Multiscale Analysis of Immune Responses

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

Abstract

To extend our Histo-cytometry we have moved in two directions over the past several years. First, we have developed a method (IBEX) that permits performing rapid iterative 4-10 color fluorescent immunohistochemistry on sections to attain images with >60 markers and have developed novel methods (RAPID and SPACE) to computationally analyze the complex data emerging from this method. To examine tissue volumes rather than sections, we have developed a novel tissue clearing method called Ce3D and have now extended this approach to multiplex imaging in a new technology called Ce3D-IBEX, achieving up to 25 parameters in tissues as thick as 300u. Most recently, we have improved the pipeline for Ce3D-IBEX, increasing the speed of sample processing using a special microwave and demonstrating that the method is suitable for mouse and humans samples from diverse tissues including lung, intestine, lymph node, spleen, retina, and various tumors, whether fixed frozen or FFPE. Finally, we have incorporated a sensitive enzymatic amplification step into these methods to permit imaging of severely fixed material from BSL4-level infected sources, while adapting all these tools to more rapid imaging using new instruments that incorporate computational clearing. With respect to the problem of autofluorescence in many types of samples we have developed a new software approach called CASPER that computationally removes the autofluorescence if the user has not collected such data for all channels throughout the imaging run. We are also working on new methods for detecting protein-protein interactions at subcellular resolution in our tissue imaging studies, testing various methods that enable a combination of RNA and protein detection in the same sample, and finishing a pipeline for a new type of correlative microscopy that combined 2P dynamic imaging with Ce3D-IBEX to enable many more cell types to be studied dynamically and the relationship of their dynamic and interactive behavior to be mapped to their subsequent cell state. These LBS-developed imaging technologies methods (Histo-cytometry, IBEX, Ce3D, Ce3D-IBEX, RAPID, SPACE, CASPER) are now being employed in multiple distinct mouse tumor models (breast, pancreatic, lung) to explore the detailed spatial organization of the tumor micro-environment and the changes that occur with immunotherapeutic intervention. By examining multiple different tumors in different tissues, we are beginning to develop insights into what aspects of immune cell presence / spatial organization are unique to a particular malignancy and which represent common features across tumor types. Our methods provide a much more comprehensive analysis of the organization of tumor cells, stromal elements, and immune cells than conventional pathology of immunohistochemical methods, and are especially valuable given the disorganized mature of tumors such that single tissue sections or limited parameter analyses fail to reveal larger scale patterns or variations in different regions of the tumor that may be criterial for understanding the differential response among patients to immunotherapeutic interventions. Preliminary data show clear differences in the localization of distinct T cell subsets within various tumors, with CD8 T cells and CD4 T cells (both conventional cells and Tregs) often located quite differently from the CD8 T cells. These patterns also change with administration of checkpoint immunotherapy, and the phenotypic state of the cells is also dramatically altered when various forms of immunotherapy are employed. Similar asymmetric localization of myeloid cell subsets, such as dendritic cells, monocytes, TAM, and so on, is also evident and changes dramatically when immunotherapy is used. Recent studies have shown that combining checkpoint inhibition blockade with agonistic anti-CD40 converts regulatory T cells into IFN- producing Tbet+ effector cells within the tumor, representing a novel way to both reduce immunosuppression and augment effector function. We have also developed a novel method that permits identification of T cell receptor-activated T cells in the polyclonal populations within an inflamed tissue or tumor and the identification of the relevant antigen presenting cell. This method will be especially valuable in human cancer studies. We are now using sophisticated spatial analytic methods, some in hand and others still under development, to better understand these patterns and how they relate to whether immunotherapeutic treatment is effective or not. These studies are not just of material from mouse experimental models, but in the context of the NIAID-NCI Center for Advanced Tissue Imaging (CAT-I), include analysis of samples from humans with various malignancies, including but to limited to follicular lymphoma (FL), ovarian cancer, and lung adenocarcinoma. The work on FL has revealed intriguing relationships between acellular matrix and malignant B cells as well as between mutationally added oligosaccharides on the B cell surface immunoglobulin of the malignant B cells and the lectin DC-SIGN expressed by unusual cells in the follicle. This study also included new methods for spatial analysis of tumor organization and for relating very high dimensional IF imaging to low dimension but larger area examined by conventional multiplex IF used in pathology laboratories. Other uses of IBEX in the field of cancer immunology include a study of responders and non-responders to a combination of checkpoint therapy and PARP inhibition, with preliminary finings suggesting a possible pre-treatment signature that correlates with outcome. Additional study of tumor organization involved the application of a new technology for spatial transcriptomics. Employing the NanoString GTX device, we were able to identify tumor cells and not immune or parenchymal cells as the hosts for intratumor bacteria. This association of bacteria with tumor cells correlated with a high degree of expression of oncogenic b-catenin pathway genes in the tumor, suggesting that bacterial association with tumor cells contributes to their malignant properties. We are completing a collaborative study connected to the Human Cell Atlas that involves creating an in-depth spatial map of the human thymus using both transcriptomic and multiplex imaging methods. Related methods are being employed to map the mouse thymus across development. As part of the development of technology, we contributed to a community effort to specify the best practices in multiplex imaging and are engaged in work on ontologies for immune tissues and cells as well as generation of specified staining panels that will enable easier application of iterative multiplex imaging by more laboratories seeking to use these methods in their research efforts. We also reported on the latest developments in optical imaging as applied to the immune system.