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Abstract Number: 2328

A Novel Method for Quantitative and Functional Analysis of Autophagy Using Flow Cytometry in Activated Human Primary T Cells

Ryu Watanabe1, Hiroshi Fujii2, Yukiko Kamogawa2, Kyohei Nakamura1, Tsuyoshi Shirai2, Yumi Tajima2, Shinichiro Saito2, Tomonori Ishii1 and Hideo Harigae1, 1Department of Hematology and Rheumatology, Tohoku University, Sendai, Japan, 2Tohoku University, Sendai, Japan

Meeting: 2012 ACR/ARHP Annual Meeting

Keywords: T cells

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Session Information

Session Title: T-cell Biology and Targets in Autoimmune Disease

Session Type: Abstract Submissions (ACR)

Background/Purpose: Autophagy is an evolutionally conserved self-degradation system. In this process, an isolation membrane engulfs cytoplasmic materials and organelles to form an autophagosome. The autophagosome then fuses with the lysosome, leading to the degradation of the enclosed materials to recycle or energy production. Autophagy is considered to play important roles in activated T cells to counteract various cellular stresses, and it would be a potential therapeutic target in chronic inflammatory diseases. Although several methods to measure autophagy were developed, it is not easy to quantify autophagic activity in small number of human primary cells. The aim of this study is to develop a high sensitive and high throughput method for quantitative and functional analysis of autophagy in human primary T cells using flow cytometry.

Methods: Human naïve and effector memory CD4+ T cells were isolated from peripheral blood and stimulated with anti-CD3 and anti-CD28 antibody. Two days after stimulation, GFP-LC3 fusion protein which can function as an autophagy sensor was overexpressed in human activated T cells using retroviral vector system. To inhibit autophagy, dominant negative form of ULK1 (ULK1-DN) was overexpressed in human activated T cells. Autophagic activity and cell death were quantified in naïve and effector memory CD4+ T cells.

 

Results: Using Western blotting and electron microscopy, we confirmed that autophagy was induced in activated T cells and the reduction of fluorescence intensity of GFP-LC3 correlated with autophagy (Fig.1). Compared to naïve T cells, effector memory T cells had significantly lower autophagic activity (p=0.025) and were susceptible to apoptotic cell death (p=0.01). Overexpression of ULK1-DN inhibited autophagic flux and induced more apoptotic cell death in naïve and effector memory T cells. In these autophagy-defective naïve CD4+ T cells, mitochondria volumes and reactive oxygen species were increased. In contrast, enhancement of autophagy by rapamycin reduced apoptotic cell death in naïve and effector memory CD4+ T cells.

 

Conclusion:  We established a novel method for measuring autophagic flux in activated human primary T cells. Using this assay, we first identified that effector memory CD4+ T cells had lower autophagic activity than naïve CD4+ T cells and this lower autophagic activity may contribute to more apoptotic cell death in effector memory CD4+ T cells. This novel method is helpful to examine pathological roles of autophagy and investigate autophagy as a potential therapeutic target in collagen diseases.

Fig 1. The GFP-LC3 fluorescence intensity was reduced in activated T cells, indicating that autophagy was induced in activated human primary T cells.

 


Disclosure:

R. Watanabe,
None;

H. Fujii,
None;

Y. Kamogawa,
None;

K. Nakamura,
None;

T. Shirai,
None;

Y. Tajima,
None;

S. Saito,
None;

T. Ishii,
None;

H. Harigae,
None.

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