Our ability to study characteristic mechanisms and patterns of T cell subset migration during the rejection process is limited by the use of static assay model systems. Also, little is known about live-time patterns of motility that result in recruitment and chemoattraction, as well as patterns that result in chemorepulsion and/or chemoinhibition. Here, we developed a novel microfluidic device that allows for live-time imaging of migrating human T cells and the analysis of directed movement, cell velocity and stop-start motility events of T cells. Human CD3+ T cells were isolated by negative selection from PBMC, and were initially evaluated either unstimulated or following 24hr activation with anti-CD3/anti-CD28 (1mcg/ml). The T cells were introduced into devices and were evaluated while migrating within a maze of 10um channels towards a chemokine gradient, either RANTES or IP-10 (both 100nM), both of which have been well-established in rejection. Patterns of motility within the device (n=14 mazes/device) were observed over 15 hours with time-elapsed video-microscopy, and cells were tracked manually with Image J software (n= 8 devices). We found that unstimulated T cells migrate at high velocity towards IP-10 (147.2 um/hr, n=96 cells) vs. RANTES (101.8 um/hr, n=201 cells),p<0.001. Mitogen-activated T cells migrated at high velocity towards RANTES (157.4 um/hr, n=227 cells) vs. IP-10 (119.3 um/hr, n=203 cells), p<0.001. Also, activated T cells migrated at a slower average velocity towards IP-10 vs. unstimulated cells (p<0.001). Importantly, whereas unstimulated T cells exhibit directed migration towards each chemokine, mitogen-activated T cells were found to exhibit extensive exploration within the device maze (movement in both x and y axes) vs. unstimulated cells (p<0.001) in response to both chemokines. In conclusion, using novel microfluidic technology, we find that activated T cells migrate in an exploratory pattern vs. unstimulated T cells. This difference in migratory pattern implies that targeting patterns of migration in activated T cells will have high potential as novel therapeutics following transplantation.

« Back to 2013 American Transplant Congress