title: Mesenchymal stem cells promote the sustained expression of CD69 on activated T lymphocytes: roles of canonical and non-canonical NF-κB 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-κB) 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 mechanisms 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 canonical and non-canonical NF-κB signalling, respectively. Flow cytometry and real-time PCR were used for analyses. Genes with immunoregulatory functions, CD69 and non-canonical NF-κB 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-κB signalling by BAY immediately following activation blocked the induction of CD69; however, inhibition of canonical NF-κB signalling on the third day further induced the expression of CD69. Furthermore, late expression of CD69 was inhibited by RELB siRNA. These results indicate that the canonical NF-κB pathway controls the early expression of CD69 after activation; however, in an immunoregulatory context, late and sustained CD69 expression is promoted by the non-canonical pathway and is inhibited by canonical NF-κB signalling.
authors.affiliation: National Institute of Science and Technology in Stem Cell and Cell Therapy, Center for Cell Therapy, Regional Blood Center and Faculty of Medicine, University of São Paulo (FMRP-USP), Ribeirão Preto, Brazil.
authors.forename: Felipe
authors.initials: F
authors.lastname: Saldanha-Araujo
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Haddad
authors.forename: Kelen C R Malmegrim de
authors.initials: KC
authors.lastname: Farias
authors.forename: Alessandra de Paula Alves
authors.initials: Ade P
authors.lastname: Souza
authors.forename: Patrícia V
authors.initials: PV
authors.lastname: Palma
authors.forename: Amélia G
authors.initials: AG
authors.lastname: Araujo
authors.forename: Maristela D
authors.initials: MD
authors.lastname: Orellana
authors.forename: Julio C
authors.initials: JC
authors.lastname: Voltarelli
authors.forename: Dimas T
authors.initials: DT
authors.lastname: Covas
authors.forename: Marco A
authors.initials: MA
authors.lastname: Zago
authors.forename: Rodrigo A
authors.initials: RA
authors.lastname: Panepucci
mesh_terms: D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism*; D000945:Antigens, Differentiation, T-Lymphocyte / Q000235:genetics / Q000378:metabolism*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D020869:Gene Expression Profiling; D006801:Humans; D037181:Lectins, C-Type / Q000235:genetics / Q000378:metabolism*; D008213:Lymphocyte Activation / Q000276:immunology*; D059630:Mesenchymal Stem Cells / Q000378:metabolism*; D046228:Microarray Analysis; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D009570:Nitriles; D036102:Peripheral Blood Stem Cell Transplantation / Q000379:methods; D010641:Phenotype; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction; D013450:Sulfones; D050378:T-Lymphocytes, Regulatory / Q000276:immunology* / Q000378:metabolism; D051841:Transcription Factor RelB / Q000235:genetics / Q000378:metabolism; D000099192:CD69 Antigens
chemical_list: D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D037181:Lectins, C-Type; D016328:NF-kappa B; D009570:Nitriles; D013450:Sulfones; D051841:Transcription Factor RelB; D000099192:CD69 Antigens; C434003:3-(4-methylphenylsulfonyl)-2-propenenitrile; C497593:RELB protein, human