Synergistic Effect Of Co-inhibitory Molecules On Induction Of Oral-tolerance

OBJECTIVES: B7-H1, a member of the B7 family of co-signaling molecules, isknown to play a role in induction of peripheral tolerance. Recently, another B7 familymember, B7-H4 was also revealed to suppress a cancer-specific T cell immunity. Herewe investigated whether B7-H1 and B7-H4 synergistically enhance the oral-toleranceinduced by feeding allogeneic dendritic cell line.METHODS: We induced oral tolerance by feeding DC2.4 wild type cell line to micewhich were hydrodynamically injected with B7-H1 or B7-H4 plasmid via tail vein.The in vivo expression of soluble B7-H1 or B7-H4 Ig was tested by enzyme linkedimmune sorbent assay (ELISA) with blood plasma drawn at indicated days. We madestable cell lines expressing B7-H1 or B7-H4 mouse dendritiC cell line DC2.4 (H-2~b).We also induced oral tolerance by feeding mice (Balb/c, H-2~d) every day for 10 dayswith indicated stable cell lines. We performed delayed type hypersensitivity(DTH)assay by immunizing and challenging the mice with Balb/c spleen cells anddetermined the ear thickness next day of challenge. We also tested a mixedlymphocyte reaction (MLR) using spleen cells from the mice. Frequency and mRNAexpression of FoxP3+CD4+ T cells and cytokine production were investigated byFACS and RT-PCRRESULTS: DTH and MLR were greatly inhibited by oral tolerance induced by oralfeeding of B7-H1 or B7-H4 stable cell lines compared to that of wild type DC2.4-fedgroup. In case of hydrodynamic injection of plasmid, there was no significantdifference in DTH between the groups; however, MLR was significantly reduced in mice injected with B7-H1 or B7-H4 plasmid. We found that simultaneous injection ofB7-H1 Ig and B7-H4 Ig plasmid greatly reduced MLR compared to mice with singleinjection of plasmid. There was a significant decrease in the production of Th1 andTh2 cytokines such as interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha,and interleukin (IL)-5 in the blood of mice injected with B7-H1 or B7-H4, Flowcytometric analysis showed that the frequency of CD4+CD25+FoxP3+ T regulatorycell (Treg) in spleen was increased in oral tolerance-induced mice with B7-H1 andB7-H4 stable cell line compared to those with mock stable cell line, and in alsoB7-H1 or B7-H4 plasmid-injected mice.CONCLUSION: Our results demonstrate that B7-H1 or B7-H4 can synergisticallyenhance oral tolerance induced by allo antigen, which may be associated withincrease in the frequency of FoxP3+CD4+CD25+ Treg and reduction of theproduction of the Th1 and Th2 cytokines.