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.}

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

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).

Read more

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.

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.

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

Read here

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.

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.

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.

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.

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.

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/

Abstract

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.

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.

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?

Read More

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.

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.

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.

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.

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

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

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.

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.

Abstract

Direct cell reprogramming is an emergent way of understanding and controlling somatic cell fate. Our group has previously described direct reprogramming of fibroblasts to induced dendritic cells (iDCs) through overexpression of the transcription factors (TFs) PU.1, IRF8, and BATF3 (PIB), providing evidence that immunity can be induced with direct
reprogramming. In this project, we took advantage of a Clec9a-based DC-specific reporter system previously used in our lab to screen for candidate transcription factors with the potential to induce pDC fate in mouse embryonic fibroblasts. We used an IRF8-based additive screening approach to identify reporter-activating factors since this factor is highly expressed in pDCs and has been previously found to be essential for pDC development. Our results showed that IRF8, in combination with SPIB, are sufficient to induce reporter activation and surface expression of MHCII and B220 molecules. Individual addition of the transcription factors BCL11A, CBFA2T3, CREB3L2, ETS1, STAT1, TCF12, TCF4, or ZEB2 to this initial combination increased reporter activation and expression of some of the selected markers. Moreover, preliminary results indicate that induced cells from some combinations can secrete type I IFN and other pro-inflammatory cytokines upon TLR7 and TLR9 challenging, thus suggesting similar functional properties to pDCs.

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.

Abstract

Cancer always plays a constant hide and seek game with the immune system. Tumor cells have developed multiple mechanisms to evade the immune system, including dendritic cells (DCs). DCs are in charge of obtaining antigens from cancer cells and present them to other cells such as Natural Killer cells and T-cells, which in turn are capable of eliciting an immune response. Unfortunately, one of the mechanisms used by cancer cells to hide from the immune system is inhibiting dendritic cells in the tumor site.

In an effort to increase DCs in the tumor microenvironment, the Pereira lab at Lund University discovered that the combination of three proteins PIB (PU.1, IRF8, and BATF3), was able to reprogram fibroblasts, a type of skin cells, into induced-dendritic cells. Further research showed that the PIB combination was capable of reprogramming cancer cells into induced tumor-antigen presenting cells (tumor-APCs) in a broad range of tumor types. Although the PIB combination showed extraordinary reprogramming efficiency in some cancer types such as melanoma and glioblastoma (up to 70%), reprogramming efficiency of other malignancies, e.g. lung carcinoma, were as low as 0.1%.

Lung carcinoma is the leading cause of cancer mortality worldwide, mainly due to the late diagnosis and lack of efficient treatment options. Therefore, increasing reprogramming efficiency of lung carcinoma would play an important role in the search for new, more effective treatment therapies. To do so, lung carcinoma and highly efficient glioblastoma cell lines were characterized at the protein, gene expression, and morphologic levels to assess the possible differences and their correlation with reprogramming efficiency. Subsequently, extrinsic factors were added to the investigated cell lines to evaluate variations in reprogramming efficiency.

Through observations of surface markers, protein levels, and gene expression changes in the cell lines, it was shown that lung carcinoma cells are capable of undergoing molecular reprogramming upon PIB treatment, however, at lower levels than the highly efficient glioblastoma cell lines. These results hinted that lung carcinoma cell lines may have a barrier that obstructs the reprogramming pathway. To further investigate this, small molecules modifying gene expression were added to the culture during the 9 days of reprogramming. The results from these experiments showcased an increase of partially reprogrammed cells from 5% to 45%, and an increase of fully reprogrammed cells from 0.1 to 12%.

Overall, increasing reprogramming efficiency of lung carcinoma cells was achieved through the addition of the small molecules. These findings contribute to a significant improvement of reprogramming efficiency that could be applied not only to lung carcinoma, but a broad range of tumour types, paving the way towards a novel immunotherapeutic approach, where the immune system would be able to win in the hide and seek game against cancer.

Abstract

Direct cell reprogramming is a process of turning one somatic cell type into another, usually achieved by overexpression of cell type-specific transcription factors (TFs) which can be combined with small molecules and microRNAs (miRNAs). Using Clec9a-tdTomato reporter mouse embryonic fibroblasts (MEFs), our group previously identified the combination PU.1, IRF8 and BATF3 (PIB) as sufficient to reprogram mouse and human fibroblasts to induced dendritic cells (iDCs). The generated DCs activate a DC type 1 (cDC1) gene expression program and cross-present antigens, albeit at low efficiency. We hypothesize that miRNAs could synergize with TFs in the DC reprogramming process, leading to increased efficiency. We started by identifying candidate miRNA based on literature analysis and DC-specific miRNA expression data. We cloned 15 candidate hairpin-containing genomic regions in a constitutive lentiviral expression system. Resulting lentiviral vectors were co-transduced alongside the polycistronic lentiviruses encoding DC-inducing TFs. We identified two miRNAs that improved iDC reprogramming by distinct mechanisms. One upregulated the population co-expressing CD45 and MHC-II while the other led to a remarkable increase in the cDC1-specific marker XCR1. These phenotypic changes are corroborated by morphologic differences as quantified by fluorescence microscopy. When combined, these two miRNAs increased iDC1 reprogramming efficiency 13-fold, demonstrating an additive impact on iDC reprogramming. Future work includes construction of a single PIB-miRNA dual expression vector and functional characterization of miRNA-assisted iDCs. We also cloned and tested bicistronic reprogramming factors to test whether miRNAs will be sufficient to substitute the action of individual TFs. In summary, our candidate screen identified miRNAs that increase iDC reprogramming efficiency and their cDC1 marker expression. Using this system to explore the underlying mechanisms may provide valuable insights into the role of miRNAs in cDC1 subtype specification. Overall, our study brings reprogramming to cross-presenting cDC1s closer to patient-tailored cancer immunotherapy.

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

Abstract

Gastric cancer remains a serious health burden with few therapeutic options. Therefore, the recognition of cancer stem cells (CSCs) as seeds of the tumorigenic process makes them a prime therapeutic target. Knowing that the transcription factors SOX2 and OCT4 promote stemness, our approach was to isolate stem-like cells in human gastric cancer cell lines using a traceable reporter system based on SOX2/OCT4 activity (SORE6-GFP). Cells transduced with the SORE6-GFP reporter system were sorted into SORE6+ and SORE6– cell populations, and their biological behavior characterized. SORE6+ cells were enriched for SOX2 and exhibited CSC features, including a greater ability to proliferate and form gastrospheres in non-adherent conditions, a larger in vivo tumor initiating capability, and increased resistance to 5-fluorouracil (5-FU) treatment. The overexpression and knockdown of SOX2 revealed a crucial role of SOX2 in cell proliferation and drug resistance. By combining the reporter system with a high-throughput screening of pharmacologically active small molecules we identified monensin, an ionophore antibiotic, displaying selective toxicity to SORE6+ cells. The ability of SORE6-GFP reporter system to recognize cancer stem-like cells facilitates our understanding of gastric CSC biology and serves as a platform for the identification of powerful therapeutics for targeting gastric CSCs.

Abstract

Cell reprogramming concepts have been classically developed in the fields of developmental and stem cell biology and are currently being explored for regenerative medicine, given its potential to generate desired cell types for replacement therapy. Cell fate can be experimentally reversed or modified by enforced expression of lineage specific transcription factors leading to pluripotency or attainment of another somatic cell type identity. The possibility to reprogram fibroblasts into induced dendritic cells (DC) competent for antigen presentation creates a paradigm shift for understanding and modulating the immune system with direct cell reprogramming. PU.1, IRF8, and BATF3 were identified as sufficient and necessary to impose DC fate in unrelated cell types, taking advantage of Clec9a, a C-type lectin receptor with restricted expression in conventional DC type 1. The identification of such minimal gene regulatory networks helps to elucidate the molecular mechanisms governing development and lineage heterogeneity along the hematopoietic hierarchy. Furthermore, the generation of patient-tailored reprogrammed immune cells provides new and exciting tools for the expanding field of cancer immunotherapy. Here, we summarize cell reprogramming concepts and experimental approaches, review current knowledge at the intersection of cell reprogramming with hematopoiesis, and propose how cell fate engineering can be merged to immunology, opening new opportunities to understand the immune system in health and disease.