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.

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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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/

Cell Fate Reprogramming in the Era of Cancer Immunotherapy

Abstract

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.

Reprogramming, The Journal

Cellular Reprogramming | Vol. 23, No. 3 | Editorial
© Mary Ann Liebert, Inc

Cellular reprogramming is a diverse and growing discipline that studies the reversal or modification of
cellular identity. The field aims to understand how cell fate is acquired, maintained, and inherited in homeostatic conditions and what happens when cell identity is hijacked in disease. Owing to the vast therapeutic potential of cellular reprogramming, efforts have also been placed to harness cell fate engineering for clinical applications. Cellular reprogramming history began addressing a fundamental question in biology: how are the myriad of cell types that compose an adult organism generated?

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Ontogenic Shifts in Cellular Fate Are Linked to Proteotype Changes in Lineage-biased Hematopoietic Progenitor Cells

Highlights

  • >4,000 proteins quantified in fetal and adult hematopoietic progenitor cells (HPCs)
  • Protein expression in HPCs separates cells based on ontogenic stage and lineage potential
  • Generic fetal features are suppressed in myeloid-restricted progenitors
  • Low Irf8 expression partially drives an impairment in monopoiesis in fetal HPCs

Abstract

The process of hematopoiesis is subject to substantial ontogenic remodeling that is accompanied by alterations in cellular fate during both development and disease. We combine state-of-the-art mass spectrometry with extensive functional assays to gain insight into ontogeny-specific proteomic mechanisms regulating hematopoiesis. Through deep coverage of the cellular proteome of fetal and adult lympho-myeloid multipotent progenitors (LMPPs), common lymphoid progenitors (CLPs), and granulocyte-monocyte progenitors (GMPs), we establish that features traditionally attributed to adult hematopoiesis are conserved across lymphoid and myeloid lineages, whereas generic fetal features are suppressed in GMPs. We reveal molecular and functional evidence for a diminished granulocyte differentiation capacity in fetal LMPPs and GMPs relative to their adult counterparts. Our data indicate an ontogeny-specific requirement of myosin activity for myelopoiesis in LMPPs. Finally, we uncover an ontogenic shift in the monocytic differentiation capacity of GMPs, partially driven by a differential expression of Irf8 during fetal and adult life.

Mononuclear Phagocyte Regulation by the Transcription Factor Blimp-1 in Health and Disease

Abstract

Blimp‐1, the transcription factor encoded by the gene Prdm1, plays a number of crucial roles in the adaptive immune system, which result in the maintenance of key effector functions of B and T cells. Emerging clinical data, as well as mechanistic evidence from mouse studies, has additionally identified critical functions of Blimp‐1 in the maintenance of immune homeostasis by the mononuclear phagocyte system. Blimp‐1 regulation of gene expression affects various aspects of mononuclear phagocyte biology, including developmental programs such as fate decisions of monocytes entering peripheral tissue, and functional programs such as activation, antigen presentation, and secretion of soluble inflammatory mediators. The highly tissue‐, subset‐, and state‐specific regulation of Blimp‐1 expression in mononuclear phagocytes suggests that Blimp‐1 is a dynamic regulator of immune activation, integrating environmental cues to fine‐tune the function of innate cells. In this review, we will discuss the current knowledge regarding Blimp‐1 regulation and function in macrophages and dendritic cells.

Direct Reprogramming of Mouse Embryonic Fibroblasts to Conventional Type 1 Dendritic Cells by Enforced Expression of Transcription Factors

Abstract

Ectopic expression of transcription factor combinations has been recently demonstrated to reprogram differentiated somatic cells towards the dendritic cell (DC) lineage without reversion to a multipotent state. DCs have the ability to induce potent and long-lasting adaptive immune responses. In particular, conventional type 1 DCs (cDC1s) excel on antigen cross-presentation, a critical step for inducing CD8+ T cell cytotoxic responses. The rarity of naturally occurring cDC1s and lack of in vitro methodologies for the generation of pure cDC1 populations strongly hinders the study of cDC1 lineage specification and function. Here, we describe a protocol for the generation of induced DCs (iDCs) by lentiviral-mediated expression of the transcription factors PU.1, IRF8 and BATF3 in mouse embryonic fibroblasts. iDCs acquire DC morphology, cDC1 phenotype and transcriptional signatures within 9 days. iDCs generated with this protocol acquire functional ability to respond to inflammatory stimuli, engulf dead cells, process and cross-present antigens to CD8+ T cells. DC reprogramming provides a simple and tractable system to generate high numbers of cDC1-like cells for high content screening, opening new avenues to better nderstand cDC1 specification and function. In the future, faithful induction of cDC1 fate in fibroblasts may lead to the generation of patient-specific DCs for vaccination