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The Journal of Immunology, 2006, 177: 5163-5168.
Copyright © 2006 by The American Association of Immunologists, Inc.

Autophagy Is Induced in CD4+ T Cells and Important for the Growth Factor-Withdrawal Cell Death1

Changyou Li*, Elizabeth Capan*, Yani Zhao*, Jianping Zhao*, Donna Stolz{ddagger}, Simon C. Watkins{ddagger}, Shengkan Jin§ and Binfeng Lu2,*,{dagger}

* Department of Immunology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213; {dagger} University of Pittsburgh Cancer Institute, Pittsburgh, PA 15261; {ddagger} Center for Biologic Imaging, University of Pittsburgh, Pittsburgh, PA 15261; and § Department of Pharmacology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, NJ 08854


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Autophagy is a tightly regulated catabolic mechanism that degrades proteins and organelles. Autophagy mediates programmed cell death under certain conditions. To determine the role of autophagy in T cells, we examined, in mouse CD4+ T cells, conditions under which autophagy is induced and alterations of the cell fate when autophagy is blocked. We have found that resting naive CD4+ T cells do not contain detectable autophagosomes. Autophagy can be observed in activated CD4+ T cells upon TCR stimulation, cytokine culturing, and prolonged serum starvation. Induction of autophagy in T cells requires JNK and the class III PI3K. Autophagy is inhibited by caspases and mammalian target of rapamycin in T cells. Interestingly, more Th2 cells than Th1 cells undergo autophagy. Th2 cells become more resistant to growth factor-withdrawal cell death when autophagy is blocked using either chemical inhibitors 3-methyladenine, or by RNA interference knockdown of beclin 1 and Atg7. Therefore, autophagy is an important mechanism that controls homeostasis of CD4+ T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Macroautophagy (hereafter referred to as autophagy) is a cellular process by which a portion of the cytosol or entire organelles become sequestered in double membrane vesicles known as autophagosomes (reviewed in Refs. 1 and 2). Autophagosomes are subsequently fused with lysosomes, thereby generating single-membrane autophagolysosomes and leading to cargo degradation. Autophagy is an important catabolic mechanism that recycles building blocks for basal macromolecular synthesis when cells are under nutrient starvation conditions (3, 4), degrades damaged organelles (5, 6), and eliminates pathogens that invade cells (7, 8, 9). The extremity of this catabolic process leads to type II programmed cell death, also called autophagic cell death (10).

Autophagy can either mediate cell death or act as a prosurvival mechanism depending on cellular contexts and stimuli (11). Autophagic cell death is independent of caspase activation and does not bear most other features of type I programmed cell death (apoptosis), including chromatin condensation and DNA laddering. The cellular hallmark of autophagic cell death is an increased autophagic vesicle number (12). During this type of cell death, the total volume of autophagic vacuoles can reach more than that of cytosol outside autophagic vesicles. It is believed that such high levels of autophagic activity can lead to destruction of major proportions of the cytosol and organelles, and, subsequently, to the demise of the cell (10). In contrast to acting as a death machine, autophagy is critical for cell survival during nutrient starvation (3, 13, 14, 15). Autophagy can also prevent cells from undergoing type I programmed cell death (16), most likely by acting as a cellular quality control process (5, 6).

The number of activated/effector T cells is tightly regulated by two separate pathways: activation-induced cell death (AICD)3 and activated T cell-autonomous death (17). Activated T cells succumb to cell death upon sustained TCR stimulation (AICD) (18). AICD is mainly mediated by Fas ligand and TNF-{alpha}-triggered death signaling (17), and is therefore a critical mechanism to eliminate hyperactivated T cells. When there is a lack of survival signals, activated T cells undergo autonomous death, which is independent of external death signals and mediated by proapoptotic members of the Bcl-2 gene family (17). Both apoptosis and necrosis are involved in the death of activated T cells (17, 19). We have found that autophagy can be induced in murine CD4+ T cells upon activation and is required for growth factor-withdrawal cell death. Therefore, autophagy is a key regulatory mechanism for CD4+ T cell homeostasis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Plasmid DNA

The cDNA encoding microtubule-associated protein 1-L chain 3 (LC3)-GFP has been cloned into a retroviral vector (a gift from R. A. Flavell, Yale University, New Haven, CT) with Thy-1.1 as a selection marker. To construct RNA interference (RNAi) expression vector, the following sequences are used: beclin 1, 5'-GATCCCGCAGTTTGGCACAATCAATATTCAAGAGATATTGATTGTGCCAAACTGTTTTTTGGAAA-3'; Atg7, 5'-GATCCCGTTTGTAGCCTCAAGTGTTTTCAAGAGAAACACTTGAGGCTACAAACTTTTTTGGAAA-3'.

Detailed cloning procedure will be available upon request.

Cell culture

Mouse CD4+ T cells were cultured in Th1 and Th2 conditions and activated by plate-bound anti-CD3 and anti-CD28 Abs (20). Briefly, mouse CD4+ T cells, purified from spleens and lymph nodes of C57BL/6 mice, were cultured on 24-well plates precoated with anti-CD3 (10 µg/ml) and anti-CD28 (5 µg/ml). Th1 conditions were formulated by using IL-12 (3.4 ng/ml), human IL-2 (20 U/ml), and anti-IL-4 (clone 11B11) Ab (2 µg/ml). Th2 conditions were formulated by using IL-4 (3000 U/ml), human IL-2 (20 U/ml), and anti-IFN-{gamma} (clone XMG2.1) Ab (2 µg/ml). Cells were transduced with retrovirus containing LC3-GFP and Thy-1.1 surface marker at 24 h after start of the culture. Forty-eight hours after starting the culture, cells were transferred to another plate, which was not coated with anti-CD3 or anti-CD28 Abs, with the original culture medium (including polarizing cytokines and anti-cytokine Abs), and supplemented with new human IL-2 (5 U/ml). At 72 h after start of the culture, Thy-1.1+ T cells were purified using MACS columns (Miltenyi Biotec), as per manufacturer’s instruction. These cells were then restimulated by plate-bound anti-CD3 (10 µg/ml). When chemical inhibitors were used, cells were incubated with 1 or 5 mM 3-methyladenine (3-MA; Sigma-Aldrich), 100 nM JNK inhibitor (Calbiochem), 20 µM zVAD-fmk (Calbiochem), or 10 ng/ml rapamycin (Calbiochem).

D10 (21), a Th2 cell line (a gift from L. Kane, University of Pittsburgh, Pittsburgh, PA) was restimulated every 3–4 wk with chicken conalbumin (Sigma-Aldrich) and irradiated or mitomycin C-treated APCs (RBC-depleted splenocytes from B10.BR mice). During the intervening periods, cells were maintained in complete RPMI 1640 medium (RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 50 µM 2-ME, 100 U/ml penicillin, 100 µg/ml streptomycin, and 20 U/ml human rIL-2 (Biological Resources Branch Preclinical Repository, National Cancer Institute, National Institutes of Health). Starvation of cells was performed in RPMI 1640-only medium (Invitrogen Life Technologies).

Quantification of autophagy

Cells with GFP-LC3 expression were fixed to slides with 2% paraformaldehyde and 0.1% Triton X-100 and counterstained with 4',6'-diamidino-2-phenylindole (DAPI) nuclear dyes. Slides were examined by fluorescence microscopy. Cells were examined in more than five fields per slide on multiple slides. Cells with two or more visible autophagosomes were considered to be expressing autophagy to allow for visualization error. The number of cells considered to be expressing autophagy was divided by the total number of cells in the same field to generate the percentage of cells undergoing autophagy. Data represent the average of all the fields.

Transmission electron microscopy (TEM)

Resting CD4+ T cells and CD4+ T cells cultured in Th1 or Th2 conditions were fixed in 3% glutaraldehyde, stained with 1% osmium tetroxide, enrobed in seaplaque agarose, dehydrated with ethanol, and embedded in Epon/Araldite resin. Thin sections were cut, placed on butvar-coated 200 mesh copper grids, poststained with 3% aqueous uranyl acetate and Reynolds lead citrate, and observed in a transmission electron microscope.

Analysis of live cells

Cells were stained with propidium iodide (10 µg/ml). Then cells were analyzed by FACS. Propidium iodide-negative cells were noted as live cells, and their frequency was calculated.

Detection of protein and mRNA

Atg7 mRNA was measured by RT-PCR using these primers: 5'-ACATCATTGCAGAAGTAGCAGCCA-3' and 5'-ATGCCAGGACACCCTGTGAACTTC-3'. Beclin 1, JNK1/2, Bcl-2, Bcl-xL, and beta-tublin proteins were measured by Western blot using anti-beclin 1 Ab (Sc-11427; Santa Cruz Biotechnology), anti-JNK1/2 Abs (554285; BD Pharmingen), anti-Bcl-2 Ab (2876; Cell Signaling Technology), anti-Bcl-xL Abs (sc-634; Santa Cruz Biotechnology), and anti-beta-tublin (32-2600; Invitrogen Life Technologies).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Autophagy is induced in activated CD4+ T cells

To determine whether autophagy is induced in mouse CD4+ T cells, we examined CD4+ T cells purified from C57BL/6 mice for the presence of autophagosomes using TEM. We did not detect autophagosomes in resting naive CD4+ T cells (Fig. 1A). However, when CD4+ T cells were activated by anti-CD3 and anti-CD28 and cultured in Th2-polarizing conditions, autophagosomes were easily detectable (Fig. 1, B and C) in ~20% of the cells. We could also see autophagy in resting Th1 cells as well (data not shown).


Figure 1
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  		FIGURE 1. Autophagy is induced in activated murine CD4+ T cells. A, TEM image of a resting CD4+ T cell. B, TEM image of an effector Th2 cell. C, Higher magnification inset shown in B. Scale bars, 500 nm (A and B); 100 nm (C). D, Murine Th2 cells expressing autophagosomes. The cDNA encoding LC3-GFP has been cloned into a retroviral vector with Thy-1.1 as a selection marker. CD4+ T cells were cultured in Th2 conditions and were transduced with retrovirus containing LC3-GFP. Thy-1.1+ T cells were then purified using MACS columns as per manufacturer’s instruction. These cells were harvested and examined for autophagosomes by fluorescence microscopy. The left panel shows a cell expressing LC3-GFP without autophagosomes. The right panel shows a cell undergoing autophagy.

 
LC3 is a mammalian homologue of yeast Atg8 (22). Upon autophagy activation, LC3 is processed and lipidated, and the lipid-modified form of LC3 translocates to the autophagosomal membrane. This protein has been useful in monitoring autophagy morphologically, and the level of this form of LC3 is a good quantification for autophagy (23). We therefore infected activated CD4+ T cells with a retroviral construct that allowed us to express a GFP-LC3 fusion protein in the target cells. Using this method, we could readily detect cells undergoing autophagy (Fig. 1D). GFP-LC3 has been used widely as a faithful marker for autophagic processes (23); therefore, it was used in all of our subsequent studies.

Autophagy predominates in Th2 vs Th1 cells

We have hypothesized that there are more Th1 cells that undergo autophagy than Th2 cells based on two lines of evidence. First, it is well established that Th1 cells are more prone to cell death than Th2 cells (20, 24). Therefore, it is likely that Th1 cells also use autophagy as a mechanism of elimination. Second, IL-13, which is an important Th2 cytokine, was shown to inhibit autophagy (25). Therefore, autophagy may be inhibited in Th2 cells. Surprisingly, we found a 2- to 3-fold increase in levels of autophagy when mouse CD4+ T cells were cultured in Th2 conditions as compared with those cells cultured in Th1 conditions (~30 vs 12%, respectively; Fig. 2). We could easily detect this difference using GFP-LC3 as a marker as well as using TEM (data not shown). Therefore, autophagy seems to be involved in both Th1 and Th2 cell maintenance.


Figure 2
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  		FIGURE 2. More Th2 cells undergo autophagy than do Th1 cells. CD4+ T cells were isolated from mice and stimulated with plate-bound anti-CD3 and anti-CD28 for 24 h. The cells were then transduced with retrovirus-expressing GFP-LC3. The cells were cultured continuously for another 48 h, and cells undergoing autophagy were determined using fluorescence microscopy. A, Percentage of cells that underwent autophagy. B, A representative fluorescence image shows more Th2 cells undergo autophagy. Blue color represents DAPI staining. Green color represents LC3-GFP signals. Data are representative of four independent experiments.

 
Autophagy is up-regulated by various stimuli in activated CD4+ T cells

To determine the signals that induce autophagy in mouse CD4+ T cells, we transduced primary cultured CD4+ T cells and D10 cells, a mouse Th2 cell line, with a GFP-LC3-expressing retroviral vector and then examined the ability of various stimuli to induce GFP-LC3 dots inside these T cells. Autophagy was induced to a high level when resting CD4+ T cells were cultured for 3 days in Th1 conditions (Fig. 3A; t = 0). These cells were washed and replated in complete medium on plastic dishes with or without precoated anti-CD3 Abs for another 24 h. Cells that were on plates precoated with anti-CD3 Abs maintained a high level of autophagy (Fig. 3A; anti-CD3). In contrast, cells plated with medium alone decreased their levels of autophagy (Fig. 3A; medium). Therefore, anti-TCR signaling can sustain autophagy in effector CD4+ T cells. Similarly, D10 cells, a Th2 cell line, were cultured with complete medium alone, or IL-2, or plate-bound anti-CD3 for 4, 24, and 48 h. D10 cells cultured with medium alone showed decreasing autophagy over time (Fig. 3B). However, D10 cells cultured with IL-2 or anti-CD3 showed increasing autophagy over time (Fig. 3B). Therefore, both cytokine and TCR signaling can further induce autophagy in activated CD4+ T cells.


Figure 3
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  		FIGURE 3. Autophagy is regulated in T cells. A, TCR stimulation sustained autophagy in primary-cultured CD4+ T cells. CD4+ T cells isolated from C57BL/6 mice were cultured and differentiated into Th1 cells for 3 days, then transduced with the LC3-GFP reporter, as described above. Some cells were fixed immediately for examination of autophagosomes (T=0). Other cells were washed and cultured further with or without plate-bound anti-CD3 at 10 µg/ml for 24 h, and were subsequently fixed and examined for the presence of autophagosomes. B, TCR stimulation and IL-2 induced autophagy in D10 cells. D10 cells expressing the LC3-GFP reporter protein were cultured with medium, or medium with human IL-2 (20 U/ml), or medium with plate-bound anti-CD3 (10 µg/ml) for the indicated time intervals. Cells with autophagosomes were determined, as described in Materials and Methods. C, Sustained growth factor starvation induced autophagy. D10 cells expressing the LC3-GFP reporter protein were cultured with RPMI 1640 without FCS for the time indicated and subsequently fixed and examined for the presence of autophagosomes (*, p < 0.05 by Student’s t test).

 
In mammalian cells, nutrient uptake is regulated by growth factor signaling pathways, including the well-studied PI3K/Akt system (15, 26). In the absence of stimuli for growth, cells stop importing nutrients and activate catabolic mechanisms such as autophagy (review in Ref. 15). To determine whether T cells can also trigger autophagy under growth factor-withdrawal conditions, we cultured D10 cells in RPMI 1640-only medium and used GFP-LC3 dots as the readout for autophagy. Interestingly, autophagy was induced over time when cells were cultured without growth factors (Fig. 3C).

Signaling pathways inhibit or mediate autophagy in T cells

Autophagy is regulated by various signaling pathways (27, 28). To determine whether these pathways are operational in mouse T cells, we measured numbers of T cells undergoing autophagy when they were cultured with chemical inhibitors of various signaling pathways. The mammalian target of rapamycin (mTOR) inhibitor rapamycin and the pan-caspase inhibitor zVAD enhanced autophagy (Fig. 4). This enhancement is more striking in Th1 cells and modest in Th2 cells (data not shown). Therefore, autophagy is inhibited by mTOR and caspases in T cells, consistent with findings in other cell types (27, 28). In contrast, both a chemical inhibitor for type III PI3K called 3-MA and a chemical inhibitor for JNK MAPKs inhibited autophagy in T cells (Fig. 5A). In addition to using chemical inhibitors, we have compared autophagy induced in T cells isolated from JNK1- or JNK2-deficient mice vs wild-type mice. Consistent with the results obtained by using chemical inhibitors, we have found that deletion of JNK1 or JNK2 significantly diminishes autophagy in CD4 T cells (Fig. 5, B and C). The incomplete reduction of autophagy in cells deficient of JNK1 or JNK2 was most likely due to the residual JNK activities in these cells (Fig. 5D). Therefore, JNK and type III PI3K pathways are required for the induction or maintenance of autophagy in T cells.


Figure 4
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  		FIGURE 4. Autophagy is negatively regulated by mTOR and caspases in T cells. A, Autophagy is negatively regulated by mTOR. CD4+ T cells isolated from C57BL/6 mice were cultured and differentiated into Th1 cells for 3 days and transduced with the LC3-GFP reporter, as mentioned above. Cells were then treated with DMSO or rapamycin for 4 h in the presence of plate-bound anti-CD3 (10 µg/ml). B, Autophagy is negatively regulated by caspases. Th1 cells transduced with LC3-GFP were treated with DMSO or zVAD-fmk for 24 h in the presence of plate-bound anti-CD3 (10 µg/ml).

 

Figure 5
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  		FIGURE 5. Autophagy is positively regulated by type III PI3Ks and JNK MAPKs. A, Autophagy induction requires the activities of type III PI3Ks and the JNK MAPKs. Th1 cells transduced with LC3-GFP were treated with DMSO, or 3-MA (1 mM) or JNK inhibitor (100 nM) for 24 h in the presence of plate-bound anti-CD3 (10 µg/ml). B and C, Autophagy is reduced in JNK1- and JNK2-deficient T cells. CD4+ T cells were isolated from wild-type, JNK1-deficient mice, and JNK2-deficient mice; cultured in Th1 conditions for 24 h; subsequently transduced with LC3-GFP; and then cultured for another 72 h (*, p < 0.05 by Student’s t test). D, Residual JNKs are present in JNK1- and JNK2-deficient T cells.

 
Autophagy mediates growth factor-withdrawal cell death in T cells

Because autophagy is induced at a higher level in mouse Th2 cells, we decided to study the role of autophagy in the mouse Th2 cell line, D10 cells. D10 cells grow in medium with FCS supplemented with IL-2 and die spontaneously when growth factors, including factors in the sera and IL-2, are withdrawn from the culture medium. Because withdrawal of growth factors can induce autophagy in CD4+ T cells, we examined whether autophagy plays a role in mediating the spontaneous death of these cells. We first blocked autophagy in D10 cells using a common autophagy inhibitor, 3-MA. D10 cells were cultured in the medium without FBS and IL-2 for 72 h. The 3-MA was added to some cultures. Cells cultured with 3-MA were much more resistant to cell death than cells cultured with only RPMI 1640 (Fig. 6A), suggesting autophagy mediated growth factor-withdrawal cell death. Similar results were obtained using primary Th1 and Th2 cells (data not shown). To further confirm this idea, we used RNAi to knock down the expression of two genes that are crucial for autophagy, namely beclin 1 and Atg7 (29, 30, 31). We generated stable cell lines derived from D10 cells with vectors expressing RNAi for either beclin 1 or Atg7. Levels of mRNA of Atg7 and beclin 1 protein were drastically diminished by these expression constructs (Fig. 6B). In addition, the autophagy is reduced in these cell lines in all culture conditions (Fig. 6C, and data not shown). We then grew these cell lines in RPMI 1640 only and examined whether knockdown autophagy affected cell survival. Consistent with the idea that autophagy mediates growth factor-withdrawal cell death, D10 cell lines expressing Atg7 and beclin 1 RNAi constructs were much more resistant to cell death than two independently generated control lines (Fig. 6D). Bcl-2 and Bcl-xL protect cells from growth factor-withdrawal apoptotic cell death (reviewed in Ref. 32). In addition, Bcl-2 blocks autophagy and autophagic cell death (33). To determine whether Bcl-2 and Bcl-xL have contributed to the differences we saw between control D10 cells and knockdown D10 cells, we performed Western blots to assay for the protein levels of Bcl-2 and Bcl-xL. We have found that Bcl-2 and Bcl-xL are expressed in D10 cells, and their levels are similar between control D10 cells and RNAi-knockdown D10 cells (Fig. 6E). Therefore, autophagy is important for mediating growth factor-withdrawal cell death.


Figure 6
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  		FIGURE 6. Autophagy mediates growth factor-withdrawal cell death in T cells. A, D10 cells were cultured in RPMI 1640 alone for 3 days with or without a chemical inhibitor for the type III PI3K (5 mM 3-MA). Cells were then stained with propidium iodide (10 µg/ml), and live cells were analyzed by FACS. B, Stable D10 cell lines expressing LC3-GFP and knockdown vectors were generated. The expression of Atg7 and beclin 1 was assayed using RT-PCR and Western blot, respectively. C, Control and knockdown cell lines were cultured with fresh human IL-2 (20 U/ml) for 24 h and were then subjected to analysis for autophagy. D, Knockdown cell lines are more resistant to growth factor-withdrawal cell death than controls. Control cell lines and knockdown cell lines were cultured in RPMI 1640-alone medium for 3 days. Cells were then stained with propidium iodide (10 µg/ml), and live cells were analyzed by FACS. E, D10 cells (lanes 1, 3, and 5) and Atg7 knockdown D10 cells (lanes 2, 4, and 6) were cultured with RPMI 1640 alone for 0 h (lanes 1 and 2), 48 h (lanes 3 and 4), and 72 h (lanes 5 and 6). The protein levels of Bcl-xL and Bcl-2 were determined by Western blot analysis.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We have demonstrated using both TEM methods and the standard marker protein GFP-LC3 that autophagy can be induced in activated primary T cells and T cell lines. We have shown the following: autophagy is actively regulated in effector T cells; autophagy is suppressed by the caspase and mTOR pathways; and the induction of autophagy requires the JNK MAPKs and type III PI3Ks. We have further shown that TCR signaling and cytokine stimulation are required for the formation of autophagosomes in T cells. Finally, we have demonstrated that autophagy is an important mechanism for T cell homeostasis and is involved in growth factor-withdrawal cell death in T cells.

T cells die by many means. During the peak of inflammation, T cells are constantly being stimulated, and hyperactivation of T cells leads to AICD, most likely caspase-mediated and mitochondrion-mediated programmed cell death. During recovery from inflammation, autonomous cell death most likely dominates. Both caspase-dependent and caspase-independent cell death pathways are involved in autonomous cell death. Our study demonstrates that autophagy-mediated cell death is involved in spontaneous cell death in T cells. Our data suggest that this mechanism might be involved in controlling homeostasis of T cells.

It is known that Th1 cells are more susceptible to death than Th2 cells (20, 24). Many genes encoding components of death machineries are more highly expressed in Th1 cells than Th2 cells (20, 24). We were surprised to find out that autophagy is generally induced at a higher level in Th2 cells than in Th1 cells. This is most likely due to the fact that the activities of caspases, which promote apoptosis and inhibit autophagy, are activated at a higher level in Th1 cells than in Th2 cells (B. Lu, unpublished observation). This is consistent with the notion that caspase-dependent cell death and autophagic cell death are mutually exclusive in T cells.

Autophagy can be important for both cell survival and cell death (4, 28, 34, 35, 36, 37). In normal physiological conditions, autophagy is critical for turnover of cytoplasmic proteins in terminally differentiated quiescent cells such as neurons. Blocking of autophagy has resulted in abnormal accumulation of protein that leads to formation of protein conjugates. Abnormal protein conjugates were thought to lead to neuronal cell death and neural degeneration (34, 35). When cells are under starvation, autophagy is a primary cellular adaptation mechanism to provide building blocks for cells basic by digesting cytoplasmic protein. Therefore, autophagy is critical for the survival of cells during energy depletion (4, 26). In contrast to its prosurvival roles, autophagy also mediates cell death (28, 36). Autophagic cell death does not seem to require caspases and is sometimes negatively regulated by the activities of caspase 8 (28). Our study has demonstrated that growth factor-withdrawal cell death of mouse Th2 cells is mediated by autophagy. Future studies will most likely reveal more roles of autophagy in T cell biology.


   Acknowledgments
 
We thank Drs. Lisa Borghesi and Lin Liu for carefully reading the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by Cancer Institute Investigator Award and the Pittsburgh Cancer and Aging Program Pilot Grant (to B.L.). B.L. is supported by National Institutes of Health Grant 1 K01 AR048854. Back

2 Address correspondence and reprint requests to Dr. Binfeng Lu, Department of Immunology, University of Pittsburgh, 200 Lothrop Street, Pittsburgh, PA 15261. E-mail address: binfeng{at}pitt.edu Back

3 Abbreviations used in this paper: AICD, activation-induced cell death; 3-MA, 3-methyladenine; LC3, microtubule-associated protein 1-L chain 3; RNAi, RNA inference; mTOR, mammalian target of rapamycin; TEM, transmission electron microscopy; DAPI, 4',6'-diamidino-2-phenylindole. Back

Received for publication December 8, 2005. Accepted for publication July 19, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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