Resistance to immune checkpoint inhibitors (ICI) that activate T cell mediated anti-tumor immunity is a key challenge in cancer therapy, yet the underlying mechanisms remain poorly understood. To further elucidate those, we developed a new approach, Perturb-CITE-seq, for pooled CRISPR perturbation screens with multi-modal RNA and protein single-cell profiling readout and applied it to screen patient-derived autologous melanoma and tumor infiltrating lymphocyte (TIL) co-cultures. We profiled RNA and 20 surface proteins in over 218,000 cells under ~750 perturbations, chosen by their membership in an immune evasion program that is associated with immunotherapy resistance in patients. Our screen recovered clinically-relevant resistance mechanisms concordantly reflected in RNA, protein and perturbation effects on susceptibility to T cell mediated killing. These were organized in eight co-functional modules whose perturbation distinctly affect four co-regulated programs associated with immune evasion. Among these were defects in the IFNγ-JAK/STAT pathway and in antigen presentation, and several novel mechanisms, including loss or downregulation of CD58, a surface protein without known mouse homolog. Leveraging the rich profiles in our screen, we found that loss of CD58 did not compromise MHC protein expression and that CD58 was not transcriptionally induced by the IFNγ pathway, allowing us to distinguish it as a novel mechanism of immune resistance.We further show that loss of CD58 on cancer cells conferred immune evasion across multiple T cell and Natural Killer cell patient co-culture models. Notably, CD58 is downregulated in tumors with resistance to immunotherapy in melanoma patients. Our work identifies novel mechanisms at the nexus of immune evasion and drug resistance and provides a general framework for deciphering complex mechanisms by large-scale perturbation screens with multi-modal singlecell profiles, including in systems consisting of multiple cell types.
Th17 cells are a heterogenous cell population consisting of non-pathogenic Th17 cells (npTh17) that contribute to tissue homeostasis and pathogenic Th17 cells (pTh17) that are potent mediators of tissue inflammation. To reveal regulatory mechanisms underlying Th17 heterogeneity, we performed combined ATAC-seq and RNA-seq and discovered substantial differences in the chromatin landscape of npTh17 and pTh17 cells both in vitro and in vivo. Compared to other CD4+ T cell subsets, npTh17 cells share accessible chromatin programs with Tregs, and pTh17 cells have an intermediate profile spanning features of npTh17 cells and Th1 cells. Integrating single-cell ATAC-seq and single-cell RNA-seq, we inferred self-reinforcing and mutually exclusive regulatory networks controlling the different cell states and predicted transcription factors (TFs) shaping the chromatin landscape of Th17 cell pathogenicity. We validated one novel TF, BACH2, which promotes immunomodulatory npTh17 programs and restrains pro-inflammatory Th1-like programs in Th17 cells and showed genetic evidence for protective variants in the human BACH2 locus associated with multiple sclerosis. Our work uncovered mechanisms that regulate Th17 heterogeneity, revealed shared regulatory programs with other CD4+ T cell subsets, and identified novel drivers of Th17 pathogenicity as potential targets to mitigate autoimmunity.
Resistance to immune checkpoint inhibitors (ICI) that activate T cell mediated anti-tumor immunity is a key challenge in cancer therapy, yet the underlying mechanisms remain poorly understood. To further elucidate those, we developed a new approach, Perturb-CITE-seq, for pooled CRISPR perturbation screens with multi-modal RNA and protein single-cell profiling readout and applied it to screen patient-derived autologous melanoma and tumor infiltrating lymphocyte (TIL) co-cultures. We profiled RNA and 20 surface proteins in over 218,000 cells under ~750 perturbations, chosen by their membership in an immune evasion program that is associated with immunotherapy resistance in patients. Our screen recovered clinically-relevant resistance mechanisms concordantly reflected in RNA, protein and perturbation effects on susceptibility to T cell mediated killing. These were organized in eight co-functional modules whose perturbation distinctly affect four co-regulated programs associated with immune evasion. Among these were defects in the IFNγ-JAK/STAT pathway and in antigen presentation, and several novel mechanisms, including loss or downregulation of CD58, a surface protein without known mouse homolog. Leveraging the rich profiles in our screen, we found that loss of CD58 did not compromise MHC protein expression and that CD58 was not transcriptionally induced by the IFNγ pathway, allowing us to distinguish it as a novel mechanism of immune resistance. We further show that loss of CD58 on cancer cells conferred immune evasion across multiple T cell and Natural Killer cell patient co-culture models. Notably, CD58 is downregulated in tumors with resistance to immunotherapy in melanoma patients. Our work identifies novel mechanisms at the nexus of immune evasion and drug resistance and provides a general framework for deciphering complex mechanisms by large-scale perturbation screens with multi-modal singlecell profiles, including in systems consisting of multiple cell types.
In vitro diagnostics (IVD) have become increasingly popular tool for tackling global health concerns, but conventional IVD tests, such as microscopy and nucleic‐acid amplification methods, are often expensive and complex to use, limiting their use in low‐resource settings. Whole‐cell biosensors in bacterial systems offer an attractive solution as an easy‐to‐use, portable device that non‐specialists could use for clinical purposes outside clinical locations. However, a strategy is needed to engineer ligand specific sensors for new targets. An appealing candidate for a sensing module is the riboswitch. Genetic RNA switches, termed riboswitches, are genetic regulatory elements found in the 5′‐UTR (untranslated region) of some prokaryotic mRNA and contain two functional components: an aptamer binding domain and an expression platform that modulates downstream gene expression. We developed an engineering platform coupling genetic selections and Fluorescence‐Activated Cell Sorting (FACS) screens to identify novel riboswitches from a 108 random‐sequence library in which we replaced the aptamer sequence of the ThiM#2 riboswitch with a degenerate 40‐nucleotide sequence. In a proof‐of‐principle validation of the platform, we identified novel riboswitches for the small molecule theophylline, which are distinct from the existing synthetic theophylline switches. Our best riboswitch hit displays a 2.3‐fold activation in response to theophylline and does not respond to the structurally unrelated molecule thiamine. We performed directed evolution on this riboswitch hit by mutagenizing its sequence and performing additional rounds of selections and screens, resulting in a variant with a 3.6‐fold activation. Current efforts are focused on assessing the efficacy of the selection platform when isolating riboswitches for dopamine. Furthermore, a variant of the engineering platform is being used side‐by‐side to compare if a dopamine riboswitch can be selected from a library composed of an in vitro selected aptamer coupled with a 15‐nucleotide degenerate sequence. We anticipate that these efforts will provide general guidelines toward the development of novel riboswitches for a variety of natural and non‐natural ligands.Support or Funding InformationNSF CBET 1258307Arnold and Mabel Beckman FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
OBJECTIVE The authors created a postoperative postanesthesia care unit (PACU) pathway to bypass routine intensive care unit (ICU) admissions of patients undergoing routine craniotomies, to improve ICU resource utilization and reduce overall hospital costs and lengths of stay while maintaining quality of care and patient satisfaction. In the present study, the authors evaluated this novel PACU-to-floor clinical pathway for a subset of patients undergoing craniotomy with a case time under 5 hours and blood loss under 500 ml. METHODS A single-institution retrospective cohort study was performed to compare 202 patients enrolled in the PACU-to-floor pathway and 193 historical controls who would have met pathway inclusion criteria. The pathway cohort consisted of all adult supratentorial brain tumor cases from the second half of January 2021 to the end of January 2022 that met the study inclusion criteria. Control cases were selected from the beginning of January 2020 to halfway through January 2021. The authors also discuss common themes of similar previously published pathways and the logistical and clinical barriers overcome for successful PACU pathway implementation. RESULTS Pathway enrollees had a median age of 61 years (IQR 49–69 years) and 53% were female. Age, sex, pathology, and American Society of Anesthesiologists physical status distributions were similar between pathway and control patients (p > 0.05). Most of the pathway cases (96%) were performed on weekdays, and 31% had start times before noon. Nineteen percent of pathway patients had 30-day readmissions, most frequently for headache (16%) and syncope (10%), whereas 18% of control patients had 30-day readmissions (p = 0.897). The average time to MRI was 6 hours faster for pathway patients (p < 0.001) and the time to inpatient physical therapy and/or occupational therapy evaluation was 4.1 hours faster (p = 0.046). The average total length of stay was 0.7 days shorter for pathway patients (p = 0.02). A home discharge occurred in 86% of pathway cases compared to 81% of controls (p = 0.225). The average total hospitalization charges were $13,448 lower for pathway patients, representing a 7.4% decrease (p = 0.0012, adjusted model). Seven pathway cases were escalated to the ICU postoperatively because of attending physician preference (2 cases), agitation (1 case), and new postoperative neurological deficits (4 cases), resulting in a 96.5% rate of successful discharge from the pathway. In bypassing the ICU, critical care resource utilization was improved by releasing 0.95 ICU days per patient, or 185 ICU days across the cohort. CONCLUSIONS The featured PACU-to-floor pathway reduces the stay of postoperative craniotomy patients and does not increase the risk of early hospital readmission.
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