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Open Access Publications from the University of California

Scholarly Works (11 results)

  • Thesis
  • Peer Reviewed
UC San Francisco Electronic Theses and Dissertations (2023)

The immune system persists in a sensitive equilibrium, necessarily avoiding overreaction to illegitimate threats while watchfully waiting for any bona fide harms that might come along. Fortunately, myriad cell extrinsic and intrinsic mechanisms have evolved to fortify this steady state. Amongst these regulatory forces, three distinct yet interdependent signals lie at the heart of immune activation. This first signal – engagement of the B or T cell receptor by cognate antigen – has significant diagnostic and therapeutic potential. After the emergence of SARS-CoV-2 and its associated clinical syndrome, COVID-19, we led a nationwide, multi-disciplinary scientific and clinical team to systematically benchmark numerous serological assays testing for effective “signal 1” responsiveness to SARS-CoV-2-associated antigens. Establishing accuracy of these tests had significant implications on governmental policies relating to the pandemic, as well as efforts to employ these serology tests to understand COVID-19 seroprevalence and protective immunity. Next, we applied computational tools to improve the performance of these serological tests through combinatorial application and companion analyses. Lastly, we followed these studies of serology diagnostics (corresponding to signal 1) with systematic molecular studies of costimulation: the second signal influencing T cell activation. This critical next step is indispensable to immune responsiveness, but the intricacies of regulatory factors influencing each step have yet to be systematically studied. Our labs have pioneered the use of functional genomic screening using CRISPR/Cas9 to thoroughly explore the regulatory landscape of various immunological phenotypes. Thus, we leveraged recent molecular advances to functionally define the cis and trans regulators converging on the human costimulatory locus harboring CD28, CTLA4, and ICOS at scale. This work uncovered gene-, cell subset-, and stimulation-specific cis-regulatory elements (CREs) bound by validated trans regulators to influence costimulatory gene expression. Finally, deep characterization of genomic architecture in the locus defined critical insulator elements reinforcing CRE activation of true targets while also preventing spurious activation of flanking genes. Ultimately, similar demonstrations of collaborative science to study the human immune system whether in the form of fundamental genomic immunology or translational immune diagnostics will aid our collective transition to the next generation of more precise biomedicine.

    Cover page: Functional genomic and profiling approaches to characterize mammalian immunity
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2020)
    Patients with cancer with liver metastasis demonstrate significantly worse outcomes than those without liver metastasis when treated with anti-PD-1 immunotherapy. The mechanism of liver metastases-induced reduction in systemic antitumor immunity is unclear. Using a dual-tumor immunocompetent mouse model, we found that the immune response to tumor antigen presence within the liver led to the systemic suppression of antitumor immunity. The immune suppression was antigen specific and associated with the coordinated activation of regulatory T cells (Tregs) and modulation of intratumoral CD11b+ monocytes. The dysfunctional immune state could not be reversed by anti-PD-1 monotherapy unless Treg cells were depleted (anti-CTLA-4) or destabilized (EZH2 inhibitor). Thus, this study provides a mechanistic understanding and rationale for adding Treg and CD11b+ monocyte targeting agents in combination with anti-PD-1 to treat patients with cancer with liver metastasis.
      Cover page: Regulatory T cell control of systemic immunity and immunotherapy response in liver metastasis
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2024)
      Cis-regulatory elements (CREs) interact with trans regulators to orchestrate gene expression, but how transcriptional regulation is coordinated in multi-gene loci has not been experimentally defined. We sought to characterize the CREs controlling dynamic expression of the adjacent costimulatory genes CD28, CTLA4 and ICOS, encoding regulators of T cell-mediated immunity. Tiling CRISPR interference (CRISPRi) screens in primary human T cells, both conventional and regulatory subsets, uncovered gene-, cell subset- and stimulation-specific CREs. Integration with CRISPR knockout screens and assay for transposase-accessible chromatin with sequencing (ATAC-seq) profiling identified trans regulators influencing chromatin states at specific CRISPRi-responsive elements to control costimulatory gene expression. We then discovered a critical CCCTC-binding factor (CTCF) boundary that reinforces CRE interaction with CTLA4 while also preventing promiscuous activation of CD28. By systematically mapping CREs and associated trans regulators directly in primary human T cell subsets, this work overcomes longstanding experimental limitations to decode context-dependent gene regulatory programs in a complex, multi-gene locus critical to immune homeostasis.
        Cover page: Systematic decoding of cis gene regulation defines context-dependent control of the multi-gene costimulatory receptor locus in human T cellsCreative Commons 'BY' version 4.0 license
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2021)
        Single-cell RNA sequencing (scRNA-seq) of tissues has revealed remarkable heterogeneity of cell types and states but does not provide information on the spatial organization of cells. To better understand how individual cells function within an anatomical space, we developed XYZeq, a workflow that encodes spatial metadata into scRNA-seq libraries. We used XYZeq to profile mouse tumor models to capture spatially barcoded transcriptomes from tens of thousands of cells. Analyses of these data revealed the spatial distribution of distinct cell types and a cell migration-associated transcriptomic program in tumor-associated mesenchymal stem cells (MSCs). Furthermore, we identify localized expression of tumor suppressor genes by MSCs that vary with proximity to the tumor core. We demonstrate that XYZeq can be used to map the transcriptome and spatial localization of individual cells in situ to reveal how cell composition and cell states can be affected by location within complex pathological tissue.
          Cover page: XYZeq: Spatially resolved single-cell RNA sequencing reveals expression heterogeneity in the tumor microenvironment
          • Article
          • Peer Reviewed
          UC San Francisco Previously Published Works (2020)
          Adoptive transfer of genetically modified immune cells holds great promise for cancer immunotherapy. CRISPR knockin targeting can improve cell therapies, but more high-throughput methods are needed to test which knockin gene constructs most potently enhance primary cell functions in vivo. We developed a widely adaptable technology to barcode and track targeted integrations of large non-viral DNA templates and applied it to perform pooled knockin screens in primary human T cells. Pooled knockin of dozens of unique barcoded templates into the T cell receptor (TCR)-locus revealed gene constructs that enhanced fitness in vitro and in vivo. We further developed pooled knockin sequencing (PoKI-seq), combining single-cell transcriptome analysis and pooled knockin screening to measure cell abundance and cell state ex vivo and in vivo. This platform nominated a novel transforming growth factor β (TGF-β) R2-41BB chimeric receptor that improved solid tumor clearance. Pooled knockin screening enables parallelized re-writing of endogenous genetic sequences to accelerate discovery of knockin programs for cell therapies.
            Cover page: Pooled Knockin Targeting for Genome Engineering of Cellular Immunotherapies
            • Article
            • Peer Reviewed
            UC San Francisco Previously Published Works (2023)
            Chronic stimulation can cause T cell dysfunction and limit the efficacy of cellular immunotherapies. Improved methods are required to compare large numbers of synthetic knockin (KI) sequences to reprogram cell functions. Here, we developed modular pooled KI screening (ModPoKI), an adaptable platform for modular construction of DNA KI libraries using barcoded multicistronic adaptors. We built two ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors (SRs). Over 30 ModPoKI screens across human TCR- and CAR-T cells in diverse conditions identified a transcription factor AP4 (TFAP4) construct that enhanced fitness of chronically stimulated CAR-T cells and anti-cancer function in vitro and in vivo. ModPoKI's modularity allowed us to generate an ∼10,000-member library of TF combinations. Non-viral KI of a combined BATF-TFAP4 polycistronic construct enhanced fitness. Overexpressed BATF and TFAP4 co-occupy and regulate key gene targets to reprogram T cell function. ModPoKI facilitates the discovery of complex gene constructs to program cellular functions.
              Cover page: Modular pooled discovery of synthetic knockin sequences to program durable cell therapies
              • Article
              • Peer Reviewed
              UC San Francisco Previously Published Works (2022)
              Decades of work have elucidated cytokine signalling and transcriptional pathways that control T cell differentiation and have led the way to targeted biologic therapies that are effective in a range of autoimmune, allergic and inflammatory diseases. Recent evidence indicates that obesity and metabolic disease can also influence the immune system1-7, although the mechanisms and effects on immunotherapy outcomes remain largely unknown. Here, using two models of atopic dermatitis, we show that lean and obese mice mount markedly different immune responses. Obesity converted the classical type 2 T helper (TH2)-predominant disease associated with atopic dermatitis to a more severe disease with prominent TH17 inflammation. We also observed divergent responses to biologic therapies targeting TH2 cytokines, which robustly protected lean mice but exacerbated disease in obese mice. Single-cell RNA sequencing coupled with genome-wide binding analyses revealed decreased activity of nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) in TH2 cells from obese mice relative to lean mice. Conditional ablation of PPARγ in T cells revealed that PPARγ is required to focus the in vivo TH response towards a TH2-predominant state and prevent aberrant non-TH2 inflammation. Treatment of obese mice with a small-molecule PPARγ agonist limited development of TH17 pathology and unlocked therapeutic responsiveness to targeted anti-TH2 biologic therapies. These studies reveal the effects of obesity on immunological disease and suggest a precision medicine approach to target the immune dysregulation caused by obesity.
                Cover page: Obesity alters pathology and treatment response in inflammatory disease
                • Article
                • Peer Reviewed
                UC San Francisco Previously Published Works (2020)
                Background:Serological tests are crucial tools for assessments of SARS-CoV-2 exposure, infection and potential immunity. Their appropriate use and interpretation require accurate assay performance data. Method:We conducted an evaluation of 10 lateral flow assays (LFAs) and two ELISAs to detect anti-SARS-CoV-2 antibodies. The specimen set comprised 128 plasma or serum samples from 79 symptomatic SARS-CoV-2 RT-PCR-positive individuals; 108 pre-COVID-19 negative controls; and 52 recent samples from individuals who underwent respiratory viral testing but were not diagnosed with Coronavirus Disease 2019 (COVID-19). Samples were blinded and LFA results were interpreted by two independent readers, using a standardized intensity scoring system. Results:Among specimens from SARS-CoV-2 RT-PCR-positive individuals, the percent seropositive increased with time interval, peaking at 81.8-100.0% in samples taken >20 days after symptom onset. Test specificity ranged from 84.3-100.0% in pre-COVID-19 specimens. Specificity was higher when weak LFA bands were considered negative, but this decreased sensitivity. IgM detection was more variable than IgG, and detection was highest when IgM and IgG results were combined. Agreement between ELISAs and LFAs ranged from 75.7-94.8%. No consistent cross-reactivity was observed. Conclusion:Our evaluation showed heterogeneous assay performance. Reader training is key to reliable LFA performance, and can be tailored for survey goals. Informed use of serology will require evaluations covering the full spectrum of SARS-CoV-2 infections, from asymptomatic and mild infection to severe disease, and later convalescence. Well-designed studies to elucidate the mechanisms and serological correlates of protective immunity will be crucial to guide rational clinical and public health policies.
                  Cover page: Test performance evaluation of SARS-CoV-2 serological assays.Creative Commons 'BY' version 4.0 license
                  • Article
                  • Peer Reviewed
                  UC San Francisco Previously Published Works (2020)
                  Background:Serological tests are crucial tools for assessments of SARS-CoV-2 exposure, infection and potential immunity. Their appropriate use and interpretation require accurate assay performance data. Method:We conducted an evaluation of 10 lateral flow assays (LFAs) and two ELISAs to detect anti-SARS-CoV-2 antibodies. The specimen set comprised 128 plasma or serum samples from 79 symptomatic SARS-CoV-2 RT-PCR-positive individuals; 108 pre-COVID-19 negative controls; and 52 recent samples from individuals who underwent respiratory viral testing but were not diagnosed with Coronavirus Disease 2019 (COVID-19). Samples were blinded and LFA results were interpreted by two independent readers, using a standardized intensity scoring system. Results:Among specimens from SARS-CoV-2 RT-PCR-positive individuals, the percent seropositive increased with time interval, peaking at 81.8-100.0% in samples taken >20 days after symptom onset. Test specificity ranged from 84.3-100.0% in pre-COVID-19 specimens. Specificity was higher when weak LFA bands were considered negative, but this decreased sensitivity. IgM detection was more variable than IgG, and detection was highest when IgM and IgG results were combined. Agreement between ELISAs and LFAs ranged from 75.7-94.8%. No consistent cross-reactivity was observed. Conclusion:Our evaluation showed heterogeneous assay performance. Reader training is key to reliable LFA performance, and can be tailored for survey goals. Informed use of serology will require evaluations covering the full spectrum of SARS-CoV-2 infections, from asymptomatic and mild infection to severe disease, and later convalescence. Well-designed studies to elucidate the mechanisms and serological correlates of protective immunity will be crucial to guide rational clinical and public health policies.
                    Cover page: Test performance evaluation of SARS-CoV-2 serological assaysCreative Commons 'BY' version 4.0 license
                    • Article
                    • Peer Reviewed
                    UC San Francisco Previously Published Works (2020)
                    Appropriate use and interpretation of serological tests for assessments of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exposure, infection and potential immunity require accurate data on assay performance. We conducted a head-to-head evaluation of ten point-of-care-style lateral flow assays (LFAs) and two laboratory-based enzyme-linked immunosorbent assays to detect anti-SARS-CoV-2 IgM and IgG antibodies in 5-d time intervals from symptom onset and studied the specificity of each assay in pre-coronavirus disease 2019 specimens. The percent of seropositive individuals increased with time, peaking in the latest time interval tested (>20 d after symptom onset). Test specificity ranged from 84.3% to 100.0% and was predominantly affected by variability in IgM results. LFA specificity could be increased by considering weak bands as negative, but this decreased detection of antibodies (sensitivity) in a subset of SARS-CoV-2 real-time PCR-positive cases. Our results underline the importance of seropositivity threshold determination and reader training for reliable LFA deployment. Although there was no standout serological assay, four tests achieved more than 80% positivity at later time points tested and more than 95% specificity.
                      Cover page: Evaluation of SARS-CoV-2 serology assays reveals a range of test performance
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