Sema4C Expression Characterization and Downstream Signaling in HEK Cells and B Cell Lines

Rationale: Semaphorin 4C (Sema4C) is an axonal guidance molecule that is expressed following Th2 activation on human memory B-cells. It is unknown if Sema4C can induce signal transduction in human B-cells following interaction with its ligand Plexin B2. We characterized Sema4C expression on B-cell lines and studied Plexin B2 induced Sema4C downstream signaling using Sema4C-expressing human embryonic kidney cells (HEK) cells and human B cell lines.

Methods: The expression of Sema4C was measured by Flow cytometry and immunofluorescence (IF). Sema4C transfected HEK (tHEK) and B-cells were stimulated with hrPlexinB2 and p38 phosphorylation was measured by flow cytometry from 1-60 minutes following activation.

Results: We detected Sema4C expression by FACS and IF on 6 immortalized B-cell lines including Ramos, U266 and IM9. We observed an increase in p38 phosphorylation following addition of Plexin B2 to tHEK after 1 min of stimulation, maximizing after 5 minutes and decreasing after 15 min. This was confirmed using the immortalized B-cell lines. 

Conclusions: Sema4C is present on activated and immortalized human B-cells. Addition of its ligand, Plexin B2, induces p38 MAPK downstream signaling in Sema4C transfected HEK cells and B cell lines. This data suggests that Semaphorin-PlexinB2 interaction can play a role in mature, memory B--cell function.