AFM- and NSOM-based force spectroscopy and distribution analysis of CD69 molecules on human CD4+ T cell membrane

Although CD69 is well known as an early T cell-activation marker, the possibility that CD69 are distributed as nano-structures on membrane for immune regulation during T cell activation has not been tested. In this study, nanoscale features of CD69 expression on activated T cells were determined using the atomic force microscopy (AFM) topographic and force-binding nanotechnology as well as near-field scanning optical microscopy (NSOM)-/fluorescence quantum dot (QD)-based nanosacle imaging. Unstimulated CD4(+) T cells showed neglectable numbers of membrane CD69 spots binding to the CD69 Ab-functinalized AFM tip, and no detectable QD-bound CD69 as examined by NSOM/QD-based imaging. In contrast, Phytohemagglutinin (PHA)-activated CD4(+) T cells expressed CD69, and displayed many force-binding spots binding to the CD69 Ab-functionalized AFM tip on about 45% of cell membrane, with mean binding-rupture forces 276 +/- 71 pN. Most CD69 molecules appeared to be expressed as 100-200 nm nanoclusters on the membrane of PHA-activated CD4(+) T cells. Meanwhile, NSOM/QD-based nanoscale imaging showed that CD69 were non-uniformly distributed as 80-200 nm nanoclusters on cell-membrane of PHA-activated CD4(+) T cells. This study represents the first demonstration of the nano-biology of CD69 expression during T cell activation.