According to the manufacturer, intracellular conversion of these non-florescent compounds to fluorescencent dyes requires esterase activity, thus the dyes stain only viable cells

According to the manufacturer, intracellular conversion of these non-florescent compounds to fluorescencent dyes requires esterase activity, thus the dyes stain only viable cells. thought to be rare [6]. Therefore, most imaging studies that focus on the motility of T cells in the lymphoid organs (during priming or secondary exposure to antigen) employ genetically altered mice. These mice are engineered to express in a large population of their T cells a TCR specific for a single epitope of a model antigen, e.g., ovalbumin (OVA), a male-specific antigen (Dby), or a glycoprotein from lymphocytic choriomeningitis virus (LCMV) [7C13]. Early imaging studies on intact LNs of mice described the motility of na?ve wild-type (WT) and TCR-transgenic (TCR-Tg) T cells as a random walk [7,8]. However, a few hours after injection of cognate antigen, TCR-Tg, but nor WT, T cells reduced their motility and Fissinolide exhibited swarming [7], consistent with prolonged APC-T cell interactions and directed (non-random) movement. Studies focusing on the Rabbit polyclonal to C-EBP-beta.The protein encoded by this intronless gene is a bZIP transcription factor which can bind as a homodimer to certain DNA regulatory regions. kinetics of T cell motility found that priming of TCR-Tg T cells by antigen-loaded DCs was accompanied by three distinct phases in the motile behavior of T cells [9]. During the first 8 hours after entry into the LN, the T cells made only short contacts with DCs, but the length of these interactions and the number of stationary (immotile) T cells increased progressively during the next 12 hours [9]. On the second day (~48 hours), disengagement of the T cells from DCs was observed, followed by rapid, near-random movement [9], suggesting that at this late phase T cells were either undergoing proliferation or searching for exit routes. Studies using a different TCR-Tg system reported that T cells could establish long-lived interactions with APCs very early after antigen injection [14], and they also repeatedly decelerated due to engagement with APCs during all phases of priming [15]. The differences between the TCR-Tg systems and the experimental strategies employed in these studies make it impossible to time-resolve the exact sequence of motility changes or establish a universal model for the motile behavior of T cells during primary or secondary exposure to antigen. However, the consensus emerging from these studies is that encounter with APCs carrying antigen should lead to the arrest of the cognate T cell in order to ensure its activation long-lived interactions with the relevant APCs, and that T cells resume their high motility when they are ready to divide or leave the LN. Our primary goal in the present study was to determine whether antigen-experienced T cells from immunized WT mice could mimic the motile behavior of T cells isolated from na?ve TCR-transgenic mice (reported in other systems) upon exposure to cognate antigen antigen challenge is linked to the initiation of autoimmune arthritis. Thus, the antigen (PG)-induced changes in the motile behavior of these antigen-experienced T cells in the LNs may provide some information about the conditions of T cell activation, ultimately leading to destructive autoimmunity. In the present study, we co-transferred T cells from na?ve and arthritic donor mice into syngeneic na? ve or arthritic recipients, and then challenged the donor cells with antigen (PG) injected into the ankle joints of the recipient mice. Using time-lapse two-photon microscopy [19], we Fissinolide then monitored the motility of transferred T cells in the ankle joint-draining (popliteal) LNs at different time points between 2 and 72 hours after the intra-articular injection of antigen. We asked the following questions: (exposure to antigen? (imaging. Fissinolide As a protein-free control, the same volume of PBS was injected into the.

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