A role for SEMA3A in termination of DC/T-cell interactions by repulsive destabilization of the conjugates on NP-1 interaction has been proposed 34, and in line with this, SEMA3A was produced only late after onset of allogeneic MLRs (34 and Fig. 4B). In contrast, SEMA3A production from MV-DC alone or in co-cultures with allogeneic T cells raised within few hours, indicating that this might contribute to destabilization of the IS as described to occur in these cultures earlier 10 and as evidenced by lower frequencies of stable conjugates on exogenous addition of SEMA3A (and also SEMA6A)(Fig.

6B). Notably, amounts of SEMA3A released from MV-DC/T-cell co-cultures several fold exceeded those determined to actively inhibit T-cell Selleck PCI32765 expansion stimulated allogeneic Fostamatinib concentration LPS-DC 34 or on αCD3/CD28 ligation 36. In line with previous reports 38, 39, we repeatedly detected especially in the co-cultures, at least two SEMA3A species (Fig. 4B), the generation of may involve intracellular or surface proteolytic processing, e.g. furin or membrane-resident metalloproteases 48. Whether production of two species in the MV-DC/T-cell cocultures relates to higher infection levels (as compared to the MV-DC only, Fig. 4A) or to the presence of allogeneic T cells remains to be resolved.

While abrogation of NP-1/SEMA3A interaction reportedly signficantly improved allogeneic T-cell expansion driven by LPS-DC 34, this and conjugate stability in MV-DC/T-cell co-cultures could not detectably be rescued by SEMA-neutralizing

antibodies (not shown). This is, however, not surprising since the presence of the MV gp complex on the DC surface within the DC/T-cell interface has previously been linked to IS destabilization and contact-mediated inhibition of T-cell expansion 10, 47, 49, 50. It is also because MV particles Sinomenine displaying the inhibitory complex were likely present in conditioned supernatants of MV-DC or MV-DC/T-cell co-cultures containing high levels of SEMA3A that we did not directly prove their activity on αCD3/CD28-stimulated T-cell expansion. In contrast to earlier studies 34, 36, SEMA6A was at least as efficient at interferring with IS stability and function as SEMA3A (Fig. 6B). As the IgG control always included at comparable levels did not have any effect on all parameters determined except for T-cell motility (Fig. 6A), and ligation of murine plexA4 by SEMA6A is known to negatively regulate T-cell responses 51, we consider the activity of SEMA6A in the assay as specific and thus, the obvious discrepancy cannot be explained at present, and needs further experimentation which would, as the identification of the cellular source of SEMA6A, exceed the present study.