The contribution of autophagy to lymphocyte survival and homeostasis

Abstract

Over the life span of a T lymphocyte, from thymic development to death, it is subjected to a variety of stresses and stimuli. Upon receipt of each stress or stimulus, a potentially life-changing fate decision must be made, namely, whether to commit to a form of programmed cell death or to make the necessary adaptations to effectively deal with the changing environment. In our laboratory, we have identified several stresses that a T lymphocyte will encounter during a normal life span. Our studies have focused on how T cells utilize autophagy to get a grasp on the situation, or in cases in which survival is untenable, how T cells use autophagy to hasten their demise. This review focuses on the functions of T-cell autophagy in maintaining homeostasis, eliminating excess or dangerous levels of mitochondria, trimming levels of endoplasmic reticulum, and promoting a healthy metabolic level to allow cells to perform as productive components of the immune system. In addition, the use of autophagy signaling molecules to perform autophagy-independent tasks involved in the maintenance of immune homeostasis is discussed.

Fig. 1. Atg7 T cells have increased levels of activated S6K and increased levels of inhibitory 4EBP-1

Lymph nodes of WT, Atg^f/fLck-cre, and Vps34^f/fLck-cre mice, aged 5-7 weeks, were harvested. Freshly isolated cell suspensions were permeabilized with 0.1% saponin and stained with anti-phosphoS6 Kinase (Thr421/Ser424) or anti-phospho4EBP-1 (Thr36/Thr45). Shown here are representative histograms from CD4⁺CD44^lo or CD8⁺CD44^lo naive cell populations.

Fig. 2. Autophagy limits mitochondrial and endoplasmic reticulum expansion

Splenocytes from Atg3^f/fLck-Cre or 4-OH Tamoxifen–treated Atg3^f/fER-Cre splenocytes and Atg3^f/f control splenocytes were incubated with 100 nM Mito-Tracker Green (Invitrogen, Molecular Probes) or 1 mM ER-Tracker Blue-White DPX (Invitrogen, Molecular Probes) in RPMI 1640 medium at 37°C for 30 min. The geometric mean intensity of fluorescence was used to represent the volume of mitochondria or ER. One tailed, paired, Student's t tests were performed. *=p<.05

Fig. 3. Vps34^f/fLck-cre T cells have normal autophagy but abnormal vesicular trafficking

(A). Splenocytes from Vps34^f/f(WT) or Vps34^f/fLck-cre(Vps34) were isolated and CD4⁺ T cells sorted. Cells were washed in HBSS to remove bound nutrients and remove residual medium. 24 h post-starvation, cells were permeabilized with 0.1% saponin and stained with anti-LC3 (MBL, Woburn, MA). Autophagosomes were visualized by fluorescence microscopy and analyzed using Metamorph (Molecular Devices, Sunnyvale, CA) to quantify pixel sizes and Autoquant X2 (MediaCybernetics, Bethesda, MD) to analyze LC3 punctate structures. (B). SSC ratios (Vps34/WT) of indicated T-cell lineages, taken from 10 independent measurements, analyzed on a FACSCanto (BD Biosciences, San Jose, CA).

Fig. 4. Autophagy's diverse role in lymphocyte homeostasis

Autophagy-inducing signals from TCR-, BCR-, CXCR4-, and S1P1R-mediated signals induce autophagy in lymphocytes. The two ubiquitin-like conjugation systems are crucial for autophagy progression and membrane elongation, to maintain mitochondrial and ER levels. Vps34 is dispensable for autophagy but crucial for IL-7Rα turnover. Beclin-1 contributes to pro-death effector molecule turnover. The TSC complex regulates both mTOR activity and T lymphocyte mitophagy, but not basal or activation-induced autophagy. In the absence of an intact apoptotic response, autophagy mediates a pro-death response.