Project description:A synergistic function screening system was established, in which a multiplexed CRIPSR knockout library targeting different checkpoint combinations was constructed and transduced into CD8+T cells to block multiple checkpoints in single cell. Based on this system, we performed a in vivo synergistic function screening of several checkpoints in tumor infiltrating CD8+T cells and find the best combined checkpoint blockade strategy to boost the CD8+T cell-mediated tumor elimination in vivo.
Project description:KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) is highly immunosuppressive and resistant to targeted therapies, immune checkpoint blockade and engineered T cells. Here, we performed a systematic high throughput combinatorial drug screen and identified a synergistic interaction between the MEK inhibitor trametinib and the multi-kinase inhibitor nintedanib. This interaction targets KRAS-directed oncogenic signaling in the aggressive and therapy resistant non-glandular mesenchymal subtype of PDAC, driven by an allelic imbalance, increased gene-dosage and expression of oncogenic KRAS. Mechanistically, the combinatorial treatment induces cell cycle arrest and cell death and initiates an interferon response. Using single cell RNA sequencing and immunophenotyping, we show that the combination therapy reprograms the immunosuppressive microenvironment and primes cytotoxic and memory T cells to infiltrate the tumors, thereby sensitizing mesenchymal PDAC to PD-L1 inhibition. This work opens new avenues to target the therapy refractory mesenchymal PDAC subtype.
Project description:KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) is highly immunosuppressive and resistant to targeted therapies, immune checkpoint blockade and engineered T cells. Here, we performed a systematic high throughput combinatorial drug screen and identified a synergistic interaction between the MEK inhibitor trametinib and the multi-kinase inhibitor nintedanib. This interaction targets KRAS-directed oncogenic signaling in the aggressive and therapy resistant non-glandular mesenchymal subtype of PDAC, driven by an allelic imbalance, increased gene-dosage and expression of oncogenic KRAS. Mechanistically, the combinatorial treatment induces cell cycle arrest and cell death and initiates an interferon response. Using single cell RNA sequencing and immunophenotyping, we show that the combination therapy reprograms the immunosuppressive microenvironment and primes cytotoxic and memory T cells to infiltrate the tumors, thereby sensitizing mesenchymal PDAC to PD-L1 inhibition. This work opens new avenues to target the therapy refractory mesenchymal PDAC subtype.
Project description:TGFBR2 was deleted in ovarian cancer TIL via CRISPR/Cas9 gene editing. Response of unmodified and TGFBR2 knockout TIL to TGF-B stimulation was evaluated via RNA sequencing.
Project description:Significant heterogeneity exists within the tumor infiltrating CD8 T cell population, and exhausted T cells harbor a subpopulation that may be replicating and retain signatures of activation, with potential functional consequences in tumor progression. Dysfunctional immunity in the tumor microenvironment is associated with poor cancer outcomes, making exploration of these exhausted but activated (Tex/act) subpopulations critical to the improvement of therapeutic approaches. To investigate mechanisms associated with Tex/act cells, we sorted and performed transcriptional profiling of CD8+ tumor infiltrating lymphocytes (TIL) coexpressing the exhaustion markers PD-1 and TIM-3, from large volume melanoma tumors. We additionally performed immunologic phenotyping and functional validation, including at the single cell level, to identify potential mechanisms that underlie their dysfunctional phenotype. We identified novel dysregulated pathways in CD8+PD-1+TIM-3+ cells that have not been well studied in TIL; these include bile acid and peroxisome pathway-related metabolism, and mammalian target of rapamycin (mTOR) signaling pathways, which are highly correlated with immune checkpoint receptor expression. Through bioinformatic integration of immunophenotypic data and network analysis, we propose unexpected targets for therapies to rescue the immune response to tumors in melanoma.
Project description:To investigate the importance of DNA damage checkpoint regulation in mammalian preimplantation embryos, we overexpressed wild type and mutant form of CHK1 kinase in mouse zygotes and performed high-throughput RNA-sequencing of zygotes, late 2-cell embryos and blastocysts. Transcriptome analysis revealed that zygotes are particular sensitive to the perturbation of Chk1 governed DNA damage checkpoint. However, once zygotes can overcome DNA damage checkpoint induced cell cycle arrest, they can still have normal development.
Project description:Regulatory T cells (Treg) are conventionally viewed to suppress endogenous and therapy-induced anti-tumor immunity; however, their role in modulating responses to immune checkpoint blockade (ICB) is unclear. In this study, we integrated single-cell RNAseq/TCRseq of >73,000 tumor-infiltrating Treg (TIL-Treg) from anti-PD-1-treated and treatment naive non-small cell lung cancers (NSCLC) with single cell analysis of tumor-associated antigen (TAA)-specific Treg derived from a murine tumor model. We identified 10 subsets of human TIL-Treg, most of which have high concordance with murine TIL-Treg subsets. Notably, only one subset selectively expresses high levels of OX40 and GITR, whose engangement by cognate ligand mediated proliferative programs and NF-kB activation, as well as multiple genes involved in Treg suppression, including LAG3. Functionally, the OX40hiGITRhi subset is the most highly suppressive ex vivo and its higher representation among total TIL-Treg correlated with resistance to PD-1 blockade. Surprisingly, in the murine tumor model, we found that virtually all TIL-Treg expressing T cell receptors that are specific for TAA fully develop a distinct Th1-like signature over a two-week period after entry into the tumor, down-regulating FoxP3 and up-regulating expression of TBX21 (Tbet), IFN and certain pro-inflammatory granzymes. Transfer learning of a gene score from the murine TAA-specific Th1-like Treg subset to the human single-cell dataset revealed a highly analogous subcluster that was enriched in anti-PD-1 responding tumors. These findings demonstrate that TIL-Treg partition into multiple distinct transcriptionally-defined subsets with potentially opposing effects on ICB-induced anti-tumor immunity and suggest that TAA-specific TIL-Treg may positively contribute to anti-tumor responses.