This research investigated the nanostructural alterations in blocking T cell activation using atomic force microscopy (AFM).
CD8+ T cells were isolated from the spleens of C57BL/6 mice by positive selection using CD8a MicroBeads. The isolated cells were activated with anti-mouse CD3 antibody and then treated with anti-mouse CD28 antibody. For blocking the activation, ZAP70 siRNA was transfected to the cells. Nanoscale morphological changes in T cells were investigated using an AFM system. The shape parameters of the cells, including perimeter and volume, were measured from the topographic images using the Scanning Probe Imaging Processor. The average surface roughness is defined as the arithmetic mean of the deviations in height from the mean value
The cell volume of activated CD8+ T cells significantly increased from 12.80 ± 2.44 Μm3 (resting CD8+ T cells without ZAP70 siRNA) to 28.50 ± 7.98 Μm3 (activated CD8+ T cells without ZAP70 siRNA) (p<0.0001, n=10). Additionally, the cell volume increased significantly, from 17.10 ± 8.06 Μm3 (resting with ZAP70 siRNA) to 37.20 ± 12.83 Μm3 (activated CD8+ T cells with ZAP70 siRNA) (p<0.001, n=10). The cell perimeter of activated CD8+ T cells not treated with siRNA was increased over those in the resting state (19.14 ± 2.52 Μm vs 35.70 ± 5.59 Μm, p<0.0001, n=10). With siRNA treatment, there was a significant increase in cell perimeter between the resting and activated state (28.33 ± 9.15 Μm vs 43.03 ± 6.81 Μm, p<0.001, n=10). In resting-state CD8+ T cells, ZAP70 siRNA treatment increased cell perimeter from 19.14 ± 2.52 Μm to 28.33 ± 9.15 Μm (p<0.05, n=10). In addition, ZAP70 siRNA treatment also increased the perimeter of activated CD8+ T cells from 35.70 ± 5.59 Μm to 43.03 ± 6.81 Μm (p<0.05, n=10). In resting CD8+ T cells, ZAP70 siRNA treatment increased the surface roughness, from 21.38 ± 7.74 Μm2 to 59.85 ± 28.81 Μm2 (p<0.0001). ZAP70 siRNA treatment of activated T cells also increased the roughness, from 28.48 ± 14.99 Μm2 to 51.61 ± 19.26 Μm2 (p<0.0001).
The activation of CD8+ T cells results in an increased cell volume and cell perimeter (that is, a more irregular rim shape) over that observed in resting CD8+ T cells. And the surface roughness and cell perimeter of CD8+ T cells increased with ZAP70 siRNA treatment that blocked the activation of CD+8 T cells.
To cite this abstract in AMA style:Ahn H, Kim B, Joo S, Lee Y, Lee G, Park H. Nanostructural Alteration of Blocking Activation of CD8+ T Cells Utilizing Atomic Force Microscopy [abstract]. Am J Transplant. 2013; 13 (suppl 5). https://atcmeetingabstracts.com/abstract/nanostructural-alteration-of-blocking-activation-of-cd8-t-cells-utilizing-atomic-force-microscopy/. Accessed April 10, 2020.
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