PI3Kγ 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 PI3Kγ provides a key migratory signal for T lymphocyte migration. Since PI3Kγ 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 PI3Kγ 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 (PI3Kγ), class II (PI3KC2α and PI3KC2β) 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 PI3Kγ. However, the role of PI3K in cell migration is context-dependent and diminishes during ex vivo maintenance.