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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Using Direct Cell Reprogramming to Uncover Plasmacytoid Dendritic Cell Specification Programs and Function

Abstract

Direct cell reprogramming through enforced expression of transcription factors (TFs) converts somatic cells into functional, differentiated cells of other lineages without transiting through pluripotency. Recently, PU.1, IRF8, and BATF3 were identified as the TF network capable of reprogramming murine and human fibroblasts into conventional dendritic cells type 1 (cDC1s). Dendritic cells (DCs) are a remarkable heterogenous lineage bridging innate and adaptive immunity. In addition to antigen-presenting conventional DCs, plasmacytoid DCs (pDCs) represent a specific subset specialized at producing type I interferons (IFN-I) during the antiviral response. pDCs’ ontogeny and the TF networks required to specify pDC-fate and function remain unclear.

This thesis aims to define the TF codes required to induce functional pDCs from unrelated cell-types, utilizing cell fate reprogramming approaches. We have employed an IRF8- based screening approach, given its key role and high expression in pDCs. 36 candidate TFs were screened with IRF8 using mouse embryonic fibroblasts carrying a Clec9a- based reporter system. Overexpression of IRF8 and SPIB was sufficient to induce reporter activation. By performing a secondary screen, we identified IKZF2, when combined with IRF8 and SPIB, that in addition to maintaining Clec9a activation, increases hCD2-based reporter expression and kick-start expression of hematopoietic and pDC- specific surface markers. Thus these three TFs were identified as the optimal network to induce pDC-fate. Induced pDCs express the surface markers B220, CCR9, Ly6C, and antigen-presentation MHC-II molecules. Functionally, induced cells acquire the ability to secrete IFN-I and inflammatory cytokines including TNF-a, IL-6, CCL5, and CXCL10, in
response to TLR9 triggering.

This study identified a combination of three TFs to induce IFN-I-producing pDCs from a non-related cell type by direct cell reprogramming. This finding provides insight into the unique developmental program and functional properties of pDCs. In the future, these results open the possibility to explore induced pDCs to induce immune responses for immunotherapy.

Reprogramming Human Monocytes Into Type 1 Dendritic Cells

Abstract

The potential of dendritic cells (DCs) for cancer immunotherapy has been highlighted for decades. Type 1 conventional DCs (cDC1s) gained recent attention due to their superior ability to perform antigen cross- presentation, a critical step for inducing antitumor cytotoxic T cell responses. However, the clinical testing of cDC1s has been hindered by their shortage in peripheral blood. We have previously identified a combination of three transcription factors PU.1, IRF8, and BATF3 able to reprogram mouse and human fibroblast to cDC1-like cells. Here, we show that the same combination of transcription factors can reprogram human monocytes isolated from peripheral blood into cDC1-like cells. Reprogramed monocytes express cDC1-specific surface markers including CLEC9A, CD141, and XCR1, show increased migratory capacity towards chemokine gradients and produce IL-12. Importantly, we show that reprogrammed monocytes acquire antigen cross presentation capacity to efficiently activate cytotoxic CD8+ T cells. This study sets the foundation to generate clinically applicable cDC1-like cells from easily accessible blood cells for DC vaccination.

Evaluating Dendritic Cell Reprogramming in Patient-derived Tumor Cells

Abstract

Despite tremendous efforts and achieved advances, cancer is still the leading cause of death worldwide, which underlines the need to develop novel curative approaches. In the past decade, we witnessed the rapid development of immunotherapy – novel treatment strategies based on harnessing the patient´s very own immune system to fight cancer. Although immunotherapy revolutionized the treatment in multiple malignancies, including advanced melanoma, in the vast majority of patients the response is very limited.

Recently, the Pereira lab at Lund University identified a combination of three proteins, PU.1, IRF8, and BATF3 (PIB) which were sufficient to generate immunogenic type 1 dendritic cells from mouse fibroblasts via direct cellular reprogramming. These new cells had a transcriptional program and cell morphology resembling conventional DC type 1 (DC1), which are specialized in antigen cross-presentation and initiating cytotoxic T cell responses. Furthermore, preliminary data has shown that the reprogramming towards dendritic cell fate can also be applied in cancer cells directly. Therefore, inducing antigen presentation directly in tumor cells may help to bypass current limitations of other immunotherapies, such as tumor cell heterogenicity, immune evasion, and neoantigen identification.

Now, to support the translational efforts of these findings, the application of dendritic cell reprogramming was tested in patient samples. I investigated the reprogramming efficiency mediated by PIB in cancer samples across several malignancies including breast, lung, bladder, pancreatic, head, and neck carcinomas and melanoma as well as cancer associated fibroblasts. I have further evaluated the global gene expression reprogramming at the single cell level with a focus on DC1- and antigen presentation-specific genes. Overall, I observed that all patient samples underwent significant changes during reprogramming with more than 50% of the cells expressing at least one of the reprogramming markers CD45 or HLA-DR. Particularly, lung carcinoma showed highest reprogramming efficiency where more than 60% of the cells were partially reprogrammed and 15% were fully reprogrammed. Interestingly the reprogramming efficiencies observed from patient samples were higher than cancer cell lines, showing a convergent global switch in the transcriptional program to the DC1 fate at the single cell level. Moreover, I also evaluated the reprogramming efficiency in cancer cells tumor organoids constructed by the forced-floating method. Tumor organoids were reprogrammed in similar patterns as their parent cells cultured through conventional 2D methods. However, in the 3D models, it was observed low transduction efficiency, hinting at the potential need to improve the delivery system of PIB into tissues.

These findings support the application of DC reprogramming in patients across multiple malignancies, thus paving the way for the development of a novel cancer gene therapy approach based on dendritic cell reprogramming.

Generating Dendritic Cells by Direct Cell Reprogramming

Abstract

Cell fate reprogramming towards pluripotency or alternative somatic cell-types has highlighted the plasticity of adult somatic cells, providing new technologies to generate desired cell types for tissue repair or for disease modeling. There is momentum to bring these concepts to immunology by specifying unique immune cellular identities that set in motion immune responses. Dendritic cells (DCs) are professional antigen presenting cells specialized in the recognition, processing and presentation of antigens to T cells, inducing adaptive immune responses. Within the DC compartment, conventional type 1 DCs (cDC1s) excel in antigen cross- presentation, a critical step to initiate cytotoxic T cell responses. Recent studies have highlighted complementary local and systemic roles of cDC1s in inducing anti-tumor immunity. Nevertheless, their rarity in peripheral blood and the lack of methodologies enabling the generation of a pure population of mature cDC1-like cells limits their study and therapeutic utility. Here, I explore direct cellular
reprogramming from non-hematopoietic cell-types as an alternative approach to generate cDC1.

In this thesis, I screened 34 transcription factors and identified PU.1, IRF8, and BATF3 as the minimal combination required to reprogram mouse and human fibroblasts into cDC1-like cells. Induced DCs (iDCs) generated by cell
reprogramming acquire a DC-like morphology and express cDC1 surface markers and surface molecules required for antigen presentation to T cells. I used single- cell mRNA sequencing to explore the gradual and asynchronous nature of DC reprogramming and demonstrated the downregulation of fibroblast and cell cycle progression genes coupled with the upregulation of cDC1 and antigen presentation genes. Importantly, this approach generated exclusively cDC1-like cells, and not DCs from other subsets. Reconstruction of successful human DC reprogramming trajectories highlighted gene modules associated with successful reprogramming and identified inflammatory cytokine signalling and the DC-inducing transcription factor network as key drivers of the process. Motivated by these observations, I
combined IFN-γ, IFN-β and TNF-α with constitutive expression of PU.1, IRF8 and BATF3 to increase human DC reprogramming efficiency by 190-fold. Functionally, iDCs respond to inflammatory stimuli, engulf dead cell material, secrete inflammatory cytokines and cross-present antigens to CD8+ T cells. Interestingly, I observed that intra-tumoral vaccination in syngeneic mouse models increased infiltration of antigen-specific CD8+ T cells, promoted a T cell cytotoxic profile in draining lymph nodes and controlled tumor growth. Mechanistically, I
show that PU.1 displays dominant and independent targeting capacity by engaging enhancer and promoter regions in open chromatin and recruiting IRF8 and BATF3 to the same binding sites. This cooperative binding allows the downregulation of fibroblast genes and activation of cDC1 genes required to achieve DC reprogramming. Finally, I adapted the DC reprogramming protocol to allow high- content screening of small molecules and identified several small molecules that increase reprogramming efficiency and potentially replace the action of PU.1, IRF8 and BATF3 in DC reprogramming.

These findings should open avenues to better understand cDC1 specification and
functional specialization. Ultimately, DC reprogramming might represent a platform for the future generation of cDC1s for therapy.

Compositions for Reprogramming Cells Into Dendritic Cells Type 2 Competent for Antigen Presentation, Methods and Uses Thereof

Abstract

The present disclosure relates to compositions for reprogramming cells into conventional dendritic cells (cDC), particularly into cDC type 2 (hereinafter referred to as “cDC2” or “CD11b-positive dendritic cells”), methods and uses thereof. The present disclosure relates to the development of methods for making conventional dendritic cells with antigen presenting capacity from differentiated, multipotent or pluripotent stem cells by introducing and expressing isolated/synthetic transcription factors. More particularly, the disclosure provides methods for obtaining conventional dendritic cells (cDC), particularly cDC type 2 or CD11b-positive dendritic cells, by direct cell reprogramming with the surprisingly use of combinations of specific transcription factors.

WO-2021-105234

Composition for Reprogramming Cells Into Plasmacytoid Dendritic Cells or Interferon Producing Cells, Methods and Uses Thereof

Abstract

The present disclosure relates to compositions, constructs and vectors for reprogramming cells into plasmacytoid dendritic cells or interferon type I-producing cells, methods and uses thereof. The present disclosure relates to the development of methods for making plasmacytoid dendritic cells or interferon type I-producing cells that promote antiviral and anti-tumoral immune responses from differentiated, multipotent or pluripotent stem cells by introducing and expressing isolated/synthetic transcription factors. More particularly, the disclosure provides methods for obtain plasmacytoid dendritic cells or interferon type I-producing cells by direct cellular reprogramming with the surprisingly use of combinations of specific transcription factors.

WO-2021-069672

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.

Harnessing the Potential of Cell Fate Reprogramming for Cancer Immunotherapy

Abstract

Despite significant advances in cancer immunotherapy, the majority of patients still do not benefit from treatment. Recent research has highlighted the potency of the dendritic cell subtype cDC1 to prime naïve CD8+ T cells by cross-presentation of tumor specific antigens and direct anti-cancer immunity. However, the dynamic process of immunoediting renders the cancer cells invisible for T cell recognition. Direct cell reprogramming offers the opportunity to resolve these challenges. Our group demonstrated that transcription factors Pu.1, Irf8 and Batf3 (PIB) convert fibroblasts into induced dendritic cells imposing a cDC1-like transcriptional program and cross-presentation capacity. In this study, I evaluate the transcription factor combination to reprogram murine cancer cells into professional tumor antigen presenting cells (tumor-APCs), a process conceptualized as functional reprogramming. Combined expression of PIB in melanoma and lung carcinoma induces hematopoietic and DC markers, CD45 and MHC-II, while upregulating MHC-I and costimulatory molecules CD80/86 and CD40. Notably, epigenetic modification by histone deacetylase inhibitor VPA improves reprogramming efficiency and accelerates the acquisition of a tumor-APC phenotype. Reprogramming successfully endows tumor-APCs with professional APC functions including exogenous antigen uptake by receptor-mediated endocytosis, phagocytosis or macropinocytosis, antigen processing capacity by lysosomal proteases and cross-presentation. Most importantly, PIB expression rescues self-antigen presentation leading to naive CD8+ T cell priming and exposes cancer cells to T cell mediated killing. Collectively, this study lays the foundation for further transcriptional, phenotypical and functional characterization of tumor-APCs, which will pave the way for the development of an immunotherapeutic gene therapy based on the concept of functional reprogramming.