Restoring antitumor immunity with dendritic cell reprogramming strategies. Reprogramming cancer cells to antigen-presenting cells

Sammanfattning: 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.