Mesenchymal stem cells promote the sustained expression of CD69 on activated T lymphocytes : roles of canonical and non-canonical NF- B signalling

Abstract

Mesenchymal stem cells (MSCs) are known to induce the conversion of activated T cells into regulatory T cells in vitro . The marker CD69 is a target of canonical nuclear factor kappa-B (NF- B) signalling and is transiently expressed upon activation; however, stable CD69 expression defines cells with immunoregulatory properties. Given its enormous therapeutic potential, we explored the molecular mecha- nisms underlying the induction of regulatory cells by MSCs. Peripheral blood CD3 T cells were activated and cultured in the presence or absence of MSCs. CD4 cell mRNA expression was then characterized by microarray analysis. The drug BAY11-7082 (BAY) and a siRNA against v-rel reticuloendotheliosis viral oncogene homolog B (RELB) were used to explore the differential roles of canoni cal and non-canonical NF- B signalling, respectively. Flow cytometry and real-time PCR were used for analyses. Genes with immunoregulatory functions, CD69 and non-canonical NF- B subunits (RELB and NFKB2) were all expressed at higher levels in lymphocytes co-cultured with MSCs. The frequency of CD69 cells among lymphocytes cultured alone progressively decreased after activation. In contrast, the frequency of CD69 cells increased significantly following activation in lymphocytes co-cultured with MSCs. Inhibition of canonical NF- B signalling by BAY immediately following activation blocked the induction of CD69; however, inhibition of canonical NF- B signalling on the third day further induced the expression of CD69. Furthermore, late expression of CD69 was inhibited by RELB siRNA. Thes e results indicate that the canonical NF- B pathway controls the early expression of CD69 after activation; however, in an immunoregulatory con- text, late and sustained CD69 expression is promoted by the non-canonical pathway and is inhibited by canonical NF- B signalling.