Using Direct Cell Reprogramming to Uncover Plasmacytoid Dendritic Cell Specification Programs and Function
Direct cell reprogramming through enforced expression of transcription factors (TFs) converts somatic cells into functional, differentiated cells of other lineages without transiting through pluripotency. Recently, PU.1, IRF8, and BATF3 were identified as the TF network capable of reprogramming murine and human fibroblasts into conventional dendritic cells type 1 (cDC1s). Dendritic cells (DCs) are a remarkable heterogenous lineage bridging innate and adaptive immunity. In addition to antigen-presenting conventional DCs, plasmacytoid DCs (pDCs) represent a specific subset specialized at producing type I interferons (IFN-I) during the antiviral response. pDCs’ ontogeny and the TF networks required to specify pDC-fate and function remain unclear.
This thesis aims to define the TF codes required to induce functional pDCs from unrelated cell-types, utilizing cell fate reprogramming approaches. We have employed an IRF8- based screening approach, given its key role and high expression in pDCs. 36 candidate TFs were screened with IRF8 using mouse embryonic fibroblasts carrying a Clec9a- based reporter system. Overexpression of IRF8 and SPIB was sufficient to induce reporter activation. By performing a secondary screen, we identified IKZF2, when combined with IRF8 and SPIB, that in addition to maintaining Clec9a activation, increases hCD2-based reporter expression and kick-start expression of hematopoietic and pDC- specific surface markers. Thus these three TFs were identified as the optimal network to induce pDC-fate. Induced pDCs express the surface markers B220, CCR9, Ly6C, and antigen-presentation MHC-II molecules. Functionally, induced cells acquire the ability to secrete IFN-I and inflammatory cytokines including TNF-a, IL-6, CCL5, and CXCL10, in
response to TLR9 triggering.
This study identified a combination of three TFs to induce IFN-I-producing pDCs from a non-related cell type by direct cell reprogramming. This finding provides insight into the unique developmental program and functional properties of pDCs. In the future, these results open the possibility to explore induced pDCs to induce immune responses for immunotherapy.
Reprogramming Human Monocytes Into Type 1 Dendritic Cells
The potential of dendritic cells (DCs) for cancer immunotherapy has been highlighted for decades. Type 1 conventional DCs (cDC1s) gained recent attention due to their superior ability to perform antigen cross- presentation, a critical step for inducing antitumor cytotoxic T cell responses. However, the clinical testing of cDC1s has been hindered by their shortage in peripheral blood. We have previously identified a combination of three transcription factors PU.1, IRF8, and BATF3 able to reprogram mouse and human fibroblast to cDC1-like cells. Here, we show that the same combination of transcription factors can reprogram human monocytes isolated from peripheral blood into cDC1-like cells. Reprogramed monocytes express cDC1-specific surface markers including CLEC9A, CD141, and XCR1, show increased migratory capacity towards chemokine gradients and produce IL-12. Importantly, we show that reprogrammed monocytes acquire antigen cross presentation capacity to efficiently activate cytotoxic CD8+ T cells. This study sets the foundation to generate clinically applicable cDC1-like cells from easily accessible blood cells for DC vaccination.
Evaluating Dendritic Cell Reprogramming in Patient-derived Tumor Cells
Despite tremendous efforts and achieved advances, cancer is still the leading cause of death worldwide, which underlines the need to develop novel curative approaches. In the past decade, we witnessed the rapid development of immunotherapy – novel treatment strategies based on harnessing the patient´s very own immune system to fight cancer. Although immunotherapy revolutionized the treatment in multiple malignancies, including advanced melanoma, in the vast majority of patients the response is very limited.
Recently, the Pereira lab at Lund University identified a combination of three proteins, PU.1, IRF8, and BATF3 (PIB) which were sufficient to generate immunogenic type 1 dendritic cells from mouse fibroblasts via direct cellular reprogramming. These new cells had a transcriptional program and cell morphology resembling conventional DC type 1 (DC1), which are specialized in antigen cross-presentation and initiating cytotoxic T cell responses. Furthermore, preliminary data has shown that the reprogramming towards dendritic cell fate can also be applied in cancer cells directly. Therefore, inducing antigen presentation directly in tumor cells may help to bypass current limitations of other immunotherapies, such as tumor cell heterogenicity, immune evasion, and neoantigen identification.
Now, to support the translational efforts of these findings, the application of dendritic cell reprogramming was tested in patient samples. I investigated the reprogramming efficiency mediated by PIB in cancer samples across several malignancies including breast, lung, bladder, pancreatic, head, and neck carcinomas and melanoma as well as cancer associated fibroblasts. I have further evaluated the global gene expression reprogramming at the single cell level with a focus on DC1- and antigen presentation-specific genes. Overall, I observed that all patient samples underwent significant changes during reprogramming with more than 50% of the cells expressing at least one of the reprogramming markers CD45 or HLA-DR. Particularly, lung carcinoma showed highest reprogramming efficiency where more than 60% of the cells were partially reprogrammed and 15% were fully reprogrammed. Interestingly the reprogramming efficiencies observed from patient samples were higher than cancer cell lines, showing a convergent global switch in the transcriptional program to the DC1 fate at the single cell level. Moreover, I also evaluated the reprogramming efficiency in cancer cells tumor organoids constructed by the forced-floating method. Tumor organoids were reprogrammed in similar patterns as their parent cells cultured through conventional 2D methods. However, in the 3D models, it was observed low transduction efficiency, hinting at the potential need to improve the delivery system of PIB into tissues.
These findings support the application of DC reprogramming in patients across multiple malignancies, thus paving the way for the development of a novel cancer gene therapy approach based on dendritic cell reprogramming.
Generating Dendritic Cells by Direct Cell Reprogramming
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.
Harnessing the Potential of Cell Fate Reprogramming for Cancer Immunotherapy
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.
Inducing Plasmacytoid Dendritic Like-Cells with Cell Reprogramming
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.
Reprogramming Lung Carcinoma Cells Into Antigen Presenting Cells
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.
miRNA-Directed Dendritic Cell Reprogramming
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.
Understanding the Genetic Program of Conventional Dendritic Cells Type 2 with Direct Cell Reprogramming
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.
Elucidating Gata2 Transcription Factor Role During DNA Replication and Epigenetic Inheritance
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.