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.

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.

Cell Fate Reprogramming in the Era of Cancer Immunotherapy.

Advances in understanding how cancer cells interact with the immune system allowed the development of immunotherapeutic strategies, harnessing patients' immune system to fight cancer. Dendritic cell-based vaccines are being explored to reactivate anti-tumor adaptive immunity. Immune checkpoint inhibitors and chimeric antigen receptor T-cells (CAR T) were however the main approaches that catapulted the therapeutic success of immunotherapy. Despite their success across a broad range of human cancers, many challenges remain for basic understanding and clinical progress as only a minority of patients benefit from immunotherapy. In addition, cellular immunotherapies face important limitations imposed by the availability and quality of immune cells isolated from donors. Cell fate reprogramming is offering interesting alternatives to meet these challenges. Induced pluripotent stem cell (iPSC) technology not only enables studying immune cell specification but also serves as a platform for the differentiation of a myriad of clinically useful immune cells including T-cells, NK cells, or monocytes at scale. Moreover, the utilization of iPSCs allows introduction of genetic modifications and generation of T/NK cells with enhanced anti-tumor properties. Immune cells, such as macrophages and dendritic cells, can also be generated by direct cellular reprogramming employing lineage-specific master regulators bypassing the pluripotent stage. Thus, the cellular reprogramming toolbox is now providing the means to address the potential of patient-tailored immune cell types for cancer immunotherapy. In parallel, development of viral vectors for gene delivery has opened the door for in vivo reprogramming in regenerative medicine, an elegant strategy circumventing the current limitations of in vitro cell manipulation. An analogous paradigm has been recently developed in cancer immunotherapy by the generation of CAR T-cells in vivo. These new ideas on endogenous reprogramming, cross-fertilized from the fields of regenerative medicine and gene therapy, are opening exciting avenues for direct modulation of immune or tumor cells in situ, widening our strategies to remove cancer immunotherapy roadblocks. Here, we review current strategies for cancer immunotherapy, summarize technologies for generation of immune cells by cell fate reprogramming as well as highlight the future potential of inducing these unique cell identities in vivo, providing new and exciting tools for the fast-paced field of cancer immunotherapy.

Direct reprogramming of fibroblasts into antigen-presenting dendritic cells.

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.