Stanford Advisors

All Publications

  • Gene regulatory network inference from CRISPR perturbations in primary CD4+ T cells elucidates the genomic basis of immune disease. bioRxiv : the preprint server for biology Weinstock, J. S., Arce, M. M., Freimer, J. W., Ota, M., Marson, A., Battle, A., Pritchard, J. K. 2023


    The effects of genetic variation on complex traits act mainly through changes in gene regulation. Although many genetic variants have been linked to target genes in cis, the trans-regulatory cascade mediating their effects remains largely uncharacterized. Mapping trans-regulators based on natural genetic variation, including eQTL mapping, has been challenging due to small effects. Experimental perturbation approaches offer a complementary and powerful approach to mapping trans-regulators. We used CRISPR knockouts of 84 genes in primary CD4+ T cells to perturb an immune cell gene network, targeting both inborn error of immunity (IEI) disease transcription factors (TFs) and background TFs matched in constraint and expression level, but without a known immune disease association. We developed a novel Bayesian structure learning method called Linear Latent Causal Bayes (LLCB) to estimate the gene regulatory network from perturbation data and observed 211 directed edges among the genes which could not be detected in existing CD4+ trans-eQTL data. We used LLCB to characterize the differences between the IEI and background TFs, finding that the gene groups were highly interconnected, but that IEI TFs were much more likely to regulate immune cell specific pathways and immune GWAS genes. We further characterized nine coherent gene programs based on downstream effects of the TFs and linked these modules to regulation of GWAS genes, finding that canonical JAK-STAT family members are regulated by KMT2A, a global epigenetic regulator. These analyses reveal the trans-regulatory cascade from upstream epigenetic regulator to intermediate TFs to downstream effector cytokines and elucidate the logic linking immune GWAS genes to key signaling pathways.

    View details for DOI 10.1101/2023.09.17.557749

    View details for PubMedID 37745614

    View details for PubMedCentralID PMC10516010

  • CRISPR screens decode cancer cell pathways that trigger γδ T cell detection. Nature Mamedov, M. R., Vedova, S., Freimer, J. W., Sahu, A. D., Ramesh, A., Arce, M. M., Meringa, A. D., Ota, M., Chen, P. A., Hanspers, K., Nguyen, V. Q., Takeshima, K. A., Rios, A. C., Pritchard, J. K., Kuball, J., Sebestyen, Z., Adams, E. J., Marson, A. 2023


    γδ T cells are potent anticancer effectors with the potential to target tumours broadly, independent of patient-specific neoantigens or human leukocyte antigen background1-5. γδ T cells can sense conserved cell stress signals prevalent in transformed cells2,3, although the mechanisms behind the targeting of stressed target cells remain poorly characterized. Vγ9Vδ2 T cells-the most abundant subset of human γδ T cells4-recognize a protein complex containing butyrophilin 2A1 (BTN2A1) and BTN3A1 (refs. 6-8), a widely expressed cell surface protein that is activated by phosphoantigens abundantly produced by tumour cells. Here we combined genome-wide CRISPR screens in target cancer cells to identify pathways that regulate γδ T cell killing and BTN3A cell surface expression. The screens showed previously unappreciated multilayered regulation of BTN3A abundance on the cell surface and triggering of γδ T cells through transcription, post-translational modifications and membrane trafficking. In addition, diverse genetic perturbations and inhibitors disrupting metabolic pathways in the cancer cells, particularly ATP-producing processes, were found to alter BTN3A levels. This induction of both BTN3A and BTN2A1 during metabolic crises is dependent on AMP-activated protein kinase (AMPK). Finally, small-molecule activation of AMPK in a cell line model and in patient-derived tumour organoids led to increased expression of the BTN2A1-BTN3A complex and increased Vγ9Vδ2 T cell receptor-mediated killing. This AMPK-dependent mechanism of metabolic stress-induced ligand upregulation deepens our understanding of γδ T cell stress surveillance and suggests new avenues available to enhance γδ T cell anticancer activity.

    View details for DOI 10.1038/s41586-023-06482-x

    View details for PubMedID 37648854

    View details for PubMedCentralID 7614706

  • Multimodal repertoire analysis unveils B cell biology in immune-mediated diseases. Annals of the rheumatic diseases Ota, M., Nakano, M., Nagafuchi, Y., Kobayashi, S., Hatano, H., Yoshida, R., Akutsu, Y., Itamiya, T., Ban, N., Tsuchida, Y., Shoda, H., Yamamoto, K., Ishigaki, K., Okamura, T., Fujio, K. 2023


    OBJECTIVES: Despite the involvement of B cells in the pathogenesis of immune-mediated diseases (IMDs), biological mechanisms underlying their function are scarcely understood. To overcome this gap, here we constructed and investigated a large-scale repertoire catalogue of five B cell subsets of patients with IMDs.METHODS: We mapped B cell receptor regions from RNA sequencing data of sorted B cell subsets. Our dataset consisted of 595 donors under IMDs and health. We characterised the repertoire features from various aspects, including their association with immune cell transcriptomes and clinical features and their response to belimumab treatment.RESULTS: Heavy-chain complementarity-determining region 3 (CDR-H3) length among naive B cells was shortened among autoimmune diseases. Strong negative correlation between interferon signature strength and CDR-H3 length was observed in naive B cells and suggested the role for interferon in premature B cell development. VDJ gene usage was skewed especially in plasmablasts and unswitched-memory B cells of patients with systemic lupus erythematosus (SLE). We developed a scoring system to quantify this skewing, and it positively correlated with peripheral helper T cell transcriptomic signatures and negatively correlated with the amount of somatic hyper mutations in plasmablasts, suggesting the association of extrafollicular pathway. Further, this skewing led to high usage of IGHV4-34 gene with 9G4 idiotypes in unswitched-memory B cells, which showed a prominent positive correlation with disease activity in SLE. Gene usage skewing in unswitched-memory B cells was ameliorated after belimumab treatment.CONCLUSIONS: Our multimodal repertoire analysis enabled us the system-level understanding of B cell abnormality in diseases.

    View details for DOI 10.1136/ard-2023-224421

    View details for PubMedID 37468219

  • Immunomics analysis of rheumatoid arthritis identified precursor dendritic cells as a key cell subset of treatment resistance ANNALS OF THE RHEUMATIC DISEASES Yamada, S., Nagafuchi, Y., Wang, M., Ota, M., Hatano, H., Takeshima, Y., Okubo, M., Kobayashi, S., Sugimori, Y., Masahiro, N., Yoshida, R., Hanata, N., Suwa, Y., Tsuchida, Y., Iwasaki, Y., Sumitomo, S., Kubo, K., Shimane, K., Setoguchi, K., Azuma, T., Kanda, H., Shoda, H., Zhang, X., Yamamoto, K., Ishigaki, K., Okamura, T., Fujio, K. 2023; 82 (6): 809-819


    Little is known about the immunology underlying variable treatment response in rheumatoid arthritis (RA). We performed large-scale transcriptome analyses of peripheral blood immune cell subsets to identify immune cells that predict treatment resistance.We isolated 18 peripheral blood immune cell subsets of 55 patients with RA requiring addition of new treatment and 39 healthy controls, and performed RNA sequencing. Transcriptome changes in RA and treatment effects were systematically characterised. Association between immune cell gene modules and treatment resistance was evaluated. We validated predictive value of identified parameters for treatment resistance using quantitative PCR (qPCR) and mass cytometric analysis cohorts. We also characterised the identified population by synovial single cell RNA-sequencing analysis.Immune cells of patients with RA were characterised by enhanced interferon and IL6-JAK-STAT3 signalling that demonstrate partial normalisation after treatment. A gene expression module of plasmacytoid dendritic cells (pDC) reflecting the expansion of dendritic cell precursors (pre-DC) exhibited strongest association with treatment resistance. Type I interferon signalling was negatively correlated to pre-DC gene expression. qPCR and mass cytometric analysis in independent cohorts validated that the pre-DC associated gene expression and the proportion of pre-DC were significantly higher before treatment in treatment-resistant patients. A cluster of synovial DCs showed both features of pre-DC and pro-inflammatory conventional DC2s.An increase in pre-DC in peripheral blood predicted RA treatment resistance. Pre-DC could have pathophysiological relevance to RA treatment response.

    View details for DOI 10.1136/ard-2022-223645

    View details for Web of Science ID 000968517000001

    View details for PubMedID 36918189

    View details for PubMedCentralID PMC10314026

  • Dynamic landscape of immune cell-specific gene regulation in immune-mediated diseases. Cell Ota, M., Nagafuchi, Y., Hatano, H., Ishigaki, K., Terao, C., Takeshima, Y., Yanaoka, H., Kobayashi, S., Okubo, M., Shirai, H., Sugimori, Y., Maeda, J., Nakano, M., Yamada, S., Yoshida, R., Tsuchiya, H., Tsuchida, Y., Akizuki, S., Yoshifuji, H., Ohmura, K., Mimori, T., Yoshida, K., Kurosaka, D., Okada, M., Setoguchi, K., Kaneko, H., Ban, N., Yabuki, N., Matsuki, K., Mutoh, H., Oyama, S., Okazaki, M., Tsunoda, H., Iwasaki, Y., Sumitomo, S., Shoda, H., Kochi, Y., Okada, Y., Yamamoto, K., Okamura, T., Fujio, K. 2021; 184 (11): 3006-3021.e17


    Genetic studies have revealed many variant loci that are associated with immune-mediated diseases. To elucidate the disease pathogenesis, it is essential to understand the function of these variants, especially under disease-associated conditions. Here, we performed a large-scale immune cell gene-expression analysis, together with whole-genome sequence analysis. Our dataset consists of 28 distinct immune cell subsets from 337 patients diagnosed with 10 categories of immune-mediated diseases and 79 healthy volunteers. Our dataset captured distinctive gene-expression profiles across immune cell types and diseases. Expression quantitative trait loci (eQTL) analysis revealed dynamic variations of eQTL effects in the context of immunological conditions, as well as cell types. These cell-type-specific and context-dependent eQTLs showed significant enrichment in immune disease-associated genetic variants, and they implicated the disease-relevant cell types, genes, and environment. This atlas deepens our understanding of the immunogenetic functions of disease-associated variants under in vivo disease conditions.

    View details for DOI 10.1016/j.cell.2021.03.056

    View details for PubMedID 33930287