The goal of this research effort is to understand how various types of white blood cells recognize and respond to the presence of a microorganism or cancer cell in the body, or inappropriately recognize a normal component of the body (an auto-antigen). Our experiments are designed to provide a detailed understanding of how the substances (antigens) making up these microorganisms, cancer cells, or normal self-components, are made visible to the defending cells of the innate (anti-unspecific) or adaptive (antigen-specific) limbs of the host defense system cells and how recognition in infections or malignant cells is linked through complex cell-cell interactions to the induction of protective or self-destruction effector responses. Our previous work has described the events within a cell that bring together the antigen and MHC molecule and the cellular distribution of antigenic complexes within the body (antigen processing and presentation). We are now conducting studies primarily at the cell and tissue level to relate the physiology of antigen recognition to the development of effector function, immune memory, or tolerance and to add to this picture the activities of other cell types such as non-hematopoietic cells like fibroblastic reticular cells (FRCs). During the past year we have used these novel imaging methods in concert with classical cellular immune assays to show that the small adapter protein SAP is essential for sustained interaction of antigen-specific T and B-cells and that such sustained binding is needed to produce robust B cell clonal expansion and the development of germinal centers. Surprisingly, the same molecule is unnecessary for effective T cell interaction with dendritic cells, revealing an unexpected dichotomy in the molecular control of antigen-dependent cell-cell interactions involving T cells. These studies also revealed that entry of activated T cells (follicular helper cells) into germinal centers is dependent on antigen-specific interactions with B cells, and that this interaction process is also dependent on SAP expression in the T cell. These findings have provided a new level of insight into molecular control of T cell dependent humoral immune responses of the type that are critical for effective vaccine responses. In a separate study, we investigated the role of signaling through a family of receptors involved in detecting the presence of micro-organisms in the body (TLRs) in the production of allergic inflammation in the lung. These new studies built on our previous report in the small bowel showing that Tars expressed on epithelial cells played a critical role in signaling to underlying dendritic cells for the sampling and capture of luminal bacteria. In the present work, we showed that there was a similar critical dependence of allergic inflammatory (asthma-like) responses in the lung on epithelial cell expression of TLR4 in both a simplified model of airway allergy and also in response to a well-known natural allergen from house dust mites. In a third major effort, we employed a combination of intravital imaging, in vitro migration analysis, and flow cytometry, along with micro-CT and related tools for assessing bone structure, to explore the role of the lipid-signaling pathway involving S1P and tits receptor S1P1. Osteoclasts are large multinucleate myeloid cells responsible for bone resorbtion. They form by fusion of bone surface-adherent osteoclast precursors that are cells in the monocytoid series. Based on preliminary work performed by Dr. Ishii in Japan, we hypothesized that signaling through the S1P1 receptor exposed to high S1P concentrations in the blood contributed to migration of recently attached monocyte osteoclast precursors from the bone surface to the blood, a process that would limit osteoclastogenesis. Using S1P1 agonists given in vivo to mice, we showed by both live imaging and changes in cell populations as assed by flow cytometry that stimulation of osteoclast precursors via S1P1 limited bone resorbtion and that this change in the rate of osteoclast formation could limit osteoporotic changes induced by oophorectomy (as model of post-menopausal osteoporosis). This study thus provides novel I sight into one pathway with a major role in the balance of bone devotion and destruction and points to a new target for interference with pro-osteoporotic processes. Finally, in collaboration with the Computational Biology Group of the PSIIM, we have developed novel tools for automated analysis of a diverse set of parameters that can be extracted from 4-dimensional imaging data (3D and time) and that typically required labor-intensive manual analysis in the past. These new algorithms have been employed in the analysis of osteoclast-bone interactions, T-B cell interactions during humoral immune responses, and the dynamics of cell movement during germinal center formation, among others. Not only are these automated computational methods time-saving, they reduce observer bias, providing more reliable data. They have been published the protocols and computer scripts are now available for use by other laboratories.
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