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Development of microwell system for the study of homotypic interactions among activating lymphocytes

Development of microwell system for the study of homotypic interactions among activating lymphocytes
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Activation of lymphocytes is an important event to mount robust immune responses against foreign pathogens. While molecules triggering the activation of various subsets of lymphocytes have been identified and detailed signaling pathways have been extensively studied, relatively less attention has been paid how such molecular level events are integrated and coordinated under various physiological contexts. Typically, chemokines released to during lymphocyte activation recruit other lymphocytes, resulting in increase in local density of lymphocytes. High density of activating lymphocytes is likely to induce homotypic interaction among activating lymphocytes. However, homotypic interactions among activating lymphocytes has not been systematically studied up to date due to lack of technology. To address this problem, we first developed lymphocyte-laden microwells that allow control of cell-cell contact among activating lymphocytes. Two different types of microwells were designed and fabricated. In ‘social’ microwells, various numbers of lymphocytes depending on the sizes of microwells were plated while in ‘lonesome’ microwells, only single lymphocytes were plated in individual microwells. Using this lymphocyte-laden microwell and quantitative fluorescence imaging, we systematically studied the roles of contact-mediated interactions among activating lymphocytes on activation, proliferation, and functions of lymphocytes. First, we demonstrated that proliferation and survival of activating T cells are significantly augmented by contact-mediated interactions. Second, we identified roles of 2B4-mediated homotypic interactions among activating natural killer (NK) cells in enhancing cytokine receptions by inducing clustering of cytokine receptors. Finally, we demonstrated that polarization of cytokine receptors toward cell-cell contacts for trans-presentation of interleukin 2 occurred among activating NK cells to further enhance cytokine signaling.
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