GITR Promotes Activation Induced Apoptosis of Human NK Cells

Abstract

Purpose:: To investigate glucocorticoid-induced tumor necrosis factor receptor (GITR)’s role in activation-induced cell death (AICD) of NK cells and possible mechanisms.

Methods:: Human PBMCs from normal donors were isolated from their buffy coat using Ficoll gradient centrifugation. Highly purified NK cells were obtained from isolated PBMCs by magnetic sorting technique using a negative NK isolation kit. Combination of IL2 and IL15 was used to stimulate NK cells. Apoptotic cells were detected by staining of cells with the combination of annexin-V-FITC and Via-probe^TM (7-Amino-actinomycin D, 7-AAD) followed by flow cytometry. SDS-PAGE and western blot analysis were used to detect apoptosis related signaling molecules.

Results:: About 45% of the NK population from human PBMCs constitutively expresses GITR. Upon activation by cytokines, NK cells greatly up-regulated GITR expression. TLR4 agonist LPS also up-regulated GITR expression in purified NK cells. A stimulating GITR monoclonal antibody reduced proliferation and enhanced apoptosis of cytokine-activated NK cells. The mitochondrial pathway, but not the Fas-FasL pathway was involved in GITR-enhanced apoptosis. Finally, we found that IL2/IL15 stimulation elevated cytoplasmic expression of p21^cip1/waf1 in NK cells while GITR activation decreased IL2/IL15-induced cytoplasmic expression of p21^cip1/waf1.

Conclusions:: The results support the notion that GITR functions as a costimulator in cytokine-induced AICD of NK cells and reveal a novel function of GITR in the regulation of innate immune response.

Keywords: apoptosis/cell death • cytokines/chemokines • signal transduction