| 1. |
- Ascic, Ervin, et al.
(author)
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Eliciting Anti-Tumor Immunity by Reprogramming Cancer Cells to Type 1 Conventional Dendritic Cells
- 2022
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Conference paper (other academic/artistic)abstract
- IntroductionAn important hallmark of cancer is escaping the immune system. Despite advances in immunotherapy, only a subset of patients experiences clinical benefits. It was shown that adoptive T cell or checkpoint inhibition therapy rely on the presence of conventional dendritic cells type 1 (cDC1). cDC1 excel in recruiting and priming protective CD8+ T cells through cross-presentation. However, in tumors cDC1 are often impaired in function. Recently, we demonstrated that overexpression of PU.1, IRF8 and BATF3 (PIB) imposes a cDC1 fate in fibroblasts by direct cell reprogramming. As such, we hypothesise that a similar combination of transcription factors would reprogram cancer cells into tumor-antigen presenting cells (tumor-APCs) and set in motion antigen-specific immunity.Material and Methods30 mouse tumor lines were selected to evaluate reprogramming into tumor-APCs. Reprogramming was induced by overexpression of PIB via lentiviral transduction. The phenotype was profiled by flow cytometry for cDC1 markers CD45, MHC-II, CLEC9A, XCR1 and APC markers MHC-I, CD80/86. Population mRNA-seq was applied to assess transcriptional changes. To assess cDC1 functions, cytokine secretion, cross-presentation and T cell cytotoxicity assays were performed. In vivo, ovalbumin expressing tumors were established and treated by adoptive transfer of tumor-APCs. Tumor growth and animal survival were monitored.Results and DiscussionsUpon transduction with PIB, 26 solid tumor and 4 leukemia lines initiated expression of CD45, MHC-II, at efficiencies ranging from 0.5-57.7%. Reprogramming was accompanied by CLEC9A, XCR1 and MHC-I, CD80/86 upregulation. Transcriptomic analysis of low immunogenic lines B16 and LLC, reveals that PIB overwrites the cancer transcriptome and imposes antigen presentation and cDC1 gene signatures. Importantly, tumor-APCs present endogenous antigens on MHC-I and become prone to T cell mediated killing. Functionally, reprogrammed tumor-APCs secrete inflammatory cytokines such as IL12p70 and strikingly, acquire the ability to crosspresent antigens and prime naïve CD8+ T cells. In vivo, adoptive transfer of cross-presenting tumor-APCs delays tumor growth and extends survival of animals.ConclusionThis approach combines cDC1 antigen presentation abilities with endogenous generation of tumor antigens. The induction of a cDC1 identity in tumor cells sets in motion T cell responses and makes them target for T cell mediated killing. Our study represents a pioneering contribution merging cell reprogramming with immunotherapy.
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| 2. |
- Ascic, Ervin, et al.
(author)
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Harnessing Dendritic Cell Reprogramming to Elucidate Mechanisms of Tumor Immunity
- 2022
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Conference paper (other academic/artistic)abstract
- The presence of conventional dendritic cells type 1 (cDC1) in the tumor correlates with positive treatment outcome. The ability to cross-present neoantigens and prime protective CD8+ T-cell responses, makes cDC1s central for tumor immunity. However, in tumors cDC1 are rare and often functionally impaired. Our group reported that overexpression of the transcription factors PU.1, IRF8 and BATF3 (PIB) converts mouse and human fibroblasts into cross-presenting cDC1-like cells. We employed the minimal gene regulatory network of highly immunogenic cDC1 and restored the immunogenicity of low immunogenic lung cancer and melanoma cell lines by reprogramming into professional tumor antigen presenting cells (tumor-APCs). Here, we report that upon transduction with PIB, 23 solid syngeneic cancer lines initiate reprogramming into cDC1-like cells expressing CD45 and MHC-II at efficiencies ranging from 0.5-57.7%. Functionally, PIB overexpression endows tumor cells with the capacity to cross-present exogenous antigen and prime naïve CD8+ T-cells. Adoptive transfer of ovalbumin cross-presenting B16 tumor-APCs into established ovalbumin expressing B16 tumors (B16-OVA) elicits tumor growth control and extends animal survival. Treated animals show a systemic antigen-specific T cell response against ovalbumin and endogenous tumor-associated antigen MuLV p15E. Intratumoral injection of reprogrammed B2905 and LLC into tumors shows differential response, correlating with their cross-presentation capacity. This approach combines cDC1 antigen cross-presentation abilities with the generation of tumor antigens. The induction of a cDC1 identity in tumor cells sets in motion T cell responses in vitro and in vivo. In the future of this project, dendritic cell reprogramming will be object in a 2-cell CRISPR/Cas9 screen using induced cDC1-like tumor cells and reporter T-cells to explore mechanistically cross-presentation regulators. The generation of cross-presenting tumor-APCs will be also used to map and characterize presented and cross-presented neoantigens. Finally, dendritic cell reprogramming of tumor cells will be explored in vivo by replenishing cDC1 within the tumor microenvironment through in vivo reprogramming. Ultimately, this project will provide insight into mechanisms of cross-presentation and pave the way for the development of novel cDC1-centric therapies.
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| 3. |
- Ferreira, Alexandra G, et al.
(author)
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Reprogramming Cancer Cells to Antigen-presenting Cells
- 2023
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In: Bio-protocol. - 2331-8325. ; 13:22, s. 1-25
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Journal article (peer-reviewed)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.
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| 4. |
- Ferreira, Alexandra Gabriela, et al.
(author)
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Restoring the immunogenicity of cancer cells with dendritic cell reprogramming
- 2021
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In: Experimental Hematology. - : Elsevier BV. - 1873-2399 .- 0301-472X. ; 100:Suppl, s. 72-72
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Conference paper (peer-reviewed)abstract
- An important hallmark of cancer is the ability to evade the immune system. Genetic mutations may result in the accumulation of tumor antigens, however, downregulation of antigen presentation in tumor cells results in decreased immunogenicity and immune surveillance evasion. Recently, we demonstrated that enforced expression of PU.1, IRF8 and BATF3 (PIB) imposes a conventional dendritic cell type 1 (cDC1) fate in fibroblasts by direct cell reprogramming. As such, we hypothesise that a similar combination of transcription factors can reprogram cancer cells into antigen presenting cells.Here, we show that expression of PIB factors is sufficient to induce hematopoietic and cDC1 markers in the mouse melanoma and lung cancer cell lines B16 and 3LL. We further show that reprogramming restores the expression of antigen presentation molecules (MHC-II, MHC-I and B2M) at cancer cell surface. This is accompanied by the activation of the co-stimulatory molecules CD80 and CD86. This reprogrammed tumor antigen presenting cell (tumor-APC) phenotype is specified gradually within the course of 9 days. PIB overwrites the cancer transcriptional program imposing global antigen presentation and cDC1 gene signatures. Functionally, tumor-APCs secrete inflammatory cytokines such as IL-12, IL-6, CXCL10 and type 1 interferons. After reprogramming they also acquire the capacity to uptake and process proteins as well as dead cells. Importantly, tumor-APCs directly prime antigen-specific naïve CD8+ T-cells after antigen loading. Finally, tumor-APCs are capable to show endogenous antigens to T cells and become prone to T cell mediated cell killing.Our approach combines cDC1’s antigen processing and presenting abilities with the endogenous generation of tumor antigens, and serves as a platform for the development of novel immunotherapies based on endowed antigen presentation in cancer cells.
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| 5. |
- Ferreira, Alexandra Gabriela, et al.
(author)
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Restoring tumor immunogenicity with dendritic cell reprogramming
- 2022
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In: Cancer immunology research. - 2326-6074. ; 10:12 suppl
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Conference paper (peer-reviewed)abstract
- Immunotherapy is revolutionizing cancer treatment, but success is limited to a fraction of patients. Tumor immunosurveillance and immunotherapy relies on presentation of tumor-associated antigens by conventional dendritic cells type 1 (cDC1). However, tumors develop mechanisms to avoid immune recognition such as downregulation of antigen presentation and exclusion of cDC1. We have previously demonstrated that enforced expression of the transcription factors PU.1, IRF8 and BATF3 (PIB) imposes the lineage conversion of fibroblasts to cDC1 by direct cell reprogramming. Here, we hypothesize that PIB reprograms cancer cells directly into functional tumor-antigen presenting cells (tumor-APCs) with enhanced immunogenicity. First, we show that enforced expression of PIB in a wide range of murine and human cancer cells from different origins is sufficient to induce surface expression of hematopoietic and DC-lineage specific markers (CD45 and Clec9a). Moreover, reprogramming restored the expression of antigen presentation complexes (MHC-I and MHC-II) and activated the expression of the co-stimulatory molecules CD40, CD80 and CD86, required for productive T cell activation. Transcriptomic analysis using mRNA-sequencing showed that PIB imposes a global cDC1 gene signature and an antigen presentation program in tumor cells as early as day 3 of reprogramming, overriding the original cancer cell program. Furthermore, Assay for Transposase-Accessible Chromatin (ATAC) sequencing analysis revealed that PIB-mediated cDC1 reprogramming elicited rapid epigenetic remodeling followed by gradual rewiring of transcriptional program and stabilization of cDC1 identity. Functionally, tumor-APCs present endogenous antigens on MHC-I, prime naïve CD8+ T and become prone to CD8+ T cell mediated killing. Tumor-APCs secrete pro-inflammatory cytokines (IL-12) and chemoattractants (CXCL10), uptake and process exogenous antigens, phagocyte dead cells, and cross-present exogenous antigens to activate naïve T-cells. In addition, reprogrammed tumor cells harboring TP53, KRAS and PTEN mutations downregulated proliferation and showed impaired tumorigenicity in vitro and in vivo. Importantly, we show that intra-tumoral injection of reprogrammed tumor-APCs elicited tumour growth control in vivo alongside increasing infiltration of CD8+ T and NK cells in B16-OVA tumors. Finally, we showed that our approach can be employed to convert primary cancer cells derived from melanoma, lung, breast, pancreatic, urothelial, and head and neck carcinomas as well as cancer associated fibroblasts. In summary, we provide evidence for the direct reprogramming of tumor cells into immunogenic cDC1-like cells, with restored antigen presentation capacity and the ability to reinstate anti-tumor immunity. Our approach elicits the immune system against cancer and counteract major tumor evasion mechanisms including tumor heterogeneity and impaired antigen presentation, laying the foundation for developing immunotherapeutic strategies based on the cellular reprogramming of human cancer cells.
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| 6. |
- Fiúza Rosa, Fábio, et al.
(author)
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Direct Reprogramming of Mouse and Human Fibroblasts into Conventional Dendritic Cells Type 1
- 2022
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In: Molecular Immunology. - : Elsevier BV. - 1872-9142 .- 0161-5890. ; 150, s. 22-22
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Conference paper (peer-reviewed)abstract
- Cell fate reprogramming of adult cells towards pluripotency or unrelated somatic cell-types has been explored in the context of regenerative medicine. Dendritic cells (DCs) are professional antigen presenting cells (APCs) specialized in the recognition, processing and presentation of antigens to T-cells, inducing adaptive immunity. In particular, the mouse conventional DCs type 1 (cDC1) subset or DC1 human equivalent excel on the ability to perform antigen cross-presentation, a critical step for inducing cytotoxic responses. We hypothesized that the unique properties of cDC1s could be induced in unrelated cell-types, allowing the direct control of immune responses with cell reprogramming.Here, the requirements to induce cDC1s were investigated using combinatorial overexpression of Transcription Factors (TFs) in Clec9a-tdTomato mouse fibroblasts. In the hematopoietic system, Clec9a specifically marks the DC lineage, including all conventional dendritic cells type 1 (cDC1). We have identified PU.1, IRF8 and BATF3 (PIB) as sufficient and necessary to induce Clec9a reporter activation, establish DC morphology and activate a cDC1 transcriptional program in mouse fibroblasts. The over- expression of PIB ignites the expression of DC markers including CD103, XCR1, MHC-I, MHC-II and co-stimulatory molecules. Functionally, Induced DCs (iDCs) secrete inflammatory cytokines and engulf, process, present and cross-present antigens to CD4+ and CD8+ T cells, respectively. Additionally, we have demonstrated that combined expression of PIB factors induces DC1 reprogramming in human fibroblasts. Human iDC1s acquire DC morphology, express DC1 markers, including Clec9a, CD141 and the co-stimulatory molecules CD40, CD80 and CD86, and acquire a DC1 transcriptional signature at the single cell level. Interestingly, DC1 reprogramming efficiency can be enhanced 70-fold by supplementing culture media with inflammatory cytokines, suggesting a regulatory role of inflammation during DC1 reprogramming.Hence, we provide evidence that antigen presentation and cross-presentation can be dynamically programmed by a small combination of TFs. These findings provide insights into cDC1 specification and a platform for developing cancer immunotherapies based on cell reprogramming.
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| 7. |
- Gomes, Andreia M., et al.
(author)
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Cooperative Transcription Factor Induction Mediates Hemogenic Reprogramming
- 2018
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In: Cell Reports. - : Elsevier BV. - 2211-1247.
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Journal article (peer-reviewed)abstract
- During development, hematopoietic stem and progenitor cells (HSPCs) arise from specialized endothelial cells by a process termed endothelial-to-hematopoietic transition (EHT). The genetic program driving human HSPC emergence remains largely unknown. We previously reported that the generation of hemogenic precursor cells from mouse fibroblasts recapitulates developmental hematopoiesis. Here, we 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, possess HSPC cell surface phenotype, and repopulate immunodeficient mice for 3 months. Mechanistically, GATA2 and GFI1B interact and co-occupy a cohort of targets. This cooperative binding is reflected by engagement of open enhancers and promoters, initiating 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 human HSC specification and support generation of reprogrammed HSCs for clinical applications. Gomes et al. show that specification of hemogenesis in human fibroblasts is mediated by cooperative transcription factor binding. GATA2 displays dominance, interacts with GFI1B, and recruits FOS to open chromatin, simultaneously silencing the fibroblast program and initiating an endothelial-to-hematopoietic transition to definitive hematopoiesis.
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| 8. |
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| 9. |
- Pires, Cristiana F., et al.
(author)
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Understanding and Modulating Immunity With Cell Reprogramming
- 2019
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In: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 10
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Research review (peer-reviewed)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.
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| 10. |
- Rosa, Fábio F., et al.
(author)
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Direct reprogramming of fibroblasts into antigen-presenting dendritic cells
- 2018
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In: Science Immunology. - : American Association for the Advancement of Science (AAAS). - 2470-9468. ; 3:30
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Journal article (peer-reviewed)abstract
- Ectopic expression of transcription factors has been used to reprogram differentiated somatic cells toward pluripotency or to directly reprogram them to other somatic cell lineages. This concept has been explored in the context of regenerative medicine. Here, we set out to generate dendritic cells (DCs) capable of presenting antigens from mouse and human fibroblasts. By screening combinations of 18 transcription factors that are expressed in DCs, we have identified PU.1, IRF8, and BATF3 transcription factors as being sufficient to reprogram both mouse and human fibroblasts to induced DCs (iDCs). iDCs acquire a conventional DC type 1-like transcriptional program, with features of interferon-induced maturation. iDCs secrete inflammatory cytokines and have the ability to engulf, process, and present antigens to T cells. Furthermore, we demonstrate that murine iDCs generated here were able to cross-present antigens to CD8+ T cells. Our reprogramming system should facilitate better understanding of DC specification programs and serve as a platform for the development of patient-specific DCs for immunotherapy.
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