation to lineage specification remains incompletely understood. The Di Stefano lab focuses on several outstanding questions: (1) How do RNA condensates orchestrate the establishment and maintenance of cell identity? (2) What mechanisms guide the crosstalk between chromatin architecture and RNA processing in cell fate? (3) How do RNA modifications instruct human cell fate decisions? By addressing these fundamental questions, research in the Di Stefano lab aims to uncover general principles of cell fate control and cancer development across tissue contexts while pioneering innovative RNA-based therapeutic strategies.

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 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.

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 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 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 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.

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.

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

Read here

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 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

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.

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.

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.

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 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.

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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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.