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

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.

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.


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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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.

Reprogramming Hematopoietic Cells Into Type 1 Dendritic Cells

Abstract

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

Abstract

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

Abstract

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

Abstract

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

Abstract

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

https://cellreprolab.shinyapps.io/human_iDC1_atlas/