Multiscale Analysis of Immune Responses

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications, trials & patents

Abstract

In the past few years, we have developed and then improved upon the IBEX multiplex tissue staining method, enabling rapid iterative 4-10 color fluorescent immunohistochemistry on sections to attain images with >60 markers and have added novel methods to computationally analyze the complex data emerging from this method. A major achievement in this computational arena was the publication of the analytic package SPACE (Spatial Patterning Analysis of Cellular Ensembles ), a tool that enables statically robust identification of cellular clusters of more than the 2 components revealed by other algorithms, along with a capacity to identify cellular gradients that cannot be detected with existing methods. More recently, we have combined our past development of a new tissue clearing method optimized for multiplex direct immunofluorescent 3D tissue imaging (Ce3D) with IBEX to create a new technology called Ce3D-IBEX, achieving up to 36 parameter staining in tissues as thick as 500u. The pipeline for Ce3D-IBEX can take advantage of a special microwave to speed up the sequential steps in staining, clearing, and imaging that are part of this technology and we have applied the method to mouse and human samples from diverse tissues including lung, intestine, lymph node, spleen, retina, and various tumors, whether fixed frozen or FFPE. Using Ce3D-IBEX we have discovered a novel immune structure in the lung consisting of CD4+ and CD8+ T cells and cDC1, clustered near CGPR+ vagal sensory neurons. Germ free animals and TCR transgenic animals lack these structures and they are markedly diminished in animals whose vagal system is poisoned with RTX or subject to vagotomy. We call these small immune cell collections VALT (vagus-associated lymphoid tissue) and are engaged in experiments seeking to define their role in host defense and disease. Initial studies show that cells in VALT rapidly enter proliferative cycle upon influenza virus infection of naïve animals. Since the mice do not have memory T cells for influenza, this raises interesting questions about whether antigen-recognition is involved or if these cells respond to other stimuli such as cytokines (e.g., type 1 interferon) to enter cell cycle and also to make their own effector molecules. We are finishing a pipeline for a new type of correlative microscopy that combines 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, SPACE) 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 pathological examination using 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. These patterns also change with administration of checkpoint blockade immunotherapy, and the phenotypic state of the cells is also dramatically altered when various forms of immunotherapy are employed. 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 new 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 is especially valuable in human cancer studies. These imaging-centric studies do not only utilize 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, colorectal cancer, and lung adenocarcinoma. Analysis of two mouse cancer models this past year has provided novel insights into how CD4+ T-cells can control tumor growth and the signals that enable the sustained production of the stem-like CD8+ memory T cells critical to effective checkpoint blockade immunotherapy. In the first case, we have shown that tumor antigen-responsive CD4+ T-cells can evoke the creation of perivascular myeloid cuffs of myeloid cells that are directed to an ”M1” phenotype and production of TNF via the action of T cell-derived IL-3 and possibly also CSF-2. The TNF does not act directly on the tumor cells but on the pericytes and endothelial cells of the tumor vasculature, leading to vascular leak and death of the neighboring tumor cells. In the second model, we have found that there is prolonged clustering of highly proliferated CD8+ T-cells around XCR1 cDC1 late after tumor implantation and that these cells are predominantly TCF-1+SLAMF6+ stem like memory cells with high PD-1 expression and the highest TCR affinity of all responding cells in the lymph node. Inhibition of the PD-1 pathway leads to loss of this critical population through differentiation or death. Such findings argue that checkpoint blockade therapy will be effective only in the early round(s) of use and that if too few effectors to clear the tumor are generated, the patient will progress. This has major implications for improving use of checkpoint therapy and these findings will be published in Nature. 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 findings of human biopsy samples suggesting a possible pre-treatment signature that correlates with outcome. We completed 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. The work on the human thymus was published in the premier biology journal Nature as part of a large series of such atlasing studies. As part of the development of technology, we contributed to a community effort to specify the best practices in multiplex imaging and worked 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.