Abstracts (complete list) - Wissenschaft Online

Anne Sappok, Anna S. Wenning, Melodie-Jo Wolfs, Tanja Mayer, Bettina Strauß,
Markus Hoth, Eva C. Schwarz
The activation and proliferation of primary human CD4+ Tcells
following focal stimulation
The immunological synapse (IS) is a highly ordered complex of molecules at the contact
area between a T-cell and an antigen presenting cell (APC). The formation of the IS is a
prerequisite for an efficient T-cell immune response. The interaction of T-cell receptor
(TCR)/CD3 complexes with the major histocompatibility complex (MHC) on APC is
central to IS formation. The activation of T-cells is characterized by a sustained Ca2+
influx through Ca2+ release activated Ca2+ (CRAC)/ORAI1 channels. To focally
stimulate primary human CD4+ T-cells, we used anti-CD3- and anti-CD28-antibodies
coated beads. To test if our antibody-coated bead-stimulation mimics the physiological
stimulation with APC, we analyzed the effector status of our bead-stimulated CD4+ Tcells
by the expression pattern of two surface proteins CD25 (interleukin-2 receptor)
and CD62L (L-selectin). We observed up-regulation of CD25 and down-regulation of
CD62L over two days, indicating T-cell activation. In addition, we observed a reorganization
of the actin-cytoskeleton at the contact zone between the CD4+ T-cells
and the antibody-coated beads, which is typical for the formation of an IS between a Tcell
and an APC. We further analyzed IL-2 secretion and proliferation and their Ca2+dependence
following bead-stimulation. To determine the doubling rate of the CD4+ Tcells
under these conditions, we counted the cells over 13 days. Proliferation of CD4+ Tcells
was preceded by an increase of the cells’ volume. From day 9 on, cell number
decreased again, probably because apoptosis and necrosis exceeded proliferation.
SiRNA technology (using Amaxa nucleofector) was established to down regulate several
membrane proteins (e.g. TRPC3 and STIM1) in bead-stimulated CD4+ T-cells. Our
results show that bead stimulation of primary human CD4+ T-cells together with siRNA
technology can be used to analyze physiological functions of membrane proteins during
T-cell activation.