Review

Immunology and Cell Biology (2015) 93, 25–34; doi:10.1038/icb.2014.81; published online 7 October 2014

Autophagy in T-cell development, activation and differentiation

Alisha W Bronietzki1,2, Marc Schuster1,2 and Ingo Schmitz1,2

  1. 1Systems-Oriented Immunology and Inflammation Research Group, Helmholtz Centre for Infection Research, Braunschweig, Germany
  2. 2Institute for Molecular and Clinical Immunology, Otto-von-Guericke University, Magdeburg, Germany

Correspondence: Professor Dr I Schmitz, Systems-Oriented Immunology and Inflammation Research Group, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany. E-mail: ingo.schmitz@helmholtz-hzi.de

Received 3 August 2014; Revised 28 August 2014; Accepted 29 August 2014
Advance online publication 7 October 2014

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Abstract

Autophagy is a vital catabolic process for degrading bulky cytosolic contents, which cannot be resorbed via the proteasome. First described as a survival mechanism during nutrient starvation conditions, recent reports have demonstrated that autophagy supports metabolic functions of T cells at various stages of maturation and effector function. Autophagy is crucial for T-cell development at the precursor stage as self-renewability and quiescence of hematopoietic stem cells depend on autophagy of the mitochondria and the endoplasmic reticulum. Later, during development in the thymus, autophagy regulates peptide presentation in stromal cells and professional antigen-presenting cells, which mediate thymocyte selection. Furthermore, the metabolic changes when mature T cells enter the periphery and when they are activated are both dependent on autophagy. Lastly, autophagy prevents early aging and, thus, ensures maintenance of memory T cells.

Autophagy is an eukaryotic, cytoplasmic (self-)recycling process, in which proteins as well as entire organelles are degraded via lysosomes. There are three different types of autophagy, namely chaperone-mediated autophagy, microautophagy and macroautophagy. Chaperone-mediated autophagy, unlike the other two types of autophagy, only degrades soluble cytosolic proteins by directly transferring heat-shock cognate protein of 70kDa-tagged proteins across the lysosomal membrane.1 Both micro- and macroautophagy can be nonselective as well as specific in their cargo selection and both are capable of degrading large-sized molecules. During microautophagy, the lysosomal membrane invaginates to sequester cytosolic content directly.2 Macroautophagy is distinct, in that a double-membrane structure, the autophagosome, is formed around its cytosolic cargo. This is also the most well-studied type of autophagy, and as this review deals with macroautophagy, it will be referred to simply as 'autophagy’ hereafter.

Autophagy is essential for degradation of aggregated proteins (aggrephagy)3 and damaged organelles including mitochondria (mitophagy), peroxisomes (pexophagy) and ER (ERphagy). These were previously reviewed in Ding and Yin,4 Sakai et al.5 and Bernales et al.,6 respectively. As autophagy is a fundamental process needed by almost all cell types to maintain cell homeostasis, a basal level of autophagy occurs constantly.7 Beyond this, autophagy is vital for cells during stress conditions such as starvation, activation, growth and proliferation, to provide cells with essential metabolic intermediates. These basic autophagic functions are relevant in diseases as well as aging, as the accumulation of aggregated proteins, damaged organelles or other molecules is an underlying problem of many diseases. For instance, autophagy has been implicated in neurodegenerative diseases,8,9 Crohn’s disease,10,11 cancer,12,13 aging4,15 and cystic fibrosis,16 as well as metabolic-related diseases such as fatty liver (macrolipophagy),17 autophagic vacuolar myopathies (glycogen degradation)18,19 and diabetes (crinophagy).20 Moreover, autophagy can directly degrade invading pathogens (xenophagy) and thus acts in cell-autonomous immunity.21

The current review focuses on the role of autophagy in T-cell biology, which is crucial for the adaptive immune system. Autophagy is vital for the maintenance of hematopoietic stem cells (HSCs) from which T cells develop, and for all types of T cells including thymocytes, naive T cells and memory T cells.

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Molecular mechanisms of autophagy

The process of autophagy consists of four distinct phases, which are nucleation, elongation, fusion and degradation. During nucleation a double-membrane structure is formed and separates from the originating membrane. This structure, the phagophore, is extended in the course of the elongation phase and engulfs cytosolic content, including organelles. Finally, the double membrane closes around its cargo, forming the autophagosome. Afterwards, it fuses with a lysosome, whereupon the autophagosomal content is degraded via proteases within the lysosome. The yielded molecules are released back to the cytoplasm to provide for the metabolic function of the cell. This process is regulated via a ‘core’ of over 30 autophagy-related (Atg) proteins, which is subdivided into five functional complexes. Most imminent to the onset of the autophagic process is the unc-51-like kinase complex. This complex contains unc-51-like kinase 1 and 2, Atg13, focal adhesion kinase family-interacting protein of 200kDa (also known as RB1-inducible coiled coil) and Atg101, and is involved in the very early marking of the autophagosome formation site.22 Next, the class III phosphatidylinositol 3-kinase complex encompassing Vps34, Vps15, Beclin1 and Atg14 is recruited to the autophagosome formation site during nucleation and recruits PtdIns3P-binding proteins to this site.23 Effector proteins that are recruited include WD-repeat protein interacting with phosphoinositides, and these, together with Atg9, form a cycling system, which delivers membrane to the growing structure.24,25 After nucleation of the phagophore, two ubiquitin-like conjugation systems have an essential role in the elongation of this structure into a closed autophagosome. The Atg12–Atg5 conjugation system is the first of these two and comprises Atg12 (ubiquitin-like protein), Atg7 (E1-like enzyme), Atg10 (E2-like enzyme), Atg5 and Atg16L1.26 Atg16L1 anchors the Atg12–Atg5 complex to the growing autophagosome membrane by binding to WD-repeat protein interacting with phosphoinositides.27 This complex in turn acts as an E3-like enzyme to the second conjugation system, the microtubule-associated protein light chain 3 (LC3) conjugation system.28 The LC3 conjugation system contains LC3 (ubiquitin-like protein), Atg7 (E1-like enzyme) and Atg3 (E2-like enzyme). Whereas in yeast there is only one LC3 protein, in mammals there are two subfamilies of LC3 protein orthologs: LC3 and GABARAPs, which function in early and late autophagosomal elongation, respectively.29 Not involved in this second complex, but important for its formation is Atg4, a cysteine protease, which recycles LC3 by cleaving it from its binding lipid to make it available for the formation of this second conjugation system.30 As mentioned, the Atg12–Atg5 complex promotes linkage of LC3 to phosphatidylethanolamine, a membrane phospholipid of the autophagosome.31

As autophagy can be selective, for example, targeting ubiquitin-tagged proteins or pathogens,32 the so-called autophagy receptor proteins link the selected cargo to LC3, and thus into the growing autophagosome. Autophagy receptors that have been described to mediate selective autophagy include p62/sequestosome 1, neighbor of BRCA1 gene 1, nuclear dot protein 52 (also known as calcium binding and coiled-coil domain 2), BCL2/adenovirus E1B-interacting protein 3-like (also known as Nix) and optineurin.33 Upon their closure, autophagosomes fuse either directly with lysosomes or with multivesicular bodies first and then with lysosomes. In autophagolysosomes, the inner membrane and autophagosomal cargo are degraded and then released back into the cytoplasm.

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Autophagy in Hematopoietic Stem Cells

T cells originate from bone marrow-derived HSCs that give rise to myeloid and common lymphoid precursors (CLPs) (Figure 1). CLPs cycle in the bloodstream and can migrate into the thymus where they develop into mature T lymphocytes. Unlike their HSC ancestors, CLPs are not self-renewing, thus they have to be constantly replenished by blood-cycling precursor cells.34 To maintain the ability to supply precursor cells for the lymphocyte compartment, HSCs have to maintain quiescence and their self-renewal capacity. To ensure that these two HSC-defining qualities are preserved, they reside in an oxygen-low niche in the bone marrow. HSCs use anaerobic metabolism, defined by low mitochondrial activity and high glycolysis, thereby producing low reactive oxygen species (ROS) levels35 and making these cells extremely stress resistant.36 However, HSCs have the ability to quickly switch their metabolic program to oxidative phosphorylation. This metabolism produces high levels of ROS and is necessary for the differentiation into CLPs.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Autophagy in T-cell development, differentiation and function. T cells originate from bone marrow-derived pluripotent HSCs. These differentiate into CLPs that enter the thymus and differentiate into thymocytes. Thymocytes undergo selection processes that are dependent on interactions with thymic APCs and TECs. Mature T cells leave the thymus and enter the periphery to become effector T cells that are activated and proliferated upon antigen encounter. After an immune response, most effecter T cells die and few survive as memory T cells. The role of autophagy is indicated at the different steps of a T cells’ life. See text for further details.

Full figure and legend (127K)

Autophagy is crucial for the production of normal T-cell numbers by regulating ROS levels in HSCs and their differentiation into CLPs. To control ROS levels, HSC maintain higher basal LC3-II and Atg5 levels than more differentiated cells,37 which are not as ROS-sensitive.38,39 Fetal HSCs are less dependent on low ROS levels37,40 and therefore show less severe effects of autophagy deletion compared with adult HSCs.41 Inhibition or deletion of autophagy both in vitro37 and in vivo40, 41, 42 resulted in the loss of HSC self-renewability, quiescence and much reduced HSC numbers. Furthermore, the frequency of differentiated progenitor cells was altered and resulted in decreased lymphoid and erythroid progenitor cell numbers, as well as severe myeloproliferation, which caused anemia40, 41, 42, 43 and malignant myeloproliferative disease in these mice.40,42 The consequence of lymphoid progenitor cell reduction is diminished CLP and T-cell numbers.42

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Autophagy in thymocyte development

Autophagy is involved in thymic T-cell development and the thymus has considerably high amounts of constitutive basal autophagy compared with other tissues.7,32,44 Cells in the thymus showing increased autophagy include mature thymic epithelial cells (TECs) of the cortex (cTEC) and medulla (mTEC).44 Even though it is still debated whether or not positive and negative selection is limited to the cortex or medulla, respectively, cTECs drive the rearrangement of the T-cell receptor (TCR) genes in developing T cells via peptide presentation. Upon positive selection, CD4+ CD8+ double-positive T cells express the αβ TCR and become either CD4+ T cells binding major histocompatibility complex class II (MHC II)-presented peptides or CD8+ T cells recognizing MHC class I (MHC I)-bound molecules. mTECs and dendritic cells drive the negative selection of thymocytes. Other antigen-presenting cells (APCs) found in the thymus include macrophages. However, they may be mainly responsible for the removal of apoptotic cells, as they degrade proteins too quickly for MHC loading.45

MHC I is expressed in all tissues, whereas MHC II is only constitutively expressed by TECs and hematopoietic APCs. It was believed that peptide–MHC I complexes are strictly of intracellular origin and that MHC II presents extracellular antigens only. However, via cross-presentation, extracellular antigens can be presented on MHC I, and cytosolic as well as nuclear peptides are also presented on MHC II via autophagy.46, 47, 48 Both APCs and mTECs present exogenous antigens on MHC II via the endocytic pathway, although APCs are more efficient. For the presentation of intracellular peptides APCs, cTECs and mTECs use the constitutive secretory pathway and the lysosomal pathway via autophagy.49,50 The role of autophagy in these selection processes during T-cell maturation will be described here.

Autophagy in MHC II antigen presentation

Autophagy regulation of MHC II peptide presentation seems to be very specific in terms of subcellular localization of the antigen, as well as in the nature of the antigenic peptide, as data from TECs and APCs demonstrate. Multiple studies have shown that autophagy only regulates the presentation of nuclear, lysosomal and mitochondrial peptides, whereas it does not promote the presentation of membrane-associated peptides.46,47 Autophagy specificity goes beyond the subcellular origin of the peptide, as Klein and co-workers44 reported that autophagy promotes the presentation of specific intracellular MHC II peptides by TECs. Also, a study investigating the presentation of citrullinated proteins, that is, proteins that are posttranslationally modified by deamination of arginine residues versus unchanged proteins found that autophagy selectively presented citrullinated proteins only.51 Furthermore, a proteomic study using differentiated B-lymphoblastoid cells reported that autophagy differentially promotes the presentation of peptides even from the same protein source.47

Before describing the rather sparse information available on the mechanism of autophagic delivery of peptides to MHC II molecules, this process will be described briefly. MHC II molecules are bound to invariant chains (Ii) in the endoplasmic reticulum (ER) and then transported in MHC II-containing compartments, where they fuse with late endosomes or with autophagolysosomes;44,50,52 these contain peptides from degraded intracellular proteins. Cathepsins then lyse the Ii until only a residue named the class II-associated invariant chain peptide is left. The MHC II-like molecule H2-DM removes class II-associated invariant chain peptide so that a peptide with higher affinity for MHC II αβ heterodimers can bind. This stabilizes the complex, which is then shuttled to the cell surface to elicit a CD4+ T-cell response.50 Colocalization experiments using thymic tissue sections from 1-day-old mice revealed that 20% of MHC II-Ii-positive compartments colocalized with LC3, 40% of H2-DM-positive compartments colocalized with LC3 and lysosomal-associated membrane protein-1-positive compartments (autolysosomes) and 60% of MHC II-class II-associated invariant chain peptide positive compartments colocalized with LC3. This suggests that autolysosomes fuse with MHC II-Ii-carrying compartments, whereupon Ii is quickly degraded to class II-associated invariant chain peptide and thereby autophagy supports loading of peptides unto MHC II.50 Moreover, autophagy downregulates cathepsins, which have varying degrees of resistance to this regulation and thereby peptides are differentially degraded.47 Reducing protease abundance and by that their activity may increase time for MHC II loading and thus promote presentation.

Autophagy regulates positive and negative selection via MHC II loading

Autophagy also has a role in MHC II peptide presentation in the thymus during positive and negative selection. The regulation of peptide loading by autophagy in cTECs for positive selection of T cells has been shown.44 Furthermore, cTEC presentation of hemagglutinin and cognate peptides of the SEP T-cell receptor (TCR) was promoted by autophagy, whereas loading of cognate peptides recognized by the AND and DEP TCRs, respectively, was not.44 Beyond that, DO11.10 TCR-specific peptide loading even seemed to be negatively regulated by autophagy.44 These results support the hypothesis that autophagy and other MHC II loading pathways shape the T-cell repertoire by promoting the presentation of specific peptides during the positive selection process (Figure 1).

Besides the role of autophagy in positive selection, a function of autophagy in negative selection of T cells is controversial. Studies have shown LC3 puncta in both cTECs and mTECs during normal conditions, although to a lesser degree in the latter,44,50 and, as stated above, colocalization of LC3 puncta and MHC II loading vesicles was also shown in these cells.50 As peptide presentation by mTECs and APCs is redundant, autophagy seems dispensable when the antigen levels are very high and APCs can indirectly induce negative selection. At low antigen levels, however, autophagy-dependent peptide presentation in mTECs becomes indispensible.46 Importantly, the transfer of Atg5-deficient thymi into athymic nude mice resulted in rapid wasting owing to autoimmune disease development, characterized by infiltration of activated CD4+ T cells into tissues.44 A subsequent study by the Klein laboratory46 demonstrated that autophagy is crucial for negative selection, that is, deletion of autoreactive CD4+ T cells, as it mediates direct presentation of self-antigens on mTECs. However, it was also reported that only cTECs and not mTECs exhibited LC3 puncta during steady-state conditions.53 Moreover, conditional knockout mice that lack Atg7 specifically in TECs had normal frequencies of T-cell sub-populations in the thymus and the periphery and did not develop signs of autoimmune disease.53 Although the normal T-cell frequencies are consistent with the experiments where Atg5-deficient thymi have been engrafted into a polyclonal T-cell environment, further experiments are needed to determine the cause of these differences observed regarding autoimmunity. Nevertheless, the accumulated evidence suggests that both cTECs and mTECs can present intracellular peptides on MHC II via autophagy and thereby shape the T-cell repertoire.

Thymocyte-intrinsic effects of autophagy

Experiments using chimeric mice have examined the contribution of autophagy to T-cell development. To this end, Rag1-deficient mice, which themselves cannot generate T and B cells, were reconstituted with autophagy-deficient stem cells, so that the lymphocyte compartment could only arise from the transferred stem cells, while all other cell types could be from both genetic origins. One study used Beclin1-deficient embryonic stem cells to complement Rag-deficient blastocysts and found highly reduced CLP and thymocyte numbers, whereas peripheral T- and B-cell numbers were comparable to chimeric mice reconstituted with wild-type embryonic stem cells.54 In contrast, transfer of fetal liver cells from Atg5-deficient mice (containing HSCs) resulted in reduced numbers of CLPs, thymocytes and peripheral T cells.55 Of note, the frequencies of the different thymocyte sub-populations were not altered by Atg5 deficiency, whereas this was the case for Beclin1 deficiency depending on the chimerism, which varied between experiments.54 A potential caveat of these experiments is that autophagy-deficient stem cells were used that, as discussed above, have altered self-renewability and differentiation capabilities. Nevertheless, the differences between Atg5 and Beclin1 deficiency might indicate that developing thymocytes and mature T cells depend on different parts of the autophagic machinery. An alternative explanation might be that Beclin1 but not Atg5 regulates sensitivity to apoptosis in thymocytes.

When Atg genes were conditionally deleted at later stages of thymic development using CD4Cre mice, thymocyte numbers were not affected.56,57 However, when deletion occurred early on in the thymus, that is, deletion via LCKCre, total thymocyte numbers were reduced (ranging from severe to modestly reduced numbers), although the CD4/CD8 subsets were normally distributed.54,58,59,60 This suggests that autophagy is important for survival or proliferation of CD4 CD8 double-negative thymocytes or for the transition from the double-negative to the double-positive stage (Figure 1 and Table 1).


Autophagy promotes iNKT and Treg differentiation in the thymus

As autophagy regulates the priming of thymocytes, this could be the means by which autophagy promotes the development of specific T-cell subtypes (Figure 1 and Table 1). Although T cells differentiate into separate subtypes upon naive T-cell activation, mice with a conditional deletion of Vps34 in T cells using the Cre recombinase under the control of the CD4 promoter had no peripheral invariant natural killer T cells (iNKTs) and their development was arrested at the earliest stage of iNKT development in the thymus, which is characterized by CD1d-tetramer+ CD24+ CD44 NK1.1.56 Also, regulatory T (Treg) cell development and priming seems to be dependent on autophagy, as there was a 20–30% reduction of Treg cells in the periphery of mice lacking Vps34 in the T-cell compartment. Furthermore, these Treg cells were non-functional, which was shown using in vitro suppression assays and in a transfer colitis model. In line with impaired Treg cell function, aged Vps34-deficient mice developed anemia, intestinal inflammation and wasting syndrome.56

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Autophagy and maintenance of T cells

Interestingly, thymocytes maintain very high ER and mitochondrial volume when compared with peripheral T cells.59,61 Upon deletion of Atg genes in thymocytes, ER56,58,61 and mitochondria56,58,59 levels are not reduced in naive T cells, but rather further expanded. The ER was also distributed throughout the cell rather than localizing at one pole, as seen in wild-type naive T cells and ER stress was induced.61 Mitochondrial expansion resulted in increased ROS production57, 58, 59 and an accumulation of membrane structures or vesicles in autophagy-deficient naive T cells was observed.61,62 Taken together, these alterations resulted in the induction of cell death and caused reduced lymphocyte counts in mice.56, 57, 58, 59, 60,62,63

Autophagy is also important for the long-term survival of naive T cells in the periphery. Survival of naive T cells in the periphery depends on TCR interactions with stromal cells and interleukin-7 (IL-7) signaling, the latter of which appears to require Atg3-dependent autophagy in an intrinsic manner. In this line, acute deletion of Atg3 in naive T cells cultured with IL-7 had no short-term effects on the survival of these cells. However, in long-term cultures over 24 days, Atg3-deficient CD4+ and CD8+ T cells exhibited a higher death rate than autophagy-proficient T cells.58 This is consistent with in vivo data obtained from lethally irradiated recipients receiving fetal liver cells from Atg5-deficient mice.55 The higher cell death rate of autophagy-deficient T cells correlated with increased ER and mitochondrial volume accumulating over time in these cells, suggesting that cell loss is because of defects in organelle homeostasis.58 Furthermore, Vps34 has been described to regulate the intracellular trafficking of the IL-7 receptor α-chain resulting in increased T-cell death and reduced IL-7 receptor expression,62 although T-cell-extrinsic factors might contribute to this effect.57 Nevertheless, Vps34 has additional autophagy-independent functions and one should keep in mind that autophagy-related proteins can function in other cellular processes as well.

T-cell-specific deletion of Beclin1 using CD4Cre did not result in mitochondrial accumulation over time.63 Despite normal mitochondrial homeostasis, apoptosis was induced in peripheral CD4+ and CD8+ T cells.63 Thus, Beclin1 might not be crucial for mitophagy, but important for the degradation of other proteins that regulate T-cell survival. Besides the increased levels of the proapoptotic proteins caspase-8, caspase-3 and Bim, also Bcl-2 levels, a prosurvival protein, were upregulated in Beclin1-deficient T cells.63 Next to its role in autophagy, Beclin1 can act in an antiapoptotic manner or, upon cleavage by caspases, as a proapoptotic BH3-only protein.64

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Autophagy in mature T cells

Transcriptional profiling and computational analysis of the gene expression profile of Atg5-deficient and -proficient thymocytes not only showed an association between mitochondria and autophagy but also demonstrated a connection with lymphocyte activation and proliferation.60 When circulating T cells bind an antigen, they proliferate, differentiate into subtypes, secrete cytokines and later die or become memory T cells. Involvement of autophagy in these mature T-cell stages will be discussed here.

T-cell activation and metabolic signaling

Full activation of T cells requires TCR stimulation, costimulation via CD28 and IL-2 signaling. Upon full activation, T cells metabolically switch from catabolic oxidative phosphorylation to anabolic glycolysis and glutaminolysis, even in the presence of oxygen (Warburg effect). T cells with TCR and without CD28 signaling cannot upregulate glycolysis65 and glutaminolysis,66 and become anergic. Anergic T cells are defined by their inability to proliferate when their TCR is engaged without costimulation and their failure to produce cytokines when rechallenged with both anti-CD3 and anti-CD28. Full T-cell activation induces AKT/mammalian target of rapamycin (mTOR) activity, which is indispensable for T-cell activation and inhibition of mTOR renders activated T cells anergic.67 mTOR directly controls glucose, amino acid, iron and low-density lipoprotein transporter expression of activated naive T cells.68 Akt/mTOR signaling also induces the activation of the transcription factors hypoxia-inducible factor-1α and c-Myc.69 c-Myc is essential for the upregulation of glycolysis and glutaminolysis69 by repressing miR-23a and miR-23b.70 Hypoxia-inducible factor-1α was reported not to be important initially during the metabolic switching in activated T cells; however, it can block oxidative phosphorylation under hypoxic conditions by inhibiting tricarboxylic acid cycle entry of pyruvate.71 Three important transcription factors that are induced upon T-cell activation include nuclear factor of activated T cells, activator protein-1 and nuclear factor-κB (NF-κB), which regulate the transcription of important effector T-cell genes.72 The levels of these transcription factors need to be well balanced, as nuclear factor of activated T cells also transcribes genes involved in anergy and increased nuclear factor of activated T cells levels in the absence of costimulation can lead to anergy in T cells.72 For the stimulation of nuclear factor of activated T cells, stable and high intracellular calcium levels are important.73 Furthermore, rapid elevation of cytoplasmic calcium levels upon TCR signaling is necessary for mitochondrial ROS production, which supports full T-cell activation.74 TCR signaling causes the rapid release of calcium from the ER via sarco/endoplasmic reticulum Ca2+ ATPase pumps, upon which stromal interaction molecule-1 is activated and causes calcium release-activated calcium channels on the cell membrane to open, resulting in the influx of extracellular calcium. These increased calcium levels in T cells shortly after receptor stimulation activate 5’ AMP-activated protein kinase (AMPK), a known inducer of catabolic metabolism and inhibitor of mTOR.75 Transient AMPK activity therefore supports the initial metabolic burst of mitochondrial ROS and ATP production in activated T cells, thereby providing the energy for full activation, growth and proliferation.

Both mTOR and AMPK regulate autophagy via phosphorylation of unc-51-like kinase complex. While mTOR inhibits, AMPK promotes autophagy.76 This might be crucial to provide activated T cells with the necessary degraded intermediates for growth and proliferation. Furthermore, autophagy maintains the ER and therefore regulates calcium mobilization in activated T cells.77 An mTOR-independent, glycolysis-upregulating pathway is also induced upon T-cell activation and signals via PIM kinases and c-Jun N-terminal kinases.78 Interestingly, T cells from c-Jun N-terminal kinase-1- and -2-deficient mice showed severely reduced autophagy,79 indicating that mTOR-independent metabolic signaling pathways can induce autophagy.

Autophagy in T-cell activation

Several studies have shown that autophagy is increased in T cells upon activation79,80 and autophagy has been shown to adjust its cargo selection upon T-cell activation. In contrast to autophagosomes of unstimulated cells, which contain many organelles, no organelles were seen in autophagosomes upon T-cell activation and rather protein degradation was induced.80 However, investigating the role of autophagy in the activation process is difficult. As mentioned, naive T cells, in which autophagy genes were deleted during thymocyte development, had increased ER and mitochondrial mass (Figure 1). And, although these naive T cells showed proliferation55,56,60,61,63,80 and cytokine secretion defects56,80 upon stimulation, this is in part because of their increased ER and mitochondrial volume. The expanded ER in these cells also stores more calcium that is released upon TCR activation, resulting in an increased net efflux of calcium from the ER in autophagy-deficient cells compared with wild-type cells. This larger ER calcium efflux causes the opening of fewer calcium release-activated calcium channels, thereby decreasing environmental calcium influx. The cytoplasmic calcium level in activated autophagy-deficient T cells with expanded ER is therefore decreased, which results in proliferation and effector function defects.61 CD25 and CD69 activation marker expression was normal in these cells, showing that activation in general was not inhibited.56,58, 59, 60 These data show that the low ER mass in naive T cells maintained by ERphagy is important for activation and subsequent proliferation and function.

To circumvent potential effects of autophagy deficiency in T-cell development that could translate to the activation of naive T cells, a tamoxifen-inducible system was applied to study loss of Atg7 in an acute manner. To this end, T cells with a floxed Atg7 allele expressing an inducible Cre recombinase were differentiated under type 1 helper T-cell (Th1) condition before deletion of Atg7.80 Upon stimulation, these cells showed reduced IL-2 transcription and secretion, which was similar to conditional deletion of Atg7 in a T cell-specific manner.80 Also chemical inhibition of autophagy resulted in decreased levels of IL-2 and IFN-γ as well as decreased AMPK phosphorylation, ATP production, lactate generation and metabolism of fatty acids.80 When methyl pyruvate, a glycolysis metabolism intermediate, was added to activated, autophagy-deficient T cells, ATP and IL-2 production were partially restored.80 Thus, the loss of autophagy inhibits lipophagy and aggrephagy, which seems vital to support the initial burst of AMPK activation and ATP and ROS production in activated T cells.

Some studies also investigated the effect of autophagy loss on levels of pro- and antiapoptotic proteins. Procaspase-8,63 procaspase-3,63 caspase-9,58,59 Bak,59 Bim63 and Bax56 protein levels were increased in autophagy-deficient activated T cells. Therefore, a function of autophagy could be the degradation of proapoptotic proteins in activated T cells (Table 1). However, autophagy can also induce apoptosis upon growth factor withdrawal and knockdown of Beclin1 or Atg7 protected T cells from apoptosis upon growth factor withdrawal.79 The molecular mechanisms are currently ill defined since conflicting data exist on the expression of pro- and antiapoptotic proteins, especially of the Bcl-2 family, in the absence or presence of autophagy.56,58,59,62,63 More studies are required to define the role of autophagy in apoptosis or apoptosis-independent cell death. This is discussed by Denton et al.81 in this issue of Immunology and Cell Biology.

Another intriguing role for autophagy in T-cell activation is the regulation of signaling pathways. Autophagy is known to induce NF-κB activity in response to various stimuli82 and NF-κB activity is also also crucial for TCR activation.83 However, in this respect, autophagy has been described to downregulate NF-κB signaling in T cells. The TCR activates the inducible transcription factor NF-κB via the so-called CBM complex, which consists of Carma-1/Card11, Bcl10 and the paracaspase Malt1.84 Interestingly, the Bcl10 subunit of the CBM complex is selectively degraded via autophagy.84 Although it is unclear how the Bcl10 specificity is brought about, Bcl10 but not the other CBM subunits colocalized with the p62/sequestrome autophagy receptor in an ubiquitin-dependent manner.84 Autophagy-dependent Bcl10 degradation occurs only in effector T cells and not in naive T cells.84 Therefore, it seems that autophagy tightly regulates NF-κB activity in T cells by both supporting the NF-κB activation in naive T cells and terminating its activity in effector T cells. In conclusion, autophagy provides a negative feedback loop for NF-κB signaling.

Autophagy in antigen presentation to naive T cells

Naive T cells are activated by APCs that present antigens to them via MHC II and MHC I. Pathogens and pattern-recognition receptors can directly activate autophagy to upregulate MHC II presentation.85 Studies of pathogens whose proteins are presented by APCs on MHC II via autophagy include viral and bacterial peptides. Viral examples include the Epstein–Barr virus nuclear antigen 186 and the influenza matrix protein 1.52 Bacterial peptides presented on MHC II via autophagy include Yersinia protein YopE,87 Mycobacterium tuberculosis antigen Ag85B88 and the bacterial transposon-derived neomycin phosphotransferase II.89 Lastly, tumor peptide presentation on MHC II is also promoted by autophagy.48,90

Autophagy regulating MHC I loading seemed implausible, as these pathways do not intersect with the source of peptide–MHC I complexes, being proteasomal substrates. Furthermore, in the thymus autophagy does not seem to regulate peptide loading onto MHC I,44,50 although it could be limited to very specific peptides that have not been among the antigens tested so far. Also in the periphery, APCs seem to use autophagy to cross-present only selected exogenous peptides to CD8+ T cells, as some reports found no involvement of autophagy in MHC I cross-presentation of the specific antigen they investigated.48,52,86 However, a few reports demonstrated that autophagy can be involved in loading peptides to MHC I. Studies on influenza A viral peptide presentation,91 human cytomegalovirus protein pUL138 presentation,92 ovalbumin and melanocyte differentiation antigen gp100 presentation90 revealed a role for autophagy in the cross-presentation of these. One study described that as peptide–MHC I complexes are recycled, they fuse with autophagolysosomes. Upon pH change the peptide–MHC I complex becomes unstable and a novel autophagy-delivered peptide binds to the MHC I and is transported to the cell surface.92

Autophagy in T-cell differentiation

There are multiple subtypes of CD4+ T cells that are functionally and metabolically distinct. The two main subtypes are Treg cells and Th cells. Further the Th cells also comprise different types, including Th1, Th2 and Th17 cells. Treg cells differ metabolically from Th cells in that they use lipid oxidation, whereas effector T cells are glycolysis-dependent.93 Therefore, if mTOR is induced in activated T cells, they differentiate into Th cells, whereas if mTOR activation is low and AMPK levels are high, the naive T cells preferentially differentiate into Treg cells.93 Moreover, although all Th cells depend on mTOR activity, Th1 and Th17 cells require Rheb-dependent mTORC1 activation, whereas Th2 differentiation is promoted by mTORC2 activation.94 Th17 cell differentiation is additionally regulated by HIF-1α.95 Therefore, environmental factors such as glucose availability and cytokines can induce preferential differentiation of naive T cells into certain Th cells or Treg cells. An early study suggested that Th2 cells exhibit more autophagy than Th1 cells and that this correlated with resistance to cell death.79 However, autophagy was solely analyzed by the presence of GFP-LC3 puncta in the steady state and no autophagic flux analyses were performed. When naive Beclin1-deficient T cells were differentiated in vitro into Th1, Th2 or Th17 cells, all subtypes except for Th17 cells had high death rates upon stimulation. Th17 differentiated cells were more resistant and therefore do not seem to depend on autophagy for survival as much as the other effector T cells.63 Surprisingly, mice lacking Beclin1 specifically in T cells were resistant toward myelin oligodendrocyte glycoprotein-peptide-induced experimental autoimmune encephalomyelitis, which is a Th1- and Th17-dependent autoimmune disease model of multiple sclerosis. Beclin1-deficient T cells did not enter the central nervous system and proliferated less in vitro upon restimulation.63 The differential impact of autophagy on Th1 versus Th17 biology in the experimental autoimmune encephalomyelitis model needs to be addressed in future experiments. Thus, although more direct evidence of the role of autophagy in different T-cell types is needed, it seems likely that T cells regulate autophagy according to their metabolic need and effector function.

Autophagy in CD8+ T cells

Almost all studies reported a stronger effect of autophagy deletion on CD8+ T cells than on CD4+ T cells. Upon impaired autophagy, the CD8+ T-cell frequency was reduced more than the CD4+ T-cell frequency.56, 57, 58,60,63 As in certain effector T-cell subtypes, autophagy might have an even more prominent role in these cells. It is difficult to decipher, but autophagy might have a role by supporting and driving the differentiation of these cells in the thymus. Additionally, it might simply be because of the fact that basal autophagy levels are higher in these cells compared with CD4+ T cells60 and they are more dependent on autophagy to maintain their metabolic environment.

Autophagy in memory T cells

CD8+ T cells also depend on glycolysis to induce effector functions. After an immune response CD8+ T cells that become long-lived memory T cells revert to oxidative phosphorylation and depend specifically on lipid oxidation for maintenance, similar to Treg cells.96 A recent study demonstrates that CD8+ memory T cells use a futile lipid cycle to serve their metabolic demands and that lipolysis involves the lysosome.97 Whether or not lipophagy might contribute to lipid delivery to the lysosome in CD8+ memory T cells is currently unknown. Of note, lipid-oxidation rates are reduced in hepatocytes when lipophagy is inhibited.17 Although it should be noted that mTOR regulates other metabolic pathways next to autophagy, inhibition of mTOR can induce memory CD8+ T-cell differentiation and, thus, is consistent with a role for autophagy in the maintenance of memory cells.98 Interestingly, the Tax oncoprotein of the human T-cell leukemia retroviruses human T-cell leukemia virus type 1 and human T-cell leukemia virus type 2 has recently been shown to induce autophagy in human CD4+ T cells and thereby promotes survival and proliferation of memory T cells.99,100 Therefore, inhibition of autophagy might be a therapeutic option to cope with T-cell transformation upon human T-cell leukemia virus infection.

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Concluding remarks

Owing to the cells fundamental necessity for autophagy, a lot of research has been carried out in this field since coining of the term in the 1960s. However, research in the field of immunology has just begun to uncover the impact of this process on immune cells. This review discussed direct and indirect roles of autophagy in T cells. T cells indirectly depend on autophagy maintaining HSC metabolism and differentiation, as well as regulating the presentation of peptides by APCs during positive and negative selection of thymocytes and during activation of mature T cells in the periphery. Autophagy directly regulates the development and survival of CD4 CD8 double-negative thymocytes, iNKT and Treg cells. Upon maturation, T-cell reduction of ER and mitochondria by autophagy ensures their survival as they enter the periphery. Autophagy further supports T-cell activation, proliferation, differentiation, function and finally memory T-cell maintenance. There are still many open questions and much research needs to be carried out on this research topic. Especially interesting in understanding autophagy and the meaning of its function is to investigate its role in the different T-cell subsets. As mentioned, autophagy affects Treg cell and iNKT cell differentiation and function and may be involved in CD8+ memory maintenance. How autophagy differentially regulates these from other types of T cells requires further research that certainly will deepen our understanding of the many facets of autophagy regulation and function.

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Acknowledgements

AWB was supported by the President's Initiative and Networking Fund of the Helmholtz Association of German Research Centers (HGF) under contract number VH-GS-202. This work was supported by the Helmholtz portfolio program Metabolic Dysfunction, the Fritz-Thyssen foundation and the Deutsche Forschungsgemeinschaft (SCHM1586/3-1) to IS.