Two-photon microscopy, together with the use of genetically encoded fluorescent proteins (e.g. GFP), has made it possible to directly visualize cellular behavior in 3- dimensional living tissues. We have been using this approach to investigate thymocyte migration and interactions with thymic stromal cells, and how these events relate to positive and negative selection of the T cell antigen receptor (TCR) repertoire. In the previous funding period we made the unexpected observation that many thymocytes undergoing negative selection do not rapidly arrest and die, but rather adopt a highly confined migration pattern while make repeated, transient contacts with dendritic cells surrounding confinement zones.
In Aim 1 we plan to 1) determine the anatomical features of confinement zones for auto-reactive medullary thymocytes, 2) test the hypothesis that a large proportion of polyclonal medullary thymocytes are undergoing confined migration as the result of negative selection, 3) explore the role of AIRE in establishing anatomical niches for negative selection and, 4) determine how the abundance of negative selecting ligands affects the behavior of auto-reactive thymocytes during negative selection.
In Aim 2, we plan to investigate the relationship between migration, chemokines and TCR repertoire selection by 1) examining the balance between thymocyte migration and TCR signaling during different modes of thymocyte selection, 2) examining the impact of modulating chemokine gradients on thymocyte-stromal cell interactions and thymocyte selection, and 3) investigating the relationship between altered chemokine response and impaired positive selection in thymocytes lacking the adaptor protein, GIT-2. By providing insight into the mechanisms that generate mature T cells that are functional and self-tolerant, these studies may lead to improved treatments for autoimmunity, immunodeficiency, and improve our ability to manipulate immune response to tumors or pathogens.

Public Health Relevance

T cells orchestrate immune responses, and defects in T cell development in the thymus can lead to auto-immunity or immunodeficiency. We are using advanced microscopy techniques to directly visualize the process of T cell development in the thymus, and to examine the impact of mutations and other manipulations that lead to defective T cell development. These studies should provide insights into how the migration of developing T cells and their interactions with support cells in the thymus leads to the formation of an appropriately regulated immune response, and thus may lead to improved treatments for a variety of immune-system related diseases and improve our ability to manipulate immune response to tumors or pathogens.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Special Emphasis Panel (ZRG1-IMM-E (02))
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Prabhudas, Mercy R
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University of California Berkeley
Schools of Arts and Sciences
United States
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Melichar, Heather J; Ross, Jenny O; Taylor, Kayleigh T et al. (2015) Stable interactions and sustained TCR signaling characterize thymocyte-thymocyte interactions that support negative selection. J Immunol 194:1057-61
Au-Yeung, Byron B; Melichar, Heather J; Ross, Jenny O et al. (2014) Quantitative and temporal requirements revealed for Zap70 catalytic activity during T cell development. Nat Immunol 15:687-94
Ross, Jenny O; Melichar, Heather J; Au-Yeung, Byron B et al. (2014) Distinct phases in the positive selection of CD8+ T cells distinguished by intrathymic migration and T-cell receptor signaling patterns. Proc Natl Acad Sci U S A 111:E2550-8
Melichar, Heather J; Ross, Jenny O; Herzmark, Paul et al. (2013) Distinct temporal patterns of T cell receptor signaling during positive versus negative selection in situ. Sci Signal 6:ra92
Dzhagalov, Ivan Lilyanov; Chen, Katherine Grace; Herzmark, Paul et al. (2013) Elimination of self-reactive T cells in the thymus: a timeline for negative selection. PLoS Biol 11:e1001566
Dzhagalov, Ivan L; Melichar, Heather J; Ross, Jenny O et al. (2012) Two-photon imaging of the immune system. Curr Protoc Cytom Chapter 12:Unit12.26
Melichar, Heather J; Li, Ou; Herzmark, Paul et al. (2011) Quantifying subcellular distribution of fluorescent fusion proteins in cells migrating within tissues. Immunol Cell Biol 89:549-57
Phee, Hyewon; Dzhagalov, Ivan; Mollenauer, Marianne et al. (2010) Regulation of thymocyte positive selection and motility by GIT2. Nat Immunol 11:503-11
Le Borgne, Marie; Ladi, Ena; Dzhagalov, Ivan et al. (2009) The impact of negative selection on thymocyte migration in the medulla. Nat Immunol 10:823-30