| 1. |
|
|
| 2. |
- Ascic, Ervin, et al.
(författare)
-
Eliciting Anti-Tumor Immunity by Reprogramming Cancer Cells to Type 1 Conventional Dendritic Cells
- 2022
-
Konferensbidrag (övrigt vetenskapligt/konstnärligt)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.
|
|
| 3. |
- Ascic, Ervin, et al.
(författare)
-
Harnessing Dendritic Cell Reprogramming to Elucidate Mechanisms of Tumor Immunity
- 2022
-
Konferensbidrag (övrigt vetenskapligt/konstnärligt)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.
|
|
| 4. |
- Barros Ferreira, Alexandra Gabriela
(författare)
-
Restoring antitumor immunity with dendritic cell reprogramming strategies. Reprogramming cancer cells to antigen-presenting cells.
- 2023
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- For the past two decades, immunotherapy revolutionized cancer treatment. However, responses vary significantly among eligible patients and some cancer types are not yet open to immunotherapy. Several mechanisms contribute to immunotherapy resistance, including loss of antigen presentation machinery and immunosuppression. Conventional dendritic cells type 1 (cDC1) are a rare population of professional antigen-presenting cells (APCs) that specialize in recognizing, processing, and cross-presenting antigens to cytotoxic CD8+ T cells and orchestrating complex immune responses. During carcinogenesis, the role of cDC1 is to capture tumor-associated antigens (TAAs) and stimulate effector immune cells to build an immune response against cancer. In cancer patients, cDC1s are dysfunctional or excluded from the tumor microenvironment (TME). Furthermore, cancer cells downregulate key components of antigen presentation pathway, including major histocompatibility complex class I (MHC-I), allowing them to evade immune surveillance. Therefore, there is a need for strategies that counteract cancers’ mechanisms of immune evasion. Cell reprogramming has highlighted the cellular plasticity of somatic cells, while direct lineage conversion promoted the identification of the transcription factor combinations that gatekeep the cell identity for various cell types. Additionally, cell reprogramming products have opened new avenues for regenerative medicine and repair. Cancer cells were shown to be amenable to cell reprogramming strategies; however, previous efforts to reprogram cancer cells aimed at decreasing tumorigenic drive. In this thesis, I leveraged direct cell reprogramming to enhance tumor immunogenicity and overcome major immune evasion mechanisms. In Study I, I participated in the identification of the transcription factors that impose a cDC1-lineage in unrelated cell types, including mouse and human fibroblasts, within 9 days of reprogramming. Overexpression of PU.1, IRF8, and BATF3 led to cDC1-like morphology and induced the expression of hematopoietic marker CD45 and professional APC marker MHC class II (MHC-II). Additionally, combining the expression of PU.1, IRF8, and BATF3 in a polycistronic cassette improved reprogramming efficiency and demonstrated that higher levels of PU.1 are required to initiate reprogramming. In Study II, single-cell RNA and chromatin immunoprecipitation (ChIP)-sequencing informed the cooperation between the cDC1-specific factors to silence the fibroblast program and kickstart the dendritic cell state as early as day 3 of reprogramming. In Studies III and IV, I demonstrated that dendritic cell reprogramming endowed mouse and human cancer cells with professional APCs machinery and function, including the secretion of cytokines (interleukin-12) and chemokines (CXCL10) with important roles in antitumor immune responses. Moreover, tumor-APCs responded to inflammatory stimuli, engulfed dead cells and other exogenous antigens, and primed naïve CD4+ and CD8+ T cells. Importantly, I have also shown that cDC1 reprogramming enhances tumor cells’ immunogenicity by increasing MHC-I molecules and, consequently, the presentation of tumor antigens at the cell surface, leading to higher cytotoxic T cell-mediated cell death in vitro. I also showed that primary cancer cells and cancer-associated fibroblasts (CAFs) are amenable to cDC1 reprogramming. Finally, intratumoral infusion of tumor-APCs in vivo synergized with immune checkpoint inhibitors to delay tumor growth, resulting in increased mice survival. The results presented here show that cDC1 reprogramming enhances antitumor immunity by combining cDC1’s antigen processing and presenting abilities with the endogenous generation of tumor antigens. This thesis lays the groundwork for generating novel immunotherapies based on endowed APC function through direct reprogramming.
|
|
| 5. |
- Ferreira, Alexandra Gabriela, et al.
(författare)
-
Restoring the immunogenicity of cancer cells with dendritic cell reprogramming
- 2021
-
Ingår i: Experimental Hematology. - : Elsevier BV. - 1873-2399 .- 0301-472X. ; 100:Suppl, s. 72-72
-
Konferensbidrag (refereegranskat)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.
|
|
| 6. |
- Ferreira, Alexandra Gabriela, et al.
(författare)
-
Restoring tumor immunogenicity with dendritic cell reprogramming
- 2022
-
Ingår i: Cancer immunology research. - 2326-6074. ; 10:12 suppl
-
Konferensbidrag (refereegranskat)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.
|
|
| 7. |
- Kattge, Jens, et al.
(författare)
-
TRY plant trait database - enhanced coverage and open access
- 2020
-
Ingår i: Global Change Biology. - : Wiley-Blackwell. - 1354-1013 .- 1365-2486. ; 26:1, s. 119-188
-
Tidskriftsartikel (refereegranskat)abstract
- Plant traits-the morphological, anatomical, physiological, biochemical and phenological characteristics of plants-determine how plants respond to environmental factors, affect other trophic levels, and influence ecosystem properties and their benefits and detriments to people. Plant trait data thus represent the basis for a vast area of research spanning from evolutionary biology, community and functional ecology, to biodiversity conservation, ecosystem and landscape management, restoration, biogeography and earth system modelling. Since its foundation in 2007, the TRY database of plant traits has grown continuously. It now provides unprecedented data coverage under an open access data policy and is the main plant trait database used by the research community worldwide. Increasingly, the TRY database also supports new frontiers of trait-based plant research, including the identification of data gaps and the subsequent mobilization or measurement of new data. To support this development, in this article we evaluate the extent of the trait data compiled in TRY and analyse emerging patterns of data coverage and representativeness. Best species coverage is achieved for categorical traits-almost complete coverage for 'plant growth form'. However, most traits relevant for ecology and vegetation modelling are characterized by continuous intraspecific variation and trait-environmental relationships. These traits have to be measured on individual plants in their respective environment. Despite unprecedented data coverage, we observe a humbling lack of completeness and representativeness of these continuous traits in many aspects. We, therefore, conclude that reducing data gaps and biases in the TRY database remains a key challenge and requires a coordinated approach to data mobilization and trait measurements. This can only be achieved in collaboration with other initiatives.
|
|
| 8. |
- Silvério-Alves, Rita, et al.
(författare)
-
GATA2 mitotic bookmarking is required for definitive haematopoiesis
- 2023
-
Ingår i: Nature Communications. - 2041-1723. ; 14:1
-
Tidskriftsartikel (refereegranskat)abstract
- In mitosis, most transcription factors detach from chromatin, but some are retained and bookmark genomic sites. Mitotic bookmarking has been implicated in lineage inheritance, pluripotency and reprogramming. However, the biological significance of this mechanism in vivo remains unclear. Here, we address mitotic retention of the hemogenic factors GATA2, GFI1B and FOS during haematopoietic specification. We show that GATA2 remains bound to chromatin throughout mitosis, in contrast to GFI1B and FOS, via C-terminal zinc finger-mediated DNA binding. GATA2 bookmarks a subset of its interphase targets that are co-enriched for RUNX1 and other regulators of definitive haematopoiesis. Remarkably, homozygous mice harbouring the cyclin B1 mitosis degradation domain upstream Gata2 partially phenocopy knockout mice. Degradation of GATA2 at mitotic exit abolishes definitive haematopoiesis at aorta-gonad-mesonephros, placenta and foetal liver, but does not impair yolk sac haematopoiesis. Our findings implicate GATA2-mediated mitotic bookmarking as critical for definitive haematopoiesis and highlight a dependency on bookmarkers for lineage commitment.
|
|
| 9. |
|
|
