Genomic deletion of Bcl6 differentially affects conventional dendritic cell subsets and compromises Tfh/Tfr/Th17 cell responses.

Conventional dendritic cells (cDC) play key roles in immune induction, but what drives their heterogeneity and functional specialization is still ill-defined. Here we show that cDC-specific deletion of the transcriptional repressor Bcl6 in mice alters the phenotype and transcriptome of cDC1 and cDC2, while their lineage identity is preserved. Bcl6-deficient cDC1 are diminished in the periphery but maintain their ability to cross-present antigen to CD8+ T cells, confirming general maintenance of this subset. Surprisingly, the absence of Bcl6 in cDC causes a complete loss of Notch2-dependent cDC2 in the spleen and intestinal lamina propria. DC-targeted Bcl6-deficient mice induced fewer T follicular helper cells despite a profound impact on T follicular regulatory cells in response to immunization and mounted diminished Th17 immunity to Citrobacter rodentium in the colon. Our findings establish Bcl6 as an essential transcription factor for subsets of cDC and add to our understanding of the transcriptional landscape underlying cDC heterogeneity.

Reprogramming Cancer Cells to Antigen-presenting Cells.

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.

High-Throughput Drug Screening Revealed That Ciclopirox Olamine Can Engender Gastric Cancer Stem-like Cells.

Cancer stem cells (CSCs) are relevant therapeutic targets for cancer treatment. Still, the molecular circuits behind CSC characteristics are not fully understood. The low number of CSCs can sometimes be an obstacle to carrying out assays that explore their properties. Thus, increasing CSC numbers via small molecule-mediated cellular reprogramming appears to be a valid alternative tool. Using the SORE6-GFP reporter system embedded in gastric non-CSCs (SORE6-), we performed a high-throughput image-based drug screen with 1200 small molecules to identify compounds capable of converting SORE6- to SORE6+ (CSCs). Here, we report that the antifungal agent ciclopirox olamine (CPX), a potential candidate for drug repurposing in cancer treatment, is able to reprogram gastric non-CSCs into cancer stem-like cells via activation of SOX2 expression and increased expression of C-MYC, HIF-1α, KLF4, and HMGA1. This reprogramming depends on the CPX concentration and treatment duration. CPX can also induce cellular senescence and the metabolic shift from oxidative phosphorylation (OXPHOS) to glycolysis. We also disclose that the mechanism underlying the cellular reprogramming is similar to that of cobalt chloride (CoCl2), a hypoxia-mimetic agent.

GATA2 mitotic bookmarking is required for definitive haematopoiesis.

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.

Restoring tumor immunogenicity with dendritic cell reprogramming.

Decreased antigen presentation contributes to the ability of cancer cells to evade the immune system. We used the minimal gene regulatory network of type 1 conventional dendritic cells (cDC1) to reprogram cancer cells into professional antigen-presenting cells (tumor-APCs). Enforced expression of the transcription factors PU.1, IRF8, and BATF3 (PIB) was sufficient to induce the cDC1 phenotype in 36 cell lines derived from human and mouse hematological and solid tumors. Within 9 days of reprogramming, tumor-APCs acquired transcriptional and epigenetic programs associated with cDC1 cells. Reprogramming restored the expression of antigen presentation complexes and costimulatory molecules on the surfaces of tumor cells, allowing the presentation of endogenous tumor antigens on MHC-I and facilitating targeted killing by CD8+ T cells. Functionally, tumor-APCs engulfed and processed proteins and dead cells, secreted inflammatory cytokines, and cross-presented antigens to naïve CD8+ T cells. Human primary tumor cells could also be reprogrammed to increase their capability to present antigen and to activate patient-specific tumor-infiltrating lymphocytes. In addition to acquiring improved antigen presentation, tumor-APCs had impaired tumorigenicity in vitro and in vivo. Injection of in vitro generated melanoma-derived tumor-APCs into subcutaneous melanoma tumors delayed tumor growth and increased survival in mice. Antitumor immunity elicited by tumor-APCs was synergistic with immune checkpoint inhibitors. Our approach serves as a platform for the development of immunotherapies that endow cancer cells with the capability to process and present endogenous tumor antigens.

Single-cell transcriptional profiling informs efficient reprogramming of human somatic cells to cross-presenting dendritic cells.

Type 1 conventional dendritic cells (cDC1s) are rare immune cells critical for the induction of antigen-specific cytotoxic CD8+ T cells, although the genetic program driving human cDC1 specification remains largely unexplored. We previously identified PU.1, IRF8, and BATF3 transcription factors as sufficient to induce cDC1 fate in mouse fibroblasts, but reprogramming of human somatic cells was limited by low efficiency. Here, we investigated single-cell transcriptional dynamics during human cDC1 reprogramming. Human induced cDC1s (hiDC1s) generated from embryonic fibroblasts gradually acquired a global cDC1 transcriptional profile and expressed antigen presentation signatures, whereas other DC subsets were not induced at the single-cell level during the reprogramming process. We extracted gene modules associated with successful reprogramming and identified inflammatory signaling and the cDC1-inducing transcription factor network as key drivers of the process. Combining IFN-γ, IFN-ß, and TNF-α with constitutive expression of cDC1-inducing transcription factors led to improvement of reprogramming efficiency by 190-fold. hiDC1s engulfed dead cells, secreted inflammatory cytokines, and performed antigen cross-presentation, key cDC1 functions. This approach allowed efficient hiDC1 generation from adult fibroblasts and mesenchymal stromal cells. Mechanistically, PU.1 showed dominant and independent chromatin targeting at early phases of reprogramming, recruiting IRF8 and BATF3 to shared binding sites. The cooperative binding at open enhancers and promoters led to silencing of fibroblast genes and activation of a cDC1 program. These findings provide mechanistic insights into human cDC1 specification and reprogramming and represent a platform for generating patient-tailored cDC1s, a long-sought DC subset for vaccination strategies in cancer immunotherapy.