Figure 4.

Knock down of Plcγ1 results in impaired CNS infiltration of MCAM+ T-cells in vivo. MCAM expressing murine T-cells were prepared from 2D2 mice, expanded and differentiated in vitro, representative data is shown in (A, B) (n = 3) and plcγ1 expression was knocked down by electroporation and transfer of specific siRNA oligonucleotides (nc, negative control; A, oligonucleotide A; ABC, mixture of oligonucleotide A, B, and C). Knock down efficiency was confirmed by western blotting (C) and quantified (D; siRNA A 24 h p = 0.02, 48 h p = 0.02; siRNA ABC 48 h p = 0.06). The abundance of MCAM+ plcγ1 knock down T-cells was then analyzed in cortex and choroid plexus by immunohistochemistry 48 h after adoptive transfer into wild-type mice as illustrated in representative images (E, choroid plexus; F, cortex). The number of transferred MCAM+ T-cells with plcγ1 knock down was significantly reduced both in choroid plexus (G, p = 0.005, p = 0.09; n = 3) and cortex (H, p = 0.006, p = 0.03; n = 3) in dependence of the knock down efficiency of the respective siRNA (nc, negative control; A, siRNA A; ABC, mixture of siRNA A, B, and C). Transplanted T-cells were stained with cell tracker green prior to transplantation (CMFDA, green), endothelium was stained using anti PECAM (Alexa647, red) and DAPI staining indicates cell nuclei (blue). Scale bar = 20 µm.