To mount an immune response, T lymphocytes must home from blood into lymph nodes, recognize specific antigens by direct contact with dendritic cells, proliferate, differentiate, exit the lymph node, and migrate to other lymph nodes as memory cells or to tissues as effectors. These processes involve motility, cell recognition dynamics, and Ca2+ signaling - events that were hidden from view until fairly recently. Two-photon (2P) microscopy now permits real-time visualization of living cells deep within lymphoid organs, revealing an elegant cellular choreography under basal conditions and during an immune response. Ion channels in T lymphocytes regulate the triggering, intensity and duration of Ca2+ signaling leading to downstream changes in gene expression and cell proliferation. In particular, a Ca2+ channel (Orai1) and a K+ channel (Kv1.3) are being developed as immunosuppressive targets for treatment of autoimmune disorders. The overall goals of this project are to investigate how the 'default'antigen search strategy of naive T cells is optimized, how tolerogenic B cells and regulatory T cells (Tregs) interfere with naive T cell activation, and how ion channels in T cells can be targeted for in vivo immunosuppression. Studies will be based on 2P imaging of human and murine immune cells in experimental models of the immune response.
In Aim 1, new techniques for long-term cell tracking in intact tissues will be developed, and applied in 3 further subaims: (i) To explore whether T cells are attracted locally and dynamically toward DCs;(ii) Characterization of cellular interactions in a model of B cell-delivered gene therapy by tolerance induction, and (iii) Regulatory T cell suppression of naive T cell activation.
Aim 2 focuses on the in vivo action of Kv1.3 and Orai1, with three subaims: (i) Development of two novel preparations for human cell immunoimaging. (ii) Imaging of effector T cells that cause inflammation and tissue damage in autoimmune disorders during treatment with a specific blocker of Kv1.3 channels in both human and murine models. (iii) Exploration of the functional roles of the Orai1 channel in vivo by specific inhibition of Ca2+ channel activity. Collectively, the proposed experiments probe the molecular and cellular mechanisms that modulate immune responses, and which represent potential therapeutic targets for improved treatment of inflammatory and autoimmune diseases.

Public Health Relevance

Our studies using two-photon microscopy reveal the dynamics of immune cells in vivo during an immune response. The first goal in this project is to understand how cellular interactions in the lymph node are modified by B cell-delivered therapy to induce immunological tolerance and by regulatory T cells that prevent autoimmunity. The second goal is to investigate immunosuppression mediated by blockade of specific ion channels in T lymphocytes - a potassium channel Kv1.3 and a calcium channel Orai1 - for treatment of autoimmune disorders.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Cellular and Molecular Immunology - B Study Section (CMIB)
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Deatherage, James F
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University of California Irvine
Schools of Medicine
United States
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Dong, Tobias X; Othy, Shivashankar; Greenberg, Milton L et al. (2017) Intermittent Ca2+ signals mediated by Orai1 regulate basal T cell motility. Elife 6:
Matheu, Melanie P; Othy, Shivashankar; Greenberg, Milton L et al. (2015) Imaging regulatory T cell dynamics and CTLA4-mediated suppression of T cell priming. Nat Commun 6:6219
Weinger, Jason G; Greenberg, Milton L; Matheu, Melanie P et al. (2015) Two-photon imaging of cellular dynamics in the mouse spinal cord. J Vis Exp :
Greenberg, Milton L; Weinger, Jason G; Matheu, Melanie P et al. (2014) Two-photon imaging of remyelination of spinal cord axons by engrafted neural precursor cells in a viral model of multiple sclerosis. Proc Natl Acad Sci U S A 111:E2349-55
Marro, Brett S; Blanc, Caroline A; Loring, Jeanne F et al. (2014) Promoting remyelination: utilizing a viral model of demyelination to assess cell-based therapies. Expert Rev Neurother 14:1169-79
Matheu, Melanie P; Teijaro, John R; Walsh, Kevin B et al. (2013) Three phases of CD8 T cell response in the lung following H1N1 influenza infection and sphingosine 1 phosphate agonist therapy. PLoS One 8:e58033
Greenberg, Milton L; Yu, Ying; Leverrier, Sabrina et al. (2013) Orai1 function is essential for T cell homing to lymph nodes. J Immunol 190:3197-206
Matheu, Melanie P; Su, Yan; Greenberg, Milton L et al. (2012) Toll-like receptor 4-activated B cells out-compete Toll-like receptor 9-activated B cells to establish peripheral immunological tolerance. Proc Natl Acad Sci U S A 109:E1258-66
Germain, Ronald N; Robey, Ellen A; Cahalan, Michael D (2012) A decade of imaging cellular motility and interaction dynamics in the immune system. Science 336:1676-81
Khorshidi, Mohammad Ali; Vanherberghen, Bruno; Kowalewski, Jacob M et al. (2011) Analysis of transient migration behavior of natural killer cells imaged in situ and in vitro. Integr Biol (Camb) 3:770-8

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