GATA2 Mitotic Bookmarking Is Required for Definitive Haematopoiesis
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.
Genome Browser for ChIP-Seq
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
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
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
Direct Reprogramming of Fibroblasts to NK Cells
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.
Reprogramming Hematopoietic Cells Into Type 1 Dendritic Cells
Dendritic cells (DCs) are professional antigen-presenting cells able to induce potent and long-lasting adaptive immune responses. Within the DC family, type 1 conventional dendritic cells (cDC1s) excel on the ability to cross-present exogenous antigens to cytotoxic T cells, a critical step for inducing antitumor immunity. However, cDC1s are rare in peripheral blood and in vitro differentiation of monocytes, CD34+ progenitors or induced pluripotent stem cells results in the generation of heterogenous DC populations with poor antigen presentation capacity and limited ability to migrate to lymph nodes. Our group has recently identified the transcription factors PU.1, IRF8 and BATF3 (PIB) as sufficient to reprogram mouse and human fibroblasts into functional cDC1-like cells. However, fibroblasts are not readily available in
sufficient numbers, in contrast to monocytes that are easily accessible in peripheral blood at high numbers.
Here, we investigated cDC1 reprogramming in THP-1 monocytic cell line and explored multiple viral and non-viral delivery systems to achieve efficient gene delivery to primary monocytes. We showed that overexpression of PIB mediated by lentiviral vectors allowed cDC1 reprogramming of THP-1 cells at high efficiency. Reprogrammed THP-1 cells expressed the cDC1 surface markers CLEC9A and CD141, and the co-stimulatory molecules CD40 and CD80. Interestingly, high levels of CLEC9A expression were detected as early as 3 days after transduction, suggesting that reprogramming progresses with fast kinetics in monocytic cells. We observed that toll-like receptor stimulation in reprogrammed THP-1 cells induced upregulation of co-stimulatory molecules and increased secretion of inflammatory cytokines. Interestingly, cDC1 reprogramming was associated with reduced tumorigenicity of THP-1 cells. Lastly, we tested different viral and non-viral delivery systems to further optimize the transduction of primary monocytes and peripheral blood mononuclear cells and observed that AAV vectors and mRNA allowed higher transgene delivery to primary monocytes when compared to lentiviral vectors.
This work demonstrates that overexpression of PIB in human monocytic cells allows efficient and fast reprogramming to cDC1-like cells and gives insights into alternative viral and non-viral systems allowing transgene delivery to primary monocytes and other hematopoietic cells. Ultimately, this study will open the opportunity to develop efficient cDC1-based vaccines for cancer immunotherapy.
Optimization of a CRISPR/Cas9 Screening Toolbox for Defining Regulators of Hemogenic Reprogramming
Hematopoietic stem cell transplantation has been used as the primary curative treatment for a variety of hematologic malignancies. Generation of patient-tailored HSCsin vitro by direct cellular reprogramming has the potential to overcome major limitations of current treatments. Using this method, somatic cells can be converted into different lineages through enforced expression of transcription factors (TFs). The expression of GATA2, FOS and GFI1B (GaFoGi) TFs reprograms human dermal fibroblasts (HDFs) into HSC-like cells. The mechanisms and regulators underlying this dynamic process remain elusive. Genome-wide engineering screening approaches provide an opportunity to map reprogramming regulators and to improve the efficiency of the process.
This project aimed to optimize a CRISPR/Cas9 screening toolbox for defining regulators of hemogenic reprogramming. We first compared knockout efficiency of conditional and constitutive Cas9 enzymes and showed that the constitutive Cas9 yields higher knockout efficiency when compared to the inducible Cas9 system. We have also demonstrated that a multiplicity of infection of 1 is both sufficient and optimal to achieve effective knockout. In parallel, we have optimized the transduction of a sgRNA library targeting 104 genes required for HSC self-renewal and proliferation. Finally, we have improved the delivery of the hemogenic TFs to fibroblasts using a single lentiviral vector. By comparing lentiviral vectors expressing GaFoGi under the control of multiple promoters, we showed that expression of GaFoGi under the spleen focus forming virus promoter generates CD34+CD9+ACE+CD49f+ hemogenic cells at highest efficiency. This strategy has allowed us to define reprogrammed and partial reprogrammed populations for cell sorting and screening of regulators.
The findings of this study provide a foundation for CRISPR/Cas9 screening to define transcriptional and epigenetic regulators of hematopoietic reprogramming. Ultimately, the identification of molecular facilitators and barriers of reprogramming will allow generation of human HSC-like cells at high efficiency and fidelity for autologous or allogenic transplantation.
Evaluating Dendritic Cell Reprogramming in Patient-Derived Cancer Organoids
Immunotherapy utilizes the patient’s own immune system for the treatment of cancer. Due to exceptional antigen-presentation capacity Dendritic Cells (DCs) have great potential for cancer immunotherapy. Using direct cellular reprogramming our group has identified a combination of transcription factors, PU.1, IRF8, and BATF3 that reprograms mouse and human fibroblasts and a range of cancer cell lines into antigen-presenting cells resembling type 1 conventional DCs (cDC1) in terms of morphology, transcriptional, epigenetic profiles, and functional features. To support clinical translation of this approach based on induced antigen presentation of cancer antigens, we addressed cDC1 reprogramming in primary cancer tissues obtained from patients with head and neck, urothelial, lung carcinoma, and melanoma. we showed that all primary samples tested were permissive to cDC1 reprogramming with varying efficiencies according to the cancer cell type of origin. Phenotypic analysis demonstrated that reprogrammed primary cancer cells upregulated expression CD45 and HLA-DR which represent well the reprogramming trajectory. Furthermore, reprogrammed cells expressed cDC1-specific marker CD226 as well as co-stimulatory molecules CD40 and CD80, suggesting that reprogrammed primary cells became competent for antigen presentation. In addition, reprogrammed tumor cells secreted pro- inflammatory cytokines- TNF-α and IL-12p70 which may further enhance anti-tumor immunity.
To model cDC1 reprogramming within the tumor microenvironment (TME), we generated cancer derived organoids in the presence or absence of fibroblasts. We showed that reprogramming was feasible in organoid 3D models, despite an overall decrease in transduction efficiency, that could be improved by combining transduction protocols with dissociation methods. Interestingly, the efficiency of cDC1 reprogramming was not hampered in cancer organoids generated with fibroblasts, suggesting that the immunosuppressive TME does not negatively impact the reprogramming process. This study brings valuable information for clinical translation of cDC1 cancer cell reprogramming and contributes to the development of novel immunotherapies based on direct reprogramming.
Harnessing Small Molecules to Facilitate Dendritic Cell Reprogramming
Conventional type 1 dendritic cells (cDC1s) are professional antigen-presenting cells with key roles in initiating and regulating potent and long-lasting anti-tumor immune responses. As such, they are critical for the response to checkpoint blockade and adoptive T-cell transfer but their rarity in peripheral blood has limited the clinical exploitation of cDC1s for cancer immunotherapy. We have recently identified PU.1, IRF8, and BATF3 transcription factors that convert mouse and human fibroblasts into functional cDC1s.
However, low reprogramming efficiency was a major roadblock for clinical translation. Here, we developed a microscopy-based high-content screening platform using the dendritic cell specific reporter system Clec9a-tdTomato, combined with flow cytometry analysis for cDC1 surface markers to identify and validate small molecules (SMs) that enhance reprogramming efficiency. We identified 358 SMs that increased Clec9a-tdTomato reporter activation, providing proof of principle that SMs can augment cDC1 reprogramming. We then validated these SMs by flow cytometry and showed that microscopy-based high-content screening results correlate with flow cytometry. We identified retinoid acid receptor (RAR) agonists, in particular RARβ and RARγ, promoting early reprogramming events as indicated by Clec9a-tdTomato reporter activation. Moreover, inhibition of WNK2 and B-RAF facilitated late events of cDC1 reprogramming as shown by increased CD45 and MHC-II marker expression. Also, WNK2 inhibition increased CD40 and XCR1 marker expression, hence enhancing cDC1 maturation and restricting a cDC1 cell fate.
These findings suggest that a stepwise activation of retinoic acid signaling followed by promoting MEK/MAPK pathway may result in an optimized cDC1 reprogramming protocol. Our work provides new insights into molecular mechanisms underlying cDC1 specification and reprogramming, paving the way to efficiently generate cDC1s for cancer immunotherapy.
Single-Cell Transcriptional Profiling Informs Efficient Reprogramming of Human Somatic Cells to Cross-Presenting Dendritic Cells
Type 1 conventional dendritic cells (cDC1s) are rare immune cells critical for the induction of antigen-specific cytotoxic CD8+ T cells, although the genetic program driving human cDC1 specification remains largely unexplored. We previously identified PU.1, IRF8, and BATF3 transcription factors as sufficient to induce cDC1 fate in mouse fibroblasts, but reprogramming of human somatic cells was limited by low efficiency. Here, we investigated single-cell transcriptional dynamics during human cDC1 reprogramming. Human induced cDC1s (hiDC1s) generated from embryonic fibroblasts gradually acquired a global cDC1 transcriptional profile and expressed antigen presentation signatures, whereas other DC subsets were not induced at the single-cell level during the reprogramming process. We extracted gene modules associated with successful reprogramming and identified inflammatory signaling and the cDC1-inducing transcription factor network as key drivers of the process. Combining IFN-γ, IFN-β, and TNF-α with constitutive expression of cDC1-inducing transcription factors led to improvement of reprogramming efficiency by 190-fold. hiDC1s engulfed dead cells, secreted inflammatory cytokines, and performed antigen cross-presentation, key cDC1 functions. This approach allowed efficient hiDC1 generation from adult fibroblasts and mesenchymal stromal cells. Mechanistically, PU.1 showed dominant and independent chromatin targeting at early phases of reprogramming, recruiting IRF8 and BATF3 to shared binding sites. The cooperative binding at open enhancers and promoters led to silencing of fibroblast genes and activation of a cDC1 program. These findings provide mechanistic insights into human cDC1 specification and reprogramming and represent a platform for generating patient-tailored cDC1s, a long-sought DC subset for vaccination strategies in cancer immunotherapy.
Web-Based Application for Processed scRNA-seq and ChIP-seq Data
Unraveling Mechanisms of Transcription Factor-Mediated cDC1 Reprogramming
Conventional dendritic cells type 1 (cDC1s) are a rare subset of circulating immune cells that
excel in cross-presentation ability and induction of antigen-specific, cytotoxic CD8+ T cells.
The transcription factor core that controls the specification of human cDC1 is still largely unexplored. PU.1, IRF8 and BATF3 were previously identified as sufficient in inducing cDC1 fate in murine and human fibroblasts, but how these reprogramming factors engage chromatin and rewire the transcriptional profile remains to be elucidated.
Here, we have dissected the reprogramming mechanisms by performing ChIP-seq analysis of the chromatin targeting by the three reprogramming factors when expressed individually or in combination in human fibroblasts. We show that PU.1 displays dominant and independent chromatin targeting capacity at early stages of reprogramming and recruits IRF8 and BATF3 to a cohort of common genomic targets, including cDC1 and antigen cross-presentation genes. This cooperative binding is reflected by the engagement of open enhancers and promoters, disrupting transcriptional initiation of fibroblast-specific genes. We show the three reprogramming factors physically interact and reprogramming is abolished by a point mutation in IRF8 that affects interaction with PU.1. While IRF8 only depends on PU.1 for chromatin engagement, BATF3 requires both PU.1 and IRF8 expression for targeting at early stages of reprogramming. Indeed, we provide evidence for physical interaction between BATF3 and PU.1, indicating a mediating role of BATF3 in the formation of the “cDC1 reprogramming complex” to fine-tune chromatin engagement.
These results shed light on the molecular mechanisms underlying specification and reprogramming of human cDC1s and provide a platform for the generation of patient-tailored cDC1s, a long-sought DC subset for vaccination strategies in cancer immunotherapy.
Cell Fate Reprogramming in the Era of Cancer Immunotherapy
Advances in understanding how cancer cells interact with the immune system allowed the development of immunotherapeutic strategies, harnessing patients’ immune system to fight cancer. Dendritic cell-based vaccines are being explored to reactivate anti-tumor adaptive immunity. Immune checkpoint inhibitors and chimeric antigen receptor T-cells (CAR T) were however the main approaches that catapulted the therapeutic success of immunotherapy. Despite their success across a broad range of human cancers, many challenges remain for basic understanding and clinical progress as only a minority of patients benefit from immunotherapy. In addition, cellular immunotherapies face important limitations imposed by the availability and quality of immune cells isolated from donors. Cell fate reprogramming is offering interesting alternatives to meet these challenges. Induced pluripotent stem cell (iPSC) technology not only enables studying immune cell specification but also serves as a platform for the differentiation of a myriad of clinically useful immune cells including T-cells, NK cells, or monocytes at scale. Moreover, the utilization of iPSCs allows introduction of genetic modifications and generation of T/NK cells with enhanced anti-tumor properties. Immune cells, such as macrophages and dendritic cells, can also be generated by direct cellular reprogramming employing lineage-specific master regulators bypassing the pluripotent stage. Thus, the cellular reprogramming toolbox is now providing the means to address the potential of patient-tailored immune cell types for cancer immunotherapy. In parallel, development of viral vectors for gene delivery has opened the door for in vivo reprogramming in regenerative medicine, an elegant strategy circumventing the current limitations of in vitro cell manipulation. An analogous paradigm has been recently developed in cancer immunotherapy by the generation of CAR T-cells in vivo. These new ideas on endogenous reprogramming, cross-fertilized from the fields of regenerative medicine and gene therapy, are opening exciting avenues for direct modulation of immune or tumor cells in situ, widening our strategies to remove cancer immunotherapy roadblocks.
Here, we review current strategies for cancer immunotherapy, summarize technologies for generation of immune cells by cell fate reprogramming as well as highlight the future potential of inducing these unique cell identities in vivo, providing new and exciting tools for the fast-paced field of cancer immunotherapy.