Multiscale Analysis of Immune Responses

National Institute Of Allergy And Infectious Diseases

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Abstract

To extend our Histo-cytometry we have moved in two directions. First, we have developed a method (IBEX) that permits performing rapid iterative 6-10 color fluorescent immunohistochemistry on sections to attain images with >60 markers and have developed novel methods 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. This is simpler than existing methods, produces superior transparency, preserves fluorescent protein signals, and also allows multiplex staining with diverse fluorochrome labeled antibodies. We have developed methods that permit detection of the oligo-conjugated antibodies used in the CITE-seq technique, facilitating direct correlation of the imaging data with RNA seq studies, while also providing an alternative to our iterative bleaching method that is especially suited to volume imaging. 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. These LBS-developed imaging technologies methods (Histo-cytometry, IBEX, Ce3D) 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 also changes dramatically when immunotherapy is used. 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, mesothelioma, and lung adenocarcinoma. Using these static imaging tools, we have discovered an unexpectedly asymmetric organization of the immune cells (both myeloid and lymphocytic) in the liver. Kupffer cells, NK T cells, and Trm CD8 T cells all concentrate in the peri-portal region. This distribution is not ontologically controlled, but induced and sustained by MyD88-dependent signaling in response to commensal TLR ligands arriving via the portal circulation. The signaling involves liver sinusoidal endothelial cells that regulate the chemical structure of the extracellular matrix to which chemokines bind, creating gradients controlling localization of the immune cells. This asymmetric distribution contributes to host defense by optimizing bacterial capture and/or residence of effectors at the sites of arrival of blood borne pathogens. The same methods used in this study of liver immune organization were also applied to unraveling the complex subpopulations of myeloid cells found in liver of animals with fat-induced liver damage (NASH). Extending our past work on neutrophil migration in vivo, we identified Grk2 as a unique regulator of the cessation of rapid neutrophil migration in response to sterile injury or bacterial infection. Detailed quantitation studies revealed that this negative regulation of the swarming response played an essential role in promoting effective clearance of bacteria. Cells deficient in Grk2 continued to move rapidly and failed to effectively phagocytize the bacteria present in the central region of an elicited neutrophil swarm. These findings emphasize that rapid migration, while key to collecting innate effectors such as neutrophils at sites of tissue damage or infection, requires local regulation to optimize the migratory behavior of the effector cells when they reach the damage / infected site with respect to clearance of debris or infectious organisms. Finally, we have pursued unexpected findings from an early effort to understand the function of diverse Nod-like receptors (NLR). There are more than 20 such proteins in mice and humans, but only a small subset have well-defined functions. We designed a strategy to probe the unknown molecules functionally that entailed removing the N-terminal auto-inhibitory domain, then expressing the molecule under doxycycline control in suitable host cells, followed by transcriptomic analysis. The loss of the auto-inhibitory domain should permit the expressed molecule to mediate its function, and we expected that several NLRs would induce RNA transitional changes we could categorize and serve as a lead to the physiologic function of the molecule. Although we have not pursued a global effort along these lines for various reasons, we did a control experiment to test this strategy using the well-studied molecule NOD-1. Truncated and full length NOD-1 was expressed under doxycycline control in the human monocyte-macrophage cell line THP-1. RNA analysis surprisingly revealed that independently of induction by doxycycline, all the cells with these constructs had high expression of proto-oncogenes and inflammatory mediators. This was not an artifact similar transduction with other NLRs did not yield this result and CRISPR removal of the NOD-1 insert reversed the transcriptional changes. Careful quantification showed that the tet cassette controlling expression was slightly leaky, resulting in a 1.5 fold over basal NOD-1 level in all the THP-1 cells. Two micro RNAs were shown to normally restrain NOD-1 expression to 1.4 maximum over the resting level and informatic studies showed that NOD-1 is the most tightly regulated intracellular sensor. We interpret these data as resulting from a sol-gel transition effect in which truncated molecules or full length molecules, which transiently become oligomerization competent all the time, achieve a density sufficient to promote the complex formation that underlies NOD-1 signaling in the absence of ligand. These findings emphasize how small changes in protein expression, even less than the 2-3 fold seen with eQTLs that control susceptibility to autoimmune diseases, can result in dramatic functional changes that can subvert normal physiological control. A similar ability of small changes of this magnitude to promote escape from Treg control of autoreactive T cells in the normal repertoire has also been found in other studies in the LBS (see AIxxxx ).