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

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

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

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

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.

Summary

Dendritic cells (DCs) constitute a remarkable heterogeneous blood lineage responsible for linking innate and adaptive immune responses. Conventional DCs (cDCs) function as professional antigen-presenting cells (APCs) by presenting captured antigens to naïve T cells and can be further divided into two major subsets: cDC type 1 (cDC1) and type 2 (cDC2). While cDC1 mainly perform antigen cross-presentation and cytotoxic CD8+ T cell priming, cDC2 are specialized in major histocompatibility complex class II (MHC-II) presentation, leading polarization towards different CD4+ helper or regulatory T cell phenotypes. Some cDC2s govern type 2 immune responses against parasites and others sense extracellular bacteria and initiate type 3 immunity. Partly due to their high heterogeneity when comparing with other DC subsets, the genetic program underlying cDC2 cell fate determination and diversity remains unclear. Epigenetic reprogramming strategies allow the induction of a somatic cell directly into different cell types. By overexpression of transcription factor (TF) combinations, this strategy is already vastly applied to the generation of multiple cell types in the context of regenerative medicine. Recently, reprogramming of fibroblasts into cDC1-like induced APCs was proven by the expression of PU.1, IRF8 and BATF3, paving the way to modulate immune responses with cell reprogramming. Here, this combinatorial TF strategy was modified to induce cDC2 lineage diversity and unravel the genetic drivers of cDC2 heterogeneity. PU.1 and IRF4 were shown to be crucial in cDC2 development but not sufficient for cDC2 reprogramming. An additional set of 33 candidate TFs to induce cDC2 cell states was identified by combining literature and bioinformatic analysis. Candidate TFs were cloned into inducible lentiviral expression vectors and were tested in mouse embryonic fibroblasts (MEFs) bearing the Clec9a-tdTomato and Zbtb46-GFP DC-reporter systems. Additional surface markers for the cDC2 lineage were validated by analysis of splenocytes and used to further confirm cDC2 reprogramming. Several combinations of TFs were shown to differentially activate the Clec9a and Zbtb46 reporters and surface marker expression. PU.1, IRF4 and TF18 dramatically increased reporters’ activation, CD11b expression and induced tdT+CD11b+ and tdT+Sirpα+ double positive cells. This combination also shown to induce CD45 and MHC-II expression, suggesting acquisition of competence for antigen presentation. TF9, and TF17 addition to PU.1 and IRF4 also resulted in reporter activation and surface marker expression although not synergistically enhancing TF18’s effect, therefore suggesting a differential cDC2 cell fate induction. Overall, several TF combinations were identified for cDC2 reprogramming. These results open new possibilities for a better understanding of the diversity of cDC2 specification and provide a platform for generating patient-tailored reprogrammed cDC2 for immunotherapy.

Abstract

Genetic and epigenetic information, involved in gene expression pattern definition and chromatin landscape are responsible for the cellular identity definition. Cell identity maintenance and cell fate decision events take place during the cell cycle, since faithful restoration of the cellular identity or differentiation into a different cell type are dependent on the persistence of epigenetic marks and gene expression patterns. During the S phase, DNA is replicated and the chromatin structure is altered to allow genes and epigenetic marks duplication. Moreover, at the same time, the transcriptional machinery remains active, suggesting that transcription factors (TFs) might play an epigenetic role. However, the mechanisms underlying epigenetic events involving TFs during S phase are not completely elucidated. The TF GATA2 is essential for definite hematopoiesis and constitutes an instructive factor for the reprogramming of fibroblasts into hematopoietic stem cells (HSCs), with epigenetic potential.
Additionally, GATA2 oscillatory pattern in cell cycle shows increased expression during the S phase. However, the role of GATA2 in epigenetic inheritance during DNA replication in hematopoietic stem cells remains unclear. We aim at the elucidation of the role of GATA2 during DNA replication in HSCs, by degrading GATA2 during S phase using transferable degradation signals. Considering the degradation of cell cycle proteins during DNA replication, we selected three proteins with a natural S phase turnover, Cdt1, Cdc6 and Cyclin E, and four degradation signals were identified Cdt130-120, Cdc61-86, CyclinE1-86 and CyclinE362-393. To validate the functionality of the sequences we have generated fusion proteins with mCherry as reporter and expressed them in HEK293T cells, creating cell lines that stably express our fusion proteins. Flow cytometry showed no association between reporter expression and cell cycle phases distribution and confocal time-lapse imaging corroborate the same findings, since no fluorescence alteration occurs throughout the different cell cycle phases. Altogether, our results suggest that these S-phase degradation systems need to be further developed. In the future, we are planning to use a spacer between the degradative and reporter sequences, an approach already described to be required in other degron-based systems. We believe that application of this degron-based technology to transcription factors, such as GATA2, will shed some light on basics mechanisms of epigenetic inheritance mediated by TFs in stem cells.

Abstract

Direct reprogramming refers to the ability of rewiring the transcriptional state of one lineage to another without passing through an intermediate pluripotent state. The ability to generate different cell types on demand with combinations of lineage-specific transcription factors (TFs) has opened new avenues for regenerative medicine and immunotherapy. It was recently established that fibroblasts transduced with PU.1, IRF8 and BATF3 results in cells with functional properties of dendritic cells (DCs). DCs are crucial cells in the immune system where they initiate adaptive immune responses, by incorporating and displaying antigens at the cell surface and present them to T-cells.
Here, I have done differential expression and pathway enrichment analysis on transcriptomic data obtained throughout reprogramming of mouse and human fibroblast into DCs. Differential expression analysis, correlation and PCA plot revealed reprogrammed mouse DCs share gene expression with “natural” DCs and t-SNE plot, heatmap and RNA velocity analysis showed reprogrammed human DCs favor a DC type 1-like fate. Pathway enrichment
analysis revealed lysosome, autophagy and inflammation as top pathways enriched throughout the reprogramming process. These pathways play an important part in DC maturation and I have identified candidate genes to address whether these pathways are important for the reprogramming process as well. Pathways that were seen decreased during reprogramming
process involved downregulation of cell division, reflecting the cell cycle status of mature DCs.
In this project, I provide evidence that the reprogramming process of fibroblasts into DCs is conserved between mouse and human based on gene expression analysis at the population and single cell levels. I further identified candidate genes in the lysosome, autophagy and inflammation pathways for future knockout/knockdown to verify their functional importance in DC reprogramming. Collectively this work increases our understanding on DC reprogramming mechanisms providing means to increase efficiency of the generation of human reprogrammed DCs for immunotherapy.

Abstract

Hematopoiesis, the process by which all blood cell types are formed, rely on self- renewing and multipotent hematopoietic stem cells (HSCs) that arise from specialized endothelial cells by a process termed endothelial-to-hematopoietic transition (EHT). The generation of autologous long-term self-renewing HSCs in vitro as replacement therapy for blood disorders has been considered the holy grail of regenerative medicine.
However, to achieve this, it is important to understand the complex genetic program driving HSC emergence during development.
The cellular reprogramming of mouse somatic cells into hematopoietic progenitors can be achieved by the enforced expression of four transcription factors – Gata2, Gfi1b, cFos, and Etv6 – and, it offers a powerful tool to better understand developmental processes and the underlying mechanisms. The induction of hematopoietic progenitors from fibroblasts progresses through hemogenic precursors that are Prom+ Sca+ CD34+ CD45- (PS34CD45-).
In this thesis, mouse placentas have shown to harbor a population with this phenotype that express endothelial and early hematopoietic markers and global gene expression profile of PS34CD45- correlates with
reprogrammed precursors. Upon stromal co-culture, PS34CD45- cells have shown to give rise to all blood cell lineages and engraft primary and secondary immunodeficient mice, establishing direct reprogramming as a model that can recapitulate developmental hematopoiesis.
Here, we also demonstrate that human fibroblasts can be reprogrammed into hemogenic cells by the same transcription factors. Induced cells display dynamic EHT transcriptional programs, generate hematopoietic progeny, express an HSC phenotype and repopulate immunodeficient mice. Mechanistically, GATA2 and GFI1B interact and co-occupy a cohort of targets. This cooperative binding is reflected by the engagement of open
enhancers and promoters, initiating the silencing of fibroblast genes and activating the hemogenic program. However, GATA2 displays dominant and independent targeting activity during the early phases of reprogramming. These findings shed light on the processes controlling HSC specification and support the generation of reprogrammed HSCs for clinical applications.

Summary

In cancer, genetic mutations result in the production of tumor specific antigens that are presented on Major Histocompatibility Complex (MHC) class I in tumor cells surface and by specialized antigen presenting cells (APCs) on MHC-I and II, mediating anticancer immune responses. However, tumor cells escape immune surveillance through mechanisms that include downregulation of MHC molecules at surface. In cancer immunotherapy, current approaches aim to restore tumor immunity, however they are either nonspecific or demand the complex identification of tumor antigens. Cellular reprogramming refers to the concept of changing the identity of cell towards another cell fate. Recently, our group has shown that three Transcription Factors (TFs) induce dendritic cell (DC) fate (professional APCs) in fibroblasts. Therefore, we aim at directly converting tumor cells into APCs with ability to present endogenous antigens, eliciting specific anticancer adaptive immune responses. Here, I have presented a strategy to induce antigen presentation in mouse melanoma and lung cancer cells through expression of DC-specific TFs. Pu.1, Irf8 and Batf3 (PIB) were expressed in 3LL carcinoma and B16 melanoma cell lines through a reprogramming proven lentiviral inducible expression system. The 3 TFs induced MHC molecules and the expression of the DC-specific marker Clec9a in tumor cell lines. Reprogramming efficiency was increased upon transduction with alternative pools of TFs or addition of cytokines essential for DC-development. In B16 and 3LL cells expressing ovalbumin (OVA), DC reprogramming induced OVA antigen presentation on MHC-I context and cohort of MHC-I/OVA+ B16 derived cells express MHC-II at the cell surface. Remarkably, purified induced MHC-II+ B16-OVA and 3LL-OVA cells were able to activate a CD8+T cell hybridoma expressing a T-cell receptor specific to the MHC-I/OVA complex. These data suggest that reprogrammed tumor cells acquired the competence for antigen presentation. Collectively, this study reports a strategy to directly reprogram tumor cells into an APC-like cell that combine DCs’ antigen processing and presenting abilities with the endogenous production of tumor antigens. These findings pave the way to generate an anticancer gene therapy to restore cancer immune surveillance.

Abstract

Cellular reprogramming strategies have highlighted the flexibility of cell fates with the possibility to use cell-type-specific transcription factors (TFs) to convert somatic cells into pluripotency. Direct lineage conversions of one differentiated cell-type into another have also been demonstrated and explored for elucidating cell biology mechanisms and for regenerative medicine purposes. Recently, we have demonstrated that antigen presenting Dendritic cells (DCs) can be reprogrammed into unrelated cell-types by a small combination of TFs. Classically, it is thought that a myeloid DC committed progenitor gives rise to the functionally different DC subsets: conventional DCs (cDCs) are professional Antigen Presenting Cells (APC) driving antigen-specific immune responses; plasmacytoid DCs (pDCs) are professional producers of type I interferons during viral infection. However, the timing and exact mechanisms regulating the divergence of the different subsets during DC development is still to be established.
We have recently identified Irf8, Pu.1 and Batf3 as sufficient and necessary to induce a cDC type 1 fate in fibroblasts. Given the important role of TFs in cell-fate decisions of the different DC subsets, the aim of this study is to investigate DC heterogeneity by fine-tuning the minimal TF network necessary to induce DC fate in order to program pDCs from fibroblasts.
By combining literature review and computational analysis, we have identified 23 pDC-inducing candidate TFs with important roles in pDC specification and restricted pDC expression. We have then validated that in Clec9a reporter mouse, pDCs are labelled with tdTomato fluorescent protein making this model suitable for screening pDC-inducing factor. Then we have transduced Clec9a reporter mouse embryonic fibroblasts (MEFs) with a set of the pDC-inducing TFs using a doxycycline-induced lentiviral system. By sequential individual elimination of each TF, we have identified Irf8 and TF2 as the minimal combination required for reporter activation. Major
histocompatibility complex (MHC) class II molecules’ expression important for DC functionality was also observed to be dependent of Irf8 and TF2. Moreover, our study highlighted the role of TF1 for pDC specification. Whilst not being intrinsically required, when combined with Irf8 and TF2, TF1 increases the expression of pDC- typical surface markers with the generation of tdTomato+ B220+ Bst2+ pDC-like cells by direct reprogramming.
In summary, we provide evidence that Irf8 when combined with TF2 and TF1 kicks-start a pDC program in fibroblasts. These findings provide valuable insights into pDC specification. In the future, the generation of pDCs by direct reprogramming opens avenues for inducing anti-viral immune responses with autologous-engineered cells.

Abstract

Hematopoietic stem cells (HSCs) are able of self-renewal and differentiation into all blood cell lineages. Due to this ability, hematopoietic stem cell transplantation (HSCT) constitutes treatment for a diversity of hematological disorders. Incompatibility between donor and host and the
insufficient number of HSCs obtained for transplantation have limited the success of this cellular therapy. To overcome these limitations, expansion of HSCs in vitro has been explored, but this process is a changeling process as HSCs quickly lose stem cell properties upon expansion. Direct reprogramming mediated by transcription factors (TFs) of somatic cells is opening new routes for regenerative medicine and personalized HSCT. Albeit at low efficiency, combined expression of Gata2, Gfi1b and cFos induces reprogramming of fibroblasts into HSC-like cells providing a novel
alternative to generate patient-specific HSCs. A better understanding of hemogenic reprogramming and the interactions between these three TFs with each other and with other players will provide valuable information to increase the efficiency of the process and to generate transplantable HSCs
that can be used in the clinic.
Here, I have defined Gata2 protein domains required to interact with Gfi1b, cFos and LSD1/CoREST1. Gata2 regulates the expression of the Kdm1a gene that encodes LSD1 and co- immunoprecipitations experiments revealed multiple domains required for the interaction of Gata2 with cFos, Gfi1b and LSD1/CoREST1. The nuclear localization sequence of Gata2 is essential for the interaction with Gfi1b but also with LSD1/CoREST1 in the nucleus. C-terminal zinc finger and the N- terminal zinc finger of Gata2 are important for the interaction with LSD1/CoREST1 and cFos, respectively. cFos also interacts with the C-transactivator domain of Gata2, highlighting multiple
regulatory pathways involved in the establishment of this “hemogenic complex”. Given this cooperation between TFs and LSD1/CoREST1 I hypothesize that this complex may be essential for the hemogenic reprogramming. LSD1 was pharmacological inhibited during hematopoietic
reprogramming into HSC-like cells. Inhibition with two structurally unrelated small molecules led to a drastic decrease of reprogramming efficiency implicating the catalytic function of LSD1/CoREST complex during the hemogenic reprogramming.
Overall, this study identified functional interactions between of Gata2, Gfi1b, cFos and LSD1/CoREST1 and the vital role of this epigenetic regulator during hematopoietic reprogramming and acquisition of the HSC fate. This study paves the way for the regulation of this hemogenic complex bringing high-efficiency hemogenic reprogramming one step closer to clinical translation.

Summary

Hematopoietic stem cell transplantation (HSCT) has been used as a treatment for a variety of haematological disorders, due to the ability of hematopoietic stem cells (HSCs) to self-renew and differentiate into all blood cell lineages. Insufficient number of cells and matching incompatibilities between donors and recipients hinder the broad application of this therapy. Expansion of HSCs has met limited success and additional strategies for the in vitro generation of HSCs are required to overcome transplant-associated limitations. Somatic cell reprogramming mediated by transcription factors (TFs) is opening new avenues for regenerative medicine and allowed the design of new approaches to convert one differentiated cell type directly into another. In the hematopoietic system, direct reprogramming of fibroblasts to HSC-like cells has been shown through ectopic expression of Gata2, Gfi1b and cFos, providing an alternative method to generate patient tailored HSCs in vitro. A better understanding of the mode of action of these three critical TFs during reprogramming is needed in order to increase the efficiency of the process.Here, I have defined potential reprogramming domains of hematopoietic TFs by homologous gene (paralog) and deletion construct substitution during hematopoietic reprogramming. First, paralogs of Gata2, Gfi1b and cFos and Gata2 deletion constructs were cloned into a lentiviral gene delivery system to induce fibroblast cell identity towards the hematopoietic lineage. Secondly, hematopoietic reprogramming efficiency was assessed by hCD34/H2BGFP reporter activation. Interestingly, Gata1 did not substitute Gata2 for hematopoietic reprogramming, despite evidences of overlapping function during hematopoiesis. Notwithstanding, Gfi1b and cFos were partially replaced by their respective paralogs, indicating a determinant role for non-homologous domains of Gata2 during reprogramming. Consistently, hematopoietic reprogramming with Gata2 deletion constructs revealed the requirement of the transactivation domains (TADs), the negative regulatory domain (NRD) and the C-terminal zinc finger (C-ZF) for successful reprogramming. Remarkably, I have also unveiled that Gata2 display mitotic bookmarking activity. This epigenetic feature may be important for the acquisition and maintenance of the HSC fate as well as the inheritance of the reprogrammed cell state to daughter cells. Overall, this study identified functional reprogramming features of Gata2, Gfi1b and cFos and sheds new light on how the HSC fate is acquired and preserved. Hereafter, these reprogramming modules will be critical for the design of enhanced synthetic TFs to increase hematopoietic reprogramming efficiency bringing this technology one step closer to clinical translation.

For a long time, cell development was thought to be a unidirectional process. Cellular identity is acquired and maintained through the expression of lineage specific transcription factors (TFs). Several studies starting in mid 19s that culminated in the complete reversion of a somatic cell to a pluripotent state with defined TFs by Shinya Yamanaka in 2006 demonstrated that cell fate could be altered and modelled at ones will. Direct cell reprogramming began to be seen an alternative strategy for the generation of autologous cells for regenerative medicine. Immune tolerance is an important process of a healthy individual to control unwanted immune responses against the self, food, environmental allergens and innocuous commensal microbiota. Regulatory T cells (Tregs) are one of the evolutive mechanisms the organism has to promote homeostasis. In autoimmune diseases, Tregs are dysfunctional and patients may benefit from a immunotherapy based on these cells. Until now, direct reprogramming of fibroblasts to Tregs has not been attempted.
In this study, we have established a strategy to induce Treg cell fate in fibroblasts. First, literature mining and computational analysis were employed to identify candidate TFs to induce Tregs. Twenty-one candidate TFs were selected based on their restricted gene expression in Tregs and implication in Treg developmental specification and function. Secondly, candidate TFs were individually cloned in a doxycycline-inducible lentiviral vector. Thirteen TFs have been used to transduce transgenic FoxP3-GFP mouse embryonic fibroblasts (MEFs). These cells harbour a Foxp3-GFP fusion protein under the control of the endogenous FoxP3 promoter. This reporter system allowed the inference of 3 candidate TFs critical for inducing FoxP3 and the Treg cell fate.
In addition to FoxP3, induced MEFs were shown to express cluster of differentiation (CD) 4 and the hematopoietic marker CD45.
Altogether, this study reports the direct reprogramming of fibroblasts to Treg cell fate and sheds light on the key players controlling Treg specification. These findings pave the way to generating a personalized immunotherapy employing induced immunosuppressive cells.

Summary

The maintenance of cellular identity relies on stable and complex gene regulatory networks. However, several studies have shown that cell fate can be reversed or modified by defined sets of lineage specific Transcription Factors (TFs). The process of direct cellular reprogramming holds promise for the generation of autologous cells for regenerative medicine. In the immunotherapy field, the use of immune modulatory cells enables the manipulation of patients ‘own immune system to target cancer cells. In this context we aim to apply direct cellular reprogramming for the generation of Dendritic Cells (DCs) as ideal antigen-presenting cells to kick-start adaptive immune responses. Here, TF-mediated direct reprogramming approach was established to generate DCs from fibroblasts. First, we employ a combination of literature mining and computational analysis to identify candidate TFs to induce DC fate in vitro. Candidate TFs were selectively expressed in DC populations in both mice and humans and their disruption caused abnormal adaptive immune phenotypes in mice. This analysis generated 19 candidate TFs with key developmental roles in the DC lineage. We have expressed a set of these TFs using a reprogramming proven Doxycycline-inducible lentivirus in mouse embryonic fibroblasts (MEFs). Employing transgenic MEFs harbouring the DC-specific reporter Clec9a-Cre X R26-stop-Tomato, a minimal combination of 4 TFs was identified. This set of 4 TFs activated the DC-specific reporter and generated tdTomato+ cells. TdTomato+ cells acquired DC-like morphology with increased size and complexity. Moreover, a percentage of tdTomato+ cells expressed Major Histocompatibility Complex (MHC) Class II at the cell surface, a critical molecule for antigen presenting function. Finally, overexpression of the 4TFs in Human Dermal Fibroblasts generated cells with DC-like morphology. These morphological changes emerged with similar timing and efficiency in mouse and Human, supporting species conservation of transcriptional regulators underlying DC commitment. Collectively, DC-like cells were generated via a TF-mediated direct reprogramming approach. The results presented in this study highlight the potential of direct reprogramming to a better understanding of transcriptional events underlying lineage specification and to generate immune modulatory cells for immunotherapy.