Project description:T cell receptor (TCR) engagement triggers T cell responses, yet how TCR-mediated activation is regulated at the plasma membrane remains unclear. Here, we report that deleting the membrane scaffolding protein Flotillin-2 (Flot2) increases T cell antigen sensitivity, resulting in enhanced TCR signaling and effector function to weak TCR stimulation. T cell-specific Flot2-deficient mice exhibited reduced tumor growth and enhanced immunity to infection. Flot2-null CD4+ T cells exhibited increased T helper 1 polarization, proliferation, Nur77 induction, and phosphorylation of ZAP70 and LCK upon weak TCR stimulation, indicating a sensitized TCR-triggering threshold. Single cell-RNA sequencing suggested that Flot2-null CD4+ T cells follow a similar route of activation as wild-type CD4+ T cells but exhibit higher occupancy of a discrete activation state under weak TCR stimulation. Given prior reports that TCR clustering influences sensitivity of T cells to stimuli, we evaluated TCR distribution with super-resolution microscopy. Flot2 ablation increased the number of surface TCR nanoclusters on naïve CD4+ T cells. Collectively, we posit that Flot2 modulates T cell functionality to weak TCR stimulation, at least in part, by regulating surface TCR clustering. Our findings have implications for improving T cell reactivity in diseases with poor antigenicity, such as cancer and chronic infections.
Project description:Targeting tumor-specific neoantigens is promising for cancer immunotherapy, yet their ultra-low expression on tumor cells poses significant challenges for T cell therapies. Here, we found that chimeric antigen receptors (CARs) exhibited 10-100 times lower sensitivity compared to T cell receptors (TCRs) when targeting p53R175H common neoantigen. To enhance CAR functionality, we introduce T cell receptor fusion construct (TRuC) and synthetic TCR and antigen receptor (STAR). Our data demonstrate that STAR, which incorporates TCR-mimic antibody fragments and complete TCR signaling machinery, optimally reproduces antigen sensitivity of TCRs. STAR outperforms both CAR and TRuC in redirecting both CD8+ and CD4+ T cells to recognize HLA class I neoantigens. In vitro, human primary T cells engineered with STAR kill multiple cancer cell lines with low neoantigen density better than CAR-T and TRuC-T cells. In tumor mouse models, STAR-T cells outperform CAR-T and TRuC-T cells in controlling neoantigen-low breast cancer and leukemia. Taken together, our findings highlight severe defects in CAR sensitivity and introduce STAR as a more sensitive synthetic receptor, providing a new framework for T cell-based immunotherapy targeting tumors with low neoantigen density.
Project description:Studies employing antigen-presenting systems at the ensemble or single cell level can provide complementary insights into T-cell activation and signaling. However, synthetic material toolkits that probe T cells at both levels are lacking, although they would be essential in advancing basic immunology and immunotherapy. Here, we develop a biomimetic antigen-presenting system (bAPS) using hexapod heterostructures (i.e., a sub-micrometer hematite core, and nanostructured silica branches with diverse surface modifications) for single-cell, single-molecule stimulation and ensemble modulation of T-cell signaling. We report with single molecule resolution of: T-cell sensing/activation by a single agonist peptide-major histocompatibility complex; distinct T-cell receptor (TCR) responses to discriminate structurally similar peptides that differ by just one amino acid; and the superior sensitivity of TCR antigen recognition compared to chimeric antigen receptors (CARs). We also show that magnetic field-induced piconewton×micrometer-level torques on the hexapods amplify immune responses in suspension T and CAR-T cells. Furthermore, by using our bAPS, we have developed a reliable and high-throughput new method for identifying neoantigen-specific TCRs at the single-cell level. The multimodal hexapod bAPS presents an unexplored, nanotechnology-based biointerface tool for investigating recognition, signaling, and the biochemical/mechanical dual sensitivity of T cells and beyond.
Project description:YH29407 with anti-PD-1 ameliorates anti-tumor effects via increased T cell functionality and antigen presenting machinery in the tumor microenvironment
Project description:Adoptive transfer of immune cells expressing chimeric antigen receptors (CARs) is an effective therapy for B-lineage malignancies. However, most patients will relapse and this therapeutic has yet to show strong efficacy in other hematologic or solid tumors. One opportunity for improvement lies in the ability to select or generate T cells that have the highest potential for potent anti-tumor responses and T cell persistence. Here, we dissect the biology of CD8+ CAR T cells by controlling whether the T cell has encountered cognate TCR antigen prior to CAR generation. We find that prior antigen experience influences multiple aspects of in vitro and in vivo CAR T cell functionality, boosting effector function and leukemia clearance in the setting of limiting target antigen density. However, this comes at the expense of proliferative capacity, resistance to dysfunction, and clearance of wildtype leukemia in the setting of limiting CAR+ cell dose. Epigenetic and transcriptomic comparisons of these cell populations uncover that modulation of the Runx2 transcription factor differentially impacts CAR T cell functionality depending on prior cell state. Collectively, our data demonstrate that prior antigen experience status determines functional attributes of a CAR T cell, as well as amenability to functional enhancement by transcription factor modulation.
Project description:Adoptive transfer of immune cells expressing chimeric antigen receptors (CARs) is an effective therapy for B-lineage malignancies. However, most patients will relapse and this therapeutic has yet to show strong efficacy in other hematologic or solid tumors. One opportunity for improvement lies in the ability to select or generate T cells that have the highest potential for potent anti-tumor responses and T cell persistence. Here, we dissect the biology of CD8+ CAR T cells by controlling whether the T cell has encountered cognate TCR antigen prior to CAR generation. We find that prior antigen experience influences multiple aspects of in vitro and in vivo CAR T cell functionality, boosting effector function and leukemia clearance in the setting of limiting target antigen density. However, this comes at the expense of proliferative capacity, resistance to dysfunction, and clearance of wildtype leukemia in the setting of limiting CAR+ cell dose. Epigenetic and transcriptomic comparisons of these cell populations uncover that modulation of the Runx2 transcription factor differentially impacts CAR T cell functionality depending on prior cell state. Collectively, our data demonstrate that prior antigen experience status determines functional attributes of a CAR T cell, as well as amenability to functional enhancement by transcription factor modulation.
Project description:Adoptive transfer of immune cells expressing chimeric antigen receptors (CARs) is an effective therapy for B-lineage malignancies. However, most patients will relapse and this therapeutic has yet to show strong efficacy in other hematologic or solid tumors. One opportunity for improvement lies in the ability to select or generate T cells that have the highest potential for potent anti-tumor responses and T cell persistence. Here, we dissect the biology of CD8+ CAR T cells by controlling whether the T cell has encountered cognate TCR antigen prior to CAR generation. We find that prior antigen experience influences multiple aspects of in vitro and in vivo CAR T cell functionality, boosting effector function and leukemia clearance in the setting of limiting target antigen density. However, this comes at the expense of proliferative capacity, resistance to dysfunction, and clearance of wildtype leukemia in the setting of limiting CAR+ cell dose. Epigenetic and transcriptomic comparisons of these cell populations uncover that modulation of the Runx2 transcription factor differentially impacts CAR T cell functionality depending on prior cell state. Collectively, our data demonstrate that prior antigen experience status determines functional attributes of a CAR T cell, as well as amenability to functional enhancement by transcription factor modulation.
Project description:Adoptive transfer of immune cells expressing chimeric antigen receptors (CARs) is an effective therapy for B-lineage malignancies. However, most patients will relapse and this therapeutic has yet to show strong efficacy in other hematologic or solid tumors. One opportunity for improvement lies in the ability to select or generate T cells that have the highest potential for potent anti-tumor responses and T cell persistence. Here, we dissect the biology of CD8+ CAR T cells by controlling whether the T cell has encountered cognate TCR antigen prior to CAR generation. We find that prior antigen experience influences multiple aspects of in vitro and in vivo CAR T cell functionality, boosting effector function and leukemia clearance in the setting of limiting target antigen density. However, this comes at the expense of proliferative capacity, resistance to dysfunction, and clearance of wildtype leukemia in the setting of limiting CAR+ cell dose. Epigenetic and transcriptomic comparisons of these cell populations uncover that modulation of the Runx2 transcription factor differentially impacts CAR T cell functionality depending on prior cell state. Collectively, our data demonstrate that prior antigen experience status determines functional attributes of a CAR T cell, as well as amenability to functional enhancement by transcription factor modulation.