All Publications

  • Highly multiplexed tissue imaging using repeated oligonucleotide exchange reaction. European journal of immunology Kennedy-Darling, J. n., Bhate, S. S., Hickey, J. W., Black, S. n., Barlow, G. L., Vazquez, G. n., Venkataraaman, V. G., Samusik, N. n., Goltsev, Y. n., Schürch, C. M., Nolan, G. P. 2021


    Multiparameter tissue imaging enables analysis of cell-cell interactions in situ, the cellular basis for tissue structure, and novel cell types that are spatially restricted, giving clues to biological mechanisms behind tissue homeostasis and disease. Here, we streamlined and simplified the multiplexed imaging method CO-Detection by indEXing (CODEX) by validating 58 unique oligonucleotide barcodes that can be conjugated to antibodies. We showed that barcoded antibodies retained their specificity for staining cognate targets in human tissue. Antibodies were visualized one at a time by adding a fluorescently labeled oligonucleotide complementary to oligonucleotide barcode, imaging, stripping, and repeating this cycle. With this we developed a panel of 46 antibodies that was used to stain five human lymphoid tissues: three tonsils, a spleen, and a lymph node. To analyze the data produced, an image processing and analysis pipeline was developed that enabled single-cell analysis on the data, including unsupervised clustering that revealed 31 cell types across all tissues. We compared cell-type compositions within and directly surrounding follicles from the different lymphoid organs and evaluated cell-cell density correlations. This sequential oligonucleotide exchange technique enables a facile imaging of tissues that leverages pre-existing imaging infrastructure to decrease the barriers to broad use of multiplexed imaging. This article is protected by copyright. All rights reserved.

    View details for DOI 10.1002/eji.202048891

    View details for PubMedID 33548142

  • Cellular neighborhoods predict pembrolizumab response in cutaneous T cell lymphoma Schurch, C. M., Phillips, D. J., Gutierrez, B., Matusiak, M., Bhate, S. S., Barlow, G. L., Fling, S. P., Ramchurren, N., Pierce, R. H., Cheever, M. A., Khodadoust, M. S., West, R., Kim, Y. H., Nolan, G. P. AMER ASSOC CANCER RESEARCH. 2020
  • Combining RNA in situ hybridization and spectral flow cytometry to investigate the leukocyte glycocalyx in autoimmunity Marshall, P. L., Kaber, G., Linde, M. H., Barlow, G. L., Haddock, N. L., Wagar, L., Nagy, N., Bollyky, P. L. AMER ASSOC IMMUNOLOGISTS. 2020
  • Hyaluronan synthesis inhibition impairs antigen presentation and delays transplantation rejection. Matrix biology : journal of the International Society for Matrix Biology Marshall, P. L., Nagy, N. n., Kaber, G. n., Barlow, G. L., Ramesh, A. n., Xie, B. J., Linde, M. H., Haddock, N. L., Lester, C. A., Tran, Q. L., de Vries, C. n., Hargil, A. n., Malkovskiy, A. n., Gurevich, I. n., Martinez, H. A., Kuipers, H. F., Yadava, K. n., Zhang, X. n., Evanko, S. P., Gebe, J. A., Wang, X. n., Vernon, R. B., de la Motte, C. n., Wight, T. N., Engleman, E. G., Krams, S. M., Meyer, E. n., Bollyky, P. L. 2020


    A coat of pericellular hyaluronan surrounds mature dendritic cells (DC) and contributes to cell-cell interactions. We asked whether 4-methylumbelliferone (4MU), an oral inhibitor of HA synthesis, could inhibit antigen presentation. We find that 4MU treatment reduces pericellular hyaluronan, destabilizes interactions between DC and T-cells, and prevents T-cell proliferation in vitro and in vivo. These effects were observed only when 4MU was added prior to initial antigen presentation but not later, consistent with 4MU-mediated inhibition of de novo antigenic responses. Building on these findings, we find that 4MU delays rejection of allogeneic pancreatic islet transplant and allogeneic cardiac transplants in mice and suppresses allogeneic T-cell activation in human mixed lymphocyte reactions. We conclude that 4MU, an approved drug, may have benefit as an adjunctive agent to delay transplantation rejection.

    View details for DOI 10.1016/j.matbio.2020.12.001

    View details for PubMedID 33290836

  • Coordinated Cellular Neighborhoods Orchestrate Antitumoral Immunity at the Colorectal Cancer Invasive Front. Cell Schürch, C. M., Bhate, S. S., Barlow, G. L., Phillips, D. J., Noti, L. n., Zlobec, I. n., Chu, P. n., Black, S. n., Demeter, J. n., McIlwain, D. R., Samusik, N. n., Goltsev, Y. n., Nolan, G. P. 2020


    Antitumoral immunity requires organized, spatially nuanced interactions between components of the immune tumor microenvironment (iTME). Understanding this coordinated behavior in effective versus ineffective tumor control will advance immunotherapies. We re-engineered co-detection by indexing (CODEX) for paraffin-embedded tissue microarrays, enabling simultaneous profiling of 140 tissue regions from 35 advanced-stage colorectal cancer (CRC) patients with 56 protein markers. We identified nine conserved, distinct cellular neighborhoods (CNs)-a collection of components characteristic of the CRC iTME. Enrichment of PD-1+CD4+ T cells only within a granulocyte CN positively correlated with survival in a high-risk patient subset. Coupling of tumor and immune CNs, fragmentation of T cell and macrophage CNs, and disruption of inter-CN communication was associated with inferior outcomes. This study provides a framework for interrogating how complex biological processes, such as antitumoral immunity, occur through concerted actions of cells and spatial domains.

    View details for DOI 10.1016/j.cell.2020.07.005

    View details for PubMedID 32763154

  • Dynamics of the Cutaneous T Cell Lymphoma Microenvironment in Patients Treated with Pembrolizumab Revealed By Highly Multiplexed Tissue Imaging Schuerch, C. M., Phillips, D. J., Bhate, S. S., Barlow, G. L., Fling, S. P., Ramchurren, N., Pierce, R., Cheever, M. A., Khodadoust, M. S., Kim, Y. H., Nolan, G. P. AMER SOC HEMATOLOGY. 2019
  • Dynamics of the Bone Marrow Microenvironment during Leukemic Progression Revealed By Codex Hyper-Parameter Tissue Imaging Schuerch, C., Barlow, G. L., Bhate, S. S., Samusik, N., Nolan, G. P., Goltsev, Y. AMER SOC HEMATOLOGY. 2018
  • 50-dimensional microenvironment analysis of human and mouse bone marrow during malignant transformation Schuerch, C. M., Barlow, G. L., Bhate, S. S., Samusik, N., Nolan, G., Goltsev, Y. NATURE PUBLISHING GROUP. 2018: 550
  • 50-dimensional microenvironment analysis of human and mouse bone marrow during malignant transformation Schuerch, C. M., Barlow, G. L., Bhate, S. S., Samusik, N., Nolan, G., Goltsev, Y. NATURE PUBLISHING GROUP. 2018: 550