PI3Kgamma is the dominant isoform involved in migratory responses of human T lymphocytes: effects of ex vivo maintenance and limitations of non-viral delivery of siRNA.
Use of mice in which individual PI3K isoforms have been deleted or mutated by gene targeting, has determined that PI3Kgamma provides a key migratory signal for T lymphocyte migration. Since PI3Kgamma can be a dispensable signal for directional migration of human T cells, we have adopted a pharmacological and siRNA strategy to assess the contribution of individual PI3K isoforms to chemokine-stimulated migration of human T cells. The broad spectrum PI3K isoform inhibitor Ly294002 inhibits CXCL12-stimulated migration of freshly isolated T lymphocytes. Use of second generation inhibitors that can discriminate between individual PI3K isoforms, revealed that PI3Kgamma was the major contributor to CXCL12-induced migration and PI3K/Akt signaling (as assessed by S6 phosphorylation). Non-viral delivery of siRNA targeting class I (PI3Kgamma), class II (PI3KC2alpha and PI3KC2beta) and class III PI3Ks, followed by 3 days ex vivo culture, reduces the levels of isoform mRNA, but is insufficient to impact on cell migration responses. However, ex vivo maintenance of T cells alone, independently of siRNA treatment, resulted in the migratory response of T cells toward CXCL12 becoming insensitive to Ly294002. Remarkably, random migration remains sensitive to Ly294002. This study therefore, highlights that the migratory response of freshly isolated human T cells is dependent on PI3K signals that are provided predominantly by PI3Kgamma. However, the role of PI3K in cell migration is context-dependent and diminishes during ex vivo maintenance.
Smith
Laura D
LD
Inflammatory Cell Biology Laboratory, Department of Pharmacy & Pharmacology, University of Bath, Claverton Down, Bath, Avon, BA2 7AY, UK.
Hickman
Emma S
ES
Parry
Richard V
RV
Westwick
John
J
Ward
Stephen G
SG
D018929:Cell Culture Techniques; D002478:Cells, Cultured; D054377:Chemokine CXCL12 / Q000378:metabolism; D002634:Chemotaxis, Leukocyte / Q000187:drug effects; D058544:Class Ib Phosphatidylinositol 3-Kinase; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D013601:T-Lymphocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection / Q000379:methods*
D054377:Chemokine CXCL12; D058544:Class Ib Phosphatidylinositol 3-Kinase; D007527:Isoenzymes; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C546760:PIK3CG protein, human; C546764:Pik3cg protein, mouse