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. Author manuscript; available in PMC: 2012 Nov 15.
Published in final edited form as: J Immunol. 2011 Oct 21;187(10):5051–5061. doi: 10.4049/jimmunol.1100710

The Class III Kinase Vps34 Promotes T Lymphocyte Survival through Regulating IL-7Rα Surface Expression

Ian X McLeod *, Xiang Zhou , Qi-Jing Li *, Fan Wang , You-Wen He *
PMCID: PMC3248807  NIHMSID: NIHMS325648  PMID: 22021616

Abstract

IL-7Rα mediated signals are essential for naive T lymphocyte survival. Recent studies show that IL-7Rα is internalized and either recycled to cell surface or degraded. However, how the intracellular process of IL-7Rα trafficking is regulated is unclear. Here we show that Vps34, the class III phosphatidylinositol 3-kinase, plays a critical role in proper IL-7Rα intracellular trafficking. Mice lacking Vps34 in T lymphocytes had a severely reduced T lymphocyte compartment. Vps34-deficient T lymphocytes exhibit increased death and reduced IL-7Rα surface expression, though three major forms of autophagy remain intact. Intracellular IL-7Rα in normal T lymphocytes at steady-state is trafficked through either early endosome/multivesicular bodies (MVB) to the late endosome-Golgi for surface expression or to the lysosome for degradation. However, Vps34-deficient T cells have mislocalized intracellular Eea1, HRS, and Vps36 protein levels, the combined consequence of which is the inability to mobilize internalized IL-7Rα into the retromer pathway for surface display. Our studies reveal that Vps34, though dispensible for autophagy induction, is a critical regulator of naïve T cell homeostasis, modulating IL-7Rα trafficking, signaling, and recycling.

Introduction

Naïve T lymphocytes are maintained by multiple homeostatic mechanisms involving TCR signaling, cytokines and nutrient factors (1). IL-7 is a central cytokine for naïve T lymphocyte survival by promoting multiple anti-apoptotic pathways (2). IL-7, produced mainly by stromal elements in secondary lymphoid organs, is unique, since its production is not dependent on exogenous signals, but is kept at constant levels (3). Thus, usage of this cytokine must be enforced on a cell autonomous basis through the regulation of IL- 7R expression. The IL-7R consists of IL-7Rα and the γc chain that is also shared by type 1 cytokines (4). The responsiveness of T lymphocytes to IL-7 is regulated primarily by IL-7Rα since the γc is ubiquitously expressed (2, 3).

Much work has been done to study the regulation of IL-7Rα expression at the transcriptional level (2, 5). Robust IL-7 signaling induces downregulation of IL-7Rα mRNA transcription (6). IL-7 induced downregulation of IL-7Rα on T lymphocytes is thought to be an important homeostatic mechanism to ensure the maximal survival of naïve T cells in an IL-7 limited environment.

Recent studies have demonstrated that IL-7Rα is also subjected to post translational regulation. IL-7Rα is internalized under steady-state and upon IL-7 stimulation through clathrin-mediated endocytosis (7). Although some of the internalized IL-7Rα is degraded in a proteasome- and lysosome-dependent manner, a significant fraction of the internalized IL-7Rα recycles back to the cell surface, which is essential to maintain proper IL-7Rα surface expression (7, 8). However, how IL-7Rα trafficking is completed intracellularly and what molecules regulate this key process remain unknown.

Along with IL-7Rα mediated signaling, several other mechanisms exist to ensure T cell homeostasis. Autophagy, a highly conserved intracellular process, has been identified as a novel mechanism regulating T lymphocyte homeostasis (9, 10). T lymphocytes lacking the autophagy gene Atg5 or Atg7 undergo large-scale apoptosis and exhibit defective intracellular mitochondria clearance, increased ROS level and impaired proliferation (9, 11). The autophagy pathway is carried out primarily through two ubiquitin-like conjugation systems that consist of 33 different autophagy-related genes in yeast and mammals (12). Among them, the sole class III phosphatidylinositol 3-kinase (PI(3) kinase) Vps34 has been shown to play a critical role in the initiation of autophagy in yeast and higher organisms (13, 14). Specifically, in S. cerevisiae, Vps34 mutants had defective turnover of late Golgi proteins after rapamycin treatment (14). Additionally, in HT-29 adenocarcinoma cells, knockdown of hVps34 led to a decrease in long-lived protein turnover (13). Vps34 has a single substrate specificity, phosphorylating the 3- hydroxyl position on phosphatidylinositol, which in turn acts as a localization signal recruiting many proteins with FYVE or GLUE domains. Vps34 is anchored to membranes by its myristoylated interaction partner, Vps15 (15), and is localized to several endomembranes, including endosomes, multivesicular bodies (MVB) and endoplasmic reticulum (16). Interaction between Vps15 and Vps34 has been shown to increase the activity of Vps34. Vps34 functions in two major complexes, both involving the tumor suppressor Beclin-1 (14, 17, 18). The first complex, involving UVRAG and Atg14L, has been proposed to function in autophagy induction (19). The second complex, involving Rubicon, seems to downregulate autophagy and control endosomal sorting functions (20). The association of Vps34 with Beclin-1 is important to recruit proteins that have membrane-flexing qualities, such as Bif-1, to induce curvature of expanding vesicles (21). Taken together, Vps34 plays a complex role in signaling and recruitment of effectors for vesicle formation and endomembrane trafficking, as well as autophagic induction in several systems (22, 23).

In studying the role of Vps34 in T lymphocytes, we have found that Vps34 is essential for T lymphocyte development and survival. Most surprisingly, the critical role of Vps34 for T cell survival is independent of autophagy. Rather, Vps34 regulates IL-7Rα expression by directing its proper intracellular trafficking at multiple steps. Our results have identified a key molecular regulator for IL-7Rα surface expression in T lymphocytes and mapped out a route for normal IL-7Rα intracellular trafficking.

Materials and Methods

Mice

Vps34-deficient T lymphocytes were generated by crossing Vps34-floxed mice (24) to Lck-Cre transgenic mice (The Jackson Laboratory). Genomic deletion was assessed by PCR detection of the Vps34 floxed allele (forward, AACTGGATCTGGGCCTATG; reverse, CACTCACCTGCTGTGAAATG) and Vps34 deleted allele (forward, AACTGGATCTGGGCCTATG; reverse, GAAGCCTGGAACGAGAAGAG). Bim−/− mice were purchased from Jackson Laboratories and hBcl2+ tg mice were a gift from Dr. Motonari Kondo. All mice were bred and housed in Duke’s specific pathogen-free facilities in accordance with IACUC regulations.

Antibodies and Reagents

FITC, PE, PE-cy5, APC, APC-cy7, or Pacific Blue conjugated anti-CD3, -CD4, -CD8, -CD44, - CD62L, -IL-7RA, -B220, -QA2, -IL-2, -CD132, -CD71, -pStat5(Y694), -Stat5, and –Bcl-2 were purchased from BioLegend, eBioscience, and BD Pharmingen. Anti-LC3 (PD015), -p62, and ZVAD- FMK were purchased from MBL. Anti-Vps34 was purchased from Abgent. Anti-HRS was purchased from Abnova. Anti-TGN46, -Vps35, and -Vps36 were purchased from Abcam. rmIL- 7 was purchased from PeproTech. Fluorokine biotin-hIL-7 was purchased from RandD Systems.

Flow Cytometry

Single cell suspensions with RBC’s lysed were incubated with FcR blocker (2.4G2; eBioscience) and were immunostained for all surface markers. For intracellular staining, cells were fixed in 4% PFA for 20 minutes, washed, and permeabilized in 0.1% saponin for 20 minutes. All stains 7 were performed in 0.1% saponin thereafter. All FACS utilized a BD Fac scan to II (BD, Franklin Lakes, NJ).

Cell Proliferation

Total single cell suspensions of splenocytes or lymphocytes at 107/ml were stained in 5% FBS at 5μM CFSE (Molecular Probes) for 5 minutes and washed 3 times. Cells were stimulated with soluble anti-CD3 at 5 μg/ml and anti-CD28 at 2 μg/ml for 48 or 72 hours.

Mitochondrial Content and ROS Production

Mitotracker Green (Molecular Probes), DHE (Sigma-Aldrich), and CM-H2DCFDA (Molecular Probes) were used as described (11).

Electron Microscopy

Mature LN T cells after EasySep negative bead selection (Stem Cell Technologies) were antibody stained and lightly fixed in 1% PFA. One million CD4+ T cells were sorted and fixed in a 4% glutaraldehyde 0.1 M sodium cacodylate buffer overnight. The samples were rinsed in 0.1 M cacodylate buffer containing7.5% sucrose three times for 15 min each and fixed in 1% osmiumin cacodylate buffer for 1 h. After being washed three times in 0.11 M veronal acetate buffer for 15 min each, the samples were incubated with 0.5% uranyl acetate in veronal acetate buffer for 1 h at room temperature. Specimens were then dehydrated in an ascending series of ethanol (35%, 70%, 95%, and two changes of 100%) for 10 min each, followed by two changes of propylene oxide for 5 min each. The samples were incubated with a 1:1mixture of 100% resin and propylene oxide for 1 h, followed by two changes of 100% resin, each for 30 min. Finally, the samples were 8 embedded in resin and polymerized at 60°C overnight. Thick sections (0.5 μm) were cut and stained with toluidine blue for light microscopy selection of the appropriate area for ultrathin sections. Thin sections (60–90 nm) were cut, mounted on copper grids, and post stained with uranyl acetate and lead citrate. Micrographs were taken with a Philips LS 410electron microscope. Images were analyzed using Axio Vision software (Zeiss).

Fluorescence Microscopy

All images were captured with a custom-built Zeiss Observer D1 using a Zeiss 100× objective lens and a 1.4 NA. Images were captured using a Photometrics Cool SNAP HQ2 and analyzed using Metamorph software for punctae number, size, and intensity. Images were deconvoluted, thresholded, and colocalization determined using Auto quant X2 software. Decon volution was done blind at 40 iterations. Flurochromes used included Pacific Blue, Cy3, FITC, and Cy5. Gamma value adjustments were made to CD127-linked flurophores to 1.25 for ease of viewing in comparison to other fluorophores.

Statistical Analysis

All statistical analysis was performed using Prism software (GraphPad Software). Tests for cell numbers, death assays, LC-3, HRS, IL-7Rα punctae formation and recovery, mitochondrial content, and ROS levels were paired, two-tailed Students’ t-tests.

Results

Reduced T lymphocyte compartment in Vps34-conditional knockout mice

To investigate the role of Vps34 in T lymphocyte development and function, we ablated Vps34 kinase activity in T cells by crossing mice containing loxP sites flanking exons 17 and 18 of Vps34 (24) to a Cre recombinase under the control of the Lck regulatory elements (25). Exons 17 and 18 correspond to the ATP-binding domain ofVps34, which were replaced by a premature stop codon. We first examined thymocyte development in Vps34-deficient mice. All four subsets of thymocytes as defined by CD4 and CD8 expression were detected in Vps34-deficient mice. However, the total thymic cellularity was reduced by >50% in Vps34-deficient mice with drasticre ductions in both the CD4+CD8+DP and CD4+or CD8+SP compartments (Figure 1A and 1B). Although the DN percentage was increased in Vps34f/fLck-crethymocytes, none of the populations were significantly increased, and since DN1 and DN2 thymocytes should not yet express the Lck promoter-driven recombinase, this difference was likely due to thymic architecture and not inherent thymocyte defects (Supplemental Figure 1A). Interestingly, there was a clear gene-dosage effect as Vps34f/+Lck-cre heterozygote mice had a significant reduction (p<0.05) in the numbers of DP, CD4+and CD8+SP thymocytes (Figure 1B). We also found that Qa2+CD4+SP or Qa2+CD8+SP mature T cells were muchreduced (Figure 1C).

Figure 1.

Figure 1

Phenotypic Analysis of Vps34f/fLck-creT cells. (A) Thymic profile of Vps34f/fLck-cre andVps34f/+Lck-crenull mice that were 5.5–6 weeks old. (B) Cell numbers from thymiof Vps34f/fLck-cremice. p values are shown in the panel. n=12. (C) QA2 expression in CD4+ or CD8+ SP thymocytes of Vps34f/fLck-cremice. (D) T cell profiles of spleen and lymph nodes from 6-week old mice of control and Vps34f/fLck-cremice. (E) Cell numbers of mature CD4+ or CD8+ T cells in Vps34f/fLck-cremice. n=12

We next examined the peripheral T lymphocyte compartment in Vps34f/fLck-cremice. The number of CD4+and CD8+T cells in the spleen and lymph nodes of Vps34f/fLck-cremice were reduced by >80% (Figure 1D and 1E). These cells expressed comparable levels of CD3 to that on wild type T cells (Figure 1D). Similar to the thymus, Vps34 haplo in sufficiency was observed for both CD4+ and CD8+ T cell compartment (Figure 1D and 1E). As expected in a lymphopenic environment, the number of T cells with an activated/memory/homeostatic proliferation phenotype was increased in T cells lacking one or both copies of Vps34 (Figure 1D).

The peripheral T cells in Vps34-deficient mice may be comprised of cells that have escaped Lck-Cre mediated deletion. CD4+peripheral T lymphocytes had almost undetectable expression of Vps34 by fluorescence microscopy, and genomic deletion of Vps34 was maximal in animals up to 6 weeks of age (Supplementary Figure 1B and 1C). Also, deletion of Vps34 assessed by western blot showed that Vps34 was 75% deleted in CD4SP thymocytes and in excess of 80% in mature, naïve CD4-lineage T cells ((Supplemental Figure 1B). Interestingly, Vps34 protein was only minimally deleted in DP thymocytes, which reflects the very long half-life of Vps34 protein. Alternatively, it is possible that DP cells which have lost Vps34 protein expression earlier in the DP stage are committed to apoptosis, while thymocytes that lose Vps34 expression later, for example, while in the DP to SP transition are viable to complete thymic development.

The mature, active/memory CD4+ T cells had minimal deletion of Vps34, and represent a population that has escaped deletion, though the genomic deletion is still clean from the total mature CD4 population. All experiments were subsequently performed using naïve cells, exclusively. Taken together, these results demonstrate that Vps34 plays an essential role for the survival of developing T lymphocytes. Taken together, these results demonstrate that Vps34 plays an essential role for the survival of developing T lymphocytes.

Vps34-deficient T cells succumb to apoptosis

The impaired T lymphocyte compartment in Vps34-deficient mice was similar to that in mice lacking other autophagy genes, Atg5 or Atg7 (9, 11). Autophagy-deficient T cells readily undergo apoptosis. To determine whether Vps34f/fLck-creT cells had increased apoptosis, we first stained freshly isolated T cells from these mice. Deletion of Vps34 in thymocytes and peripheral T cells resulted in a >2-fold increase in apoptosis of these cells (Figure 2A). To further characterize the defect of Vps34f/fLck-cre T cells, we examined the survival of Vps34f/fLck-creSP thymocytes and peripheral CD44low naïve T cells in vitro culture with or without IL-7 or the caspase inhibitor zVAD. Vps34f/fLck-creCD4+ or CD8+ naive T cells did not respond to IL-7 while wild type T cells survived well in the presence of IL-7 (Figure 2B). However, Vps34f/fLck-creCD4 T lymphocytes showed a modest but significant improvement in survival in the presence of zVAD (Figure 2B). Additionally, the administration of other common gamma chain cytokines, IL-4 and IL-15, gave no significant increase in the survival of Vps34f/fLck-creT cells, despite supporting WT T cells (Supplemental Figure 2G). These results suggest that an important function of Vps34 is to promote Tlymphocyte survival. Given that the number of naïve CD8+T cells was very low in Vps34f/fLck-cremice and that many of these cells had either escaped deletion or were CD8+cells of a non-T lineage, we focused our following analysis on CD4+ T cells.

Figure 2.

Figure 2

Enhanced apoptosis but normal autophagy in Vps34-deficient T cells. (A) Apoptosis staining of freshly isolated control and Vps34f/fLck-creT cells from 6-week old mice. (B) Increased apoptosis of Vps34f/fLck-cre T cells in vitro culture. Cells were cultured in RPMI medium alone or with IL-7 (1ng/ml)or zVAD (20μM). Peripheral cells were gated on CD44low populations and AnnexinV/7AAD exclusion was measured at 0, 24 and48hrs. Shown are mean+SD from four individual experiments. P values provided correspond to cRPMI compared to IL-7 treatment, and IL-7 treatment compared to zVAD treatment (C) Activation-induced autophagy in Vps34f/fLck-creT cells. 48 hours post anti-CD3/CD28 stimulation, control, Vps34f/fLck-cre, and Atg3-deficientCD4+ cells were stained with anti-LC3 antibody and quantified for LC3 punctae formation by microscopy. Shown are representative pictures of the LC3 staining. A total of 30 cells were counted and shown. (D) Starvation-induced autophagy. 16 hours post-starvation, autophagy was measured in the indicated cells under different culture media. A total of 30 cells were quantified. (E) TEM of Vps34f/fLck-creCD4+ T cells. Left panels are representative of WT T cells with ordered Golgi stacks and budding vesicles. Right panels are representative of Vps34f/fLck-creT cells with asymmetric vesicle traffic, but intact autophagosomes. Arrows indicate autophagosomal structures in Vps34f/fLck-cre T cells. A total of 25 cells from each genotype have been examined with TEM.

Normal autophagy induction in Vps34-deficient T cells

Similar to autophagy-deficient T cells (Atg5- or Atg7-null), Vps34f/fLck-cre T cells exhibited reduced number and increased apoptosis. Since Vps34 has been shown to be critical in autophagy induction in almost all organisms and cells types investigated (16, 26), we tested whether there were any discernable defects in several forms of autophagy induction inVps34f/fLck-cre T cells. Autophagy is induced in T lymphocytes by stimulation through the TCR complex (9). We stimulated wild type and Vps34f/fLck-cre naïve CD4+T cells with anti-CD3 plus anti-CD28 and examined LC-3 punctae formation. Although there was a significant increase in cell death in the TCR-stimulated Vps34f/fLck-cre T cells (data not shown), live cells were sorted after 48 hours of stimulation and analyzed forLC3 mobilization by fluorescence microscopy. Qualitatively, these punctae had various sizes and varied in their cytoplasmic location considerably (Figure 2C). However, there was no quantitative difference in the number of punctae between activated wild type andVps34f/fLck-cre T cells (Figure 2C). Sorted, activated T lymphocytes from an Atg3f/fLck-cre (Atg3−/−) were used as a negative control for induction. We next examined starvation-induced autophagy. Since many Vps34f/fLck-cre T cells are committed to apoptosis by 24 hr, we examined shorter starvation periods. Naïve CD4+ T lymphocytes were withdrawn from serum (iRPMI) or amino acids (HBSS) for 16hr and cultured in vitro. Cells were then stained for LC3 and punctae were quantified. Consistent with TCR stimulation induced autophagy, no defect in starvation-induced autophagy in Vps34f/fLck-cre T cells was found (Figure 2D). LC3 processing assessed by western blot was also normal in T cells lacking Vps34 (Supplemental Figure 2D). In T lymphocytes, amino acid withdrawal (HBSS) did not seem to induce autophagy to levels over that of serum withdrawal (iRPMI), but was readily inhibited by the addition of 5mM 3-Methyladenine (3MA) (Figure 2D). Finally, we investigated the basal levels of autophagy in Vps34f/fLck-cre T cells using transmission electron microscopy. Although many abnormal vesicles were present in naïve Vps34f/fLck-cre lymphocytes, autophagosomes, with characteristic double-membrane bound morphology and cytoplasmic contents, were clearly observed (Figure 2E). When both electron scarce double-membrane bound structures (autophagosomes) and electron dense structures (lysosomes) were quantified in WT and Vps34 T cell cross sections, no difference was observed, indicating that autophagy initiation was not only intact in the absence of Vps34, but maturation of autolysosomes appeared to be normal as well (Supplemental Figure 2F). Thus, no obvious defects in the three major forms of autophagy (basal, TCR- and starvation-induced) in T cells were observed in Vps34f/fLck-creT cells.

Autophagy-deficient T lymphocytes exhibit several other important defects including impaired clearance of mitochondria, increased production of reactive oxidative species (ROS), and an inability to proliferate upon TCR stimulation (9, 11). However, Vps34f/fLck-cre T cells had comparable levels of mitochondria and ROS to those in wild type T cells (Supplemental Figure 2A and 2B), indicating that both mitophagy and pexophagy are intact in maintaining the intracellular redox state in the absence of Vps34. Furthermore, Vps34f/fLck-cre T cells had equal proliferative capacity to wild type T cells from littermate controls (Supplemental Figure 2C). Finally, in order to measure autophagic flux of a specific substrate, we measured the levels of p62 (SQSTM1), which is selectively degraded during autophagy. In fresh Vps34f/fLck-cre CD4 T cells, levels of p62 were slightly higher than WT controls, and this difference was more pronounced 6hrs after TCR stimulation. However, 20hrs post stimulation, Vps34f/fLck-cre T cells had efficiently downregulated p62 to WT levels, which were considerably lower than p62 levels in resting T cells (Supplemental Figure 2E). Collectively, these results demonstrate that Vps34 is not required for auto phagic initiation nor substrate inclusion into autophagosomes in T lymphocytes.

Decreased Bcl-2 expression in Vps34f/fLck-cre T cells

A potential explanation for the defective survival of Vps34f/fLck-creT cells could be altered levels of Bcl-2 family members. Bcl-2 and Mcl-1 are crucial for the survival of naïve T 14 lymphocytes in vivo (27). We examined Bcl-2 levels in CD4+ SP thymocytes and naïve T cells lacking Vps34 by intracellular staining. The levels of Bcl-2 in CD4+ T cells from Vps34f/fLck-cre mice were reduced by 30–80% (Figure 3A). The Bcl-2 level was also slightly reduced in Vps34f/+Lck-cre CD4+naive T cells (Figure 3A). Interestingly, Mcl-1 expression level was higher in Vps34f/fLck-creT cells than that in control T cells (Figure 3B). These results are in contrast to those observed in Atg7-deficient T cells that Bcl-2 expression level was 2-fold higher while Mcl- 1 expression was not altered compared to control T cells (11). These data suggest that the reduced Bcl-2 expression levels may cause the survival defect of Vps34f/fLck-cre T cells.

Figure 3.

Figure 3

Impaired IL-7Rα expression and signaling in Vps34f/fLck-creT cells. (A) Levels of Bcl-2 protein in Vps34f/fLck-cre T cells. Intracellular Bcl-2 staining of Vps34f/fLck-cre T cells was normalized to that of WT T cells (n≥3). Student’s T tests compare the MFI’s of the Bcl-2 levels to those of the WT controls before normalization. (B) Intracellular staining of Mcl-1 in Vps34f/fLck-cre T cells. Shaded histograms represent Ig-matched isotype controls. (C) IL-7Rα expression on Vps34f/fLck-crethymocytes. IL-7Rα surface expression was examined on CD4+SP thymocytes that were freshly isolated, starved for 6 hours in HBSS, or incubated withrIL-7 (5ng/ml) for 6 hours. Numbers represent MFI’s of indicated proteins. (D) IL-7Rα mRNA expression in Vps34f/fLck-cre T cells. Freshly isolated naïve CD4+ T cells from WT and Vps34f/fLck-cre mice were measured for IL-7Rα mRNA expression with two primer sets, one 5′ and one 3′, and normalized to β-actin. Student’s T tests compare the mRNA levels to those of the WT controls before normalization. (E) IL-7 induced Stat 5 phosphorylation in Vps34f/fLck-cre T cells. Naïve CD4+ T cells were stimulated with IL-7 and assessed for Stat 5 phosphorylation by intracellular staining. Student’s T tests compare the pStat 5 MFI’s of Vps34f/fLck-cre T cells under each stimulation condition to the corresponding WT MFI. Numbers indicate MFI’s of pStat 5. (C, E) Un shaded histograms represent WT T cells, dark gray histograms represent Vps34f/fLck-creT cells, lightly shaded histograms are isotype controls. All data are representative of 3 experiments unless otherwise specified.

Impaired IL-7Rα dynamics in Vps34f/fLck-cre T cells

One place that Vps34 may mediate its effect is on intracellular sorting. SinceVps34 is localized largely to early endosomes, multivesicular bodies, and the Golgi (18, 28) and Vps34f/fLck-cre T cells have reduced survival and Bcl-2 expression, we hypothesized that Vps34 may regulate homeostatic sorting of pro-survival cytokine receptor(s) in T lymphocytes. IL-7/IL- 7R interaction provides critical survival signals to naïve T cells (2) and recent data suggest that IL-7Rα is internalized and a fraction of the internalized receptor recycles back to the surface on human T lymphocytes (7, 8). To investigate whether Vps34 regulates IL-7Rα surface display, we examined IL-7Rα levels on freshly isolated wild type and Vps34f/fLck-cre thymocytes or CD4+ naïve T cells. Interestingly, IL-7Rα levels on Vps34−/− CD4+ SP thymocytes or naïve T cells were lower than wild type controls (Figure 3C and 5A). In these experiments, DP thymocytes were used as a baseline, since they express very little IL-7Rα, indistinguishable from isotype controls. When thymocytes were placed in media without exogenous IL- 7, Vps34f/fLck-cre CD4+ SP thymocytes were unable to maintain IL-7Rα expression (Figure 3C). 15 However, Vps34f/fLck-cre CD4+ SP thymocytes efficiently downregulated IL-7Rα surface expression upon IL-7 stimulation (Figure 3C). The reduced IL-7Rα surface expression in Vps34f/fLck-cre T cells was not due to reduced mRNA of IL-7Rα (Figure 3D). These results demonstrate that IL-7Rα internalization and transcription are not impaired.

Figure 5.

Figure 5

Intracellular trafficking of IL-7Rα in Vps34f/fLck-cre T cells. (A) IL-7Rα expression in permeabilized(blue) or surface (red) stained of freshly isolated CD4+CD44lo T cells in WT and Vps34f/fLck-cre T lymphocytes. Numbers indicate MFI’s of IL-7Rα. (B) Proportion of surface IL-7Rα expression on Vps34f/fLck-cre T cells. The results are derived from staining shown in (A) from 4 experiments. (C) Expression of HRS, an ESCRT-0 protein, in WT and Vps34f/fLck-cre T cells. Intracellular staining of HRS was measured by FACS. (D) Average volume of HRS containing MVB in WT and Vps34f/fLck-cre T cells as measured by fluorescence microscopy. NRH-PE was used to label endocytotic vesicles and normalize HRS fluorescence. (E) Expression of Vps35 (retromer subunit) andVps36 (ESCRT-II subunit) in Vps34f/fLck-cre T cells. Intracellular staining of Vps35 and Vps36 were performed on WT and Vps34f/fLck-cre T cells. Accumulation of these proteins indicates lack of MVB maturation in the absence of Vps34. Dotted lines in (A, C and E) are isotype matched antibody control. Data are representative of three experiments. (C, D, and E) Vps34=Vps34f/fLck-cre

To test the functional consequence of impaired IL-7Rα surface expression, we examined Stat5 phosphorylation in Vps34f/fLck-cre T cells. Lymphocytes from Vps34f/fLck-cre and wild type littermate control mice were starved for 6 hrs and cultured with various doses of rIL-7 for 20 minutes. Cells were fixed and levels of phosphorylated Stat5 were measured. Phosphorylation of Stat5 in Vps34f/fLck-cre CD4+ lymphocytes in response to IL-7 was impaired and the defect could not be overcome by a high level of IL-7 (10 ng/ml) (Figure 3E). B220+ B cells present in the same sample served as positive control had a maximal upregulation of phosphorylated-Stat5 (Figure 3E). Together, these results demonstrate that Vps34 regulates IL- 7Rα surface expression in T lymphocytes through a transcription independent mechanism.

Disrupted IL-7Rα intracellular trafficking in Vps34f/fLck-cre T lymphocytes

Given the defective IL-7Rα surface expression dynamics and Vps34’s previous description as an inducer of autophagy as well as normal lysosomal trafficking, we examined the intracellular structure of Vps34-deficient T cells by TEM. In wild type T cells, vesicular structure was ordered and symmetric (Figure 2E), with clear endoplasmic reticulum and Golgi structures. Vesicles budding off the ER were visible, as were autophagic vacuoles. In contrast, the vesicles in Vps34-deficient T cells were completely entropic and asymmetric, suggesting a defect in vesicle maturation and efficient trafficking (Figure 2E).

Recent studies suggest that IL-7Rα is constitutively internalized via clathrin-coated pits and the internalization is increased upon IL-7 stimulation (7). Upon internalization, a fraction of IL-7Rα is degraded while a significant fraction recycles back to the cell surface. We tested IL- 7Rα intracellular trafficking and degradation using fluorophore-labeled recombinant IL-7. When added to in vitro cultures of primary T lymphocytes, wild type naïve CD4+ lymphocytes efficiently took up the IL-7, and over a period of 20 hr, gradually degraded the fluorophore signal (Figure 4A and 4B), presumably via the lysosome and proteasome (7, 8). However, although Vps34-deficient T cells only took up ~50% of the amount of labeled IL-7 and some only had background levels of the cytokine (Figure 4A), by 4 hr, the amount of labeled IL-7 on these cells was equal to that on control T cells on a per cell basis (Figure 4B). By 20 hr, all the initially bound IL-7 was still present in Vps34f/fLck-cre T cells (Figure 4A and 4B). In contrast, B220+ cells from Vps34f/fLck-cre mice took up similar levels of IL-7 (Figure 4A) and degraded the cytokine within 20 hr (data not shown). These results suggest that Vps34f/fLck-cre T cells had a defect in proper trafficking of the cytokine-receptor complex so it cannot recycle back to the cell surface or traffic to degradative compartments.

Figure 4.

Figure 4

Altered IL-7R dynamics in Vps34f/fLck-cre T cells. (A) Uptake of FITC-conjugated rhIL-7 in freshly isolated, non-permeabilized, control and Vps34f/fLck-cre CD4+ T cells. B cells were used as controls. (B) IL-7 degradation in Vps34f/fLck-cre T cells. FITC-rhIL-7 was measured on control and Vps34f/fLck-creT cells at 4 and 20 hours. Data were normalized to the MFI of bound and internalized rhIL-7 on these T cells at 0 hours. (C) Expression of CD132 on the surface of freshly isolated Vps34f/fLck-creT cells. Data are representative of three experiments.

The inability to respond to cytokine was specific to IL-7Rα, as CD132 was expressed normally on Vps34f/fLck-cre T cells at all stages of development (Figure 4C). CD132 has a very simple trafficking pattern. After signaling and endocytosis, it is targeted to late endosomes and degraded by lysosomes (29). The differential effect on IL-7Rα and CD132 expression on Vps34f/fLck-cre T cells suggests that IL-7Rα is regulated in a different manner. We have also tested the receptor levels for several other cell surface markers, some of which have known kinetics and trafficking patterns. We measured the expression of these markers in permeabilized and non-permeabilized cells. CD3, CD25 (IL-2Rα) and TCRβ, which all have very rapid recycling patterns dependent on early endosomal function, as well as CD124 (IL-4Rα), CD5, and α4β7 integrin, all had identical expression regardless of Vps34 expression (Supplemental Figure 3). These results suggest that the defective IL-7Rα expression on Vps34f/fLck-cre T cells is not due to a global defect in cell surface receptor expression.

A significant proportion IL-7Rα is intracellular

To further investigate the regulation of IL-7Rα intracellular trafficking by Vps34, we measured the levels of IL-7Rα before and after permeabilization of control and Vps34f/fLck-cre T cells. When T cells were permeabilized, the levels of IL-7Rα were doubled from cell surface staining (Figure 5A). In Vps34f/fLck-cre T cells, the levels of intracellular IL-7Rα (Total staining minus cell surface staining) were comparable to those in wild type T cells (Figure 5A). When compared to wild type T cells, the proportion of membrane-bound IL-7Rα in Vps34f/fLckcre T cells was reduced by 60–70% (Figure 5B). Since MVB’s are a major subcellular sorting site, and several ESCRT subunits have either FYVE or GLUE domains, both of which bind PI(3)P (30, 31), these organelles were quantified. Although the expression levels of the GLUE domain-containing HGF-regulated tyrosine kinase substrate (HRS), an ESCRT-0 coreprotein, were similar in Vps34f/fLck-cre T cells (Figure 5C), the size of the MVB as measured by HRS volume under fluorescence microscopy was vastly increased (Figure 5D). We next examined the expression levels of Vps35, a retromer subunit, responsible for retrograde translocation of post endocytic components back to the Golgi for surface expression, and Vps36, a GLUE domain-bearing ESCRT-II subunit that can bind ubiquitinated proteins. The expression levels of Vps35 and Vps36 in Vps34f/fLck-cre T cells were increased (Figure 5E). These results suggest that MVBs in Vps34f/fLck-creT cells are less dynamic and have a deficiency in maturing towards either Golgi or lysosomal compartments.

Vps34 localizes to MVB to promote IL-7Rα recycling via Vps36 containing vesicles

To investigate the role of Vps34 in MVB sorting in T lymphocytes, naïve CD4+Tcells were stained with Vps34 and HRS. There was a consistent co-localization between the two markers, indicating that Vps34 localizes to early-stage MVB in T lymphocytes (Figure 6A). However, there was little co-localization between IL-7Rα and HRS (Figure 6A). Much of the IL-7Rα localized in compartments in close proximity to the Golgi enzyme mannosidase (Figure 6B). This brings up the possibility that intracellular IL-7Rα trafficking requires a retrograde transport to the Golgi for surface re-expression. Since Vps34f/fLck-cre T cell shad increased MVB size (Figure 5D), we hypothesized that Vps34f/fLck-cre T cells have impaired inward vesiculation mediated by ESCRT proteins. Using the ESCRT-II proteinVps36 as a marker, which contains a GLUE domain, WT and Vps34f/fLck-creTcells were co-stained for IL- 7Rα and TGN46, a trans-Golgi marker. A majority of the punctate intracellular IL- 7Rα colocalized with Vps36 in WT T cells, but not in Vps34f/fLck-cre T cells (Figure 6C and 6D). We hypothesize that Vps36 needs PI(3)P for proper localization in order to mediate the inward vesiculation of IL-7Rα containing vesicles from the MVB. Consistent with results seen in Figure 6B, a significant portion of both IL-7Rα and Vps36 also localized to the Golgi in WT but not Vps34f/fLck-creT cells (Figure 6C). This suggests that IL-7Rα requires MVB to Golgi sorting for proper recycling.

Figure 6.

Figure 6

InternalizedIL-7Rα localizes primarily within Vps36 vesicles. Freshly isolated T cells were fixed, permeabilized and stained with indicated antibodies for fluorescence microscopy. (A) Vps34 colocalizes with HRS in MVB. (B) IL-7Rα is enriched near the Golgi apparatus. (C) Vps36 colocalizes with IL-7Rα intracellularly, and to a lesser extent, with TGN46 in WT T cells. (D) Quantification of IL-7Rα and Vps36 colocalization (n=20). (E) Colocalization between IL- 7Rα and Vps35 (n=20). Data are representative of two experiments. (C, D, and E) Vps34−/− = Vps34f/fLck-cre

To further investigate the possibility of a retrograde endosome to Golgi recycling event necessary for optimal IL-7Rα surface expression, naïve CD4+T cells were starved for 2 hr, and co-stained for Vps35 and IL-7Rα. Vps35 is involved in the retromer complex that has been shown to mediate retrograde transport of early- or late-endosomal cargos for secretion or surface re-expression (32), that would otherwise be degraded in the late-endosomal to lysosomal pathway (33). A significant portion of intracellular IL-7Rα co-localized with Vps35 positive compartments (Figure 6E), although not nearly as well as with Vps36. Very little IL-7Rα was observed colocalizing with Vps35 in the absence of Vps34. This observation is consistent with previous data that at steady-state some internalized IL-7Rα is not recycled (8).

Defective IL-7Rα recovery in Vps34f/fLck-creT cells

To investigate the kinetics of IL-7Rα surface expression in the absence of Vps34, pronase was administered to purified naïve CD4+ cells and the recovery of surface IL-7Rα was measured as described (34). In short, this proteinase was administered to T cells to cleave surface receptors and the cells were allowed to recover in media for the indicated time points. The receptor was detectable on the surface of WT cells within 2 hr post cleavage, and reached peak levels by 6 hr under starvation conditions (Figure 7A). However, Vps34f/fLck-cre T cells had minimal recovery up to 6 hrs post cleavage and not until 8 hrs post treatment did a majority of IL-7Rα recover (Figure 7A). The recovery of IL-7Rα surface expression was inhibited by the microfilament polymerization inhibitor, cytochalasin B, up to 4 hours post pronase treatment (Figure 7B and 7C). However, addition of the protein biosynthesis inhibitor cycloheximide (CHX), had minimal effect on IL-7Rα recovery in wild type T cells until 6 hr post treatment (Figure 7B and 7C). These results suggest that naïve T cells have an intracellular store of IL-7Rα sufficient to withstand at least several hours of starvation without the need to translate new protein. Such a mechanism would allow naïve T cells to rapidly adapt autonomously to changes in local cytokine concentration, perhaps even by polarizing receptor surface display towards 20 higher local concentrations. In contrast, Vps34f/fLck-creT cells have minimal ability to mobilize intracellular IL-7Rα and must rely on de novo synthesized receptor at later time points, putting them at a disadvantage.

Figure 7.

Figure 7

Defective IL-7Rα recovery in Vps34f/fLck-cre T cells that is dependent on vesicle trafficking. (A) IL-7Rα expression on Vps34f/fLck-cre T cells post-pronase treatment. Vps34f/fLck-cre CD4+ T cells were treated twice with 0.08% pronase for 15 minutes, washed, and allowed to recover for indicated times, in the presence or absence (B) of cycloheximide (CHX) or cytochalasin B (cyto B). Gray histograms represent IL-7Rα surface levels at time 0, immediately after pronase treatment and indicative of pronase efficacy. (C) Values from (A) and (B) are calculated, normalized to starting IL-7Rα surface expression. n=4; ND=Not determined. Data are representative of two independent experiments. (A, B, and C) Vps34−/− = Vps34f/fLck-cre

Bcl-2 can partially rescue Vps34-deficiency

Given that Vps34f/fLck-creT cells have defective survival, reduced IL-7Rα and Bcl-2 expression, we asked whether genomic deletion of several pro-apoptotic Bcl-2 family members or over expression of Bcl-2 itself, could rescue Vps34 deficiency in T lymphocytes. Deletion of Bim, a BH3-only pro-apoptotic protein that is induced upon cytokine withdrawal, had no effect on Vps34 deficiency (Supplemental Figure 4A). Similarly, deletion of Bax did not rescue the survival defect of Vps34f/fLck-cre T cells (data not shown). However, the expression of a human Bcl-2 transgene (35) significantly promoted early Vps34f/fLck-creT cell survival at the CD4+ SP lineage (Supplemental Figure 4B and 4C). These results suggest that the defective survival ofVps34f/fLck-creT cells is not solely due to a reduced expression of Bcl-2.

Discussion

Phosphatidylinositol 3-kinases consist of three classes and play important roles in a wide variety of cellular functions (36). Although extensive studies have been performed on the function of class I and II PI3Ks, relatively little is known about the specific function of Vps34, the sole class III PI3K in mammals in vivo. By specifically deleting Vps34 in T lymphocytes, we have found that Vps34 is essential for thymocyte survival and naïve T lymphocyte homeostasis. Vps34 regulates T cell homeostasis by promoting their survival via cell surface expression of IL- 7Rα. It is clear that IL-7Rα is regulated at both the transcriptional and post translational level. IL-7Rα is continuously internalized for recycling and degradation. Our results have mapped out a detailed route for IL-7Rα intracellular trafficking that is critically regulated by Vps34 at multiple points.

Although Vps34 is critical for autophagy induction in other types of cells (13, 14), our data demonstrate that it is not required for autophagy induction in T lymphocytes. This conclusion is supported by our findings that three major forms of autophagy including TCR-induced, starvation, and basal level autophagy in Vps34f/fLck-creT cells are comparable to those in wild type T lymphocytes. Additionally, the turnover of an autophagy-specific substrate, p62/SQSTM1, is normal in Vps34f/fLck-cre T cells. Importantly, there are significant differences on the phenotypes exhibited by autophagy-deficient (Atg5−/− or Atg7f/fLck-cre) and Vps34f/fLck-creT lymphocytes. Both types of T lymphocytes undergo apoptosis. However, autophagy-deficient T lymphocytes have increased mitochondria, elevated ROS production and Bcl-2 expression, and normal IL-7Rα surface expression (9, 11). This phenotype in autophagy deficient T cells is caused by a failure in clearance of excess mitochondria when thymocytes exit to the periphery, consistent with an essential role for autophagy to regulate intracellular organelle homeostasis. In contrast, Vps34f/fLck-cre T cells do not have detectable defects in mitochondria clearance. These results suggest that Vps34 is either not involved or functionally compensated for in the autophagy induction pathway by other molecules in T lymphocytes, perhaps even by other classes of PI3K’s or degradation products of PI3KI substrates. The ability of 3-MA to successfully limit autophagy induction in T lymphocytes would indicate that Vps34 and class I/II PI3K are all involved in this process, either in compensating for Vps34 kinase activity, contributing to PI3K scaffolding, or by generating higher ordered PIP substrates that could be devolved to PI(3)P.

The inability to maintain IL-7Rα surface expression on Vps34f/fLck-creT cells likely plays a predominant role in their increased apoptosis. Consistent with the lowered IL- 7Rα surface expression, Vps34f/fLck-creT cells have lowered expression of Bcl-2 and defective signaling as reflected by reduced phospho-Stat5 upon IL-7 stimulation. Interestingly, Mcl-1, another member of the Bcl-2 family that is critical for naïve T cell survival, is expressed at a higher level in Vps34f/fLck-creT cells than that in control T cells. This result suggests that Mcl-1 may be subjected to additional level of regulation that depends on Vps34. Nevertheless, IL- 7Rα mediated signaling to promote T cell survival is not just restricted to its role in promoting the expression of the anti-apoptotic Bcl-2 family members (2). IL-7R stimulation also activates PI3K-AKT-mTOR pathway and sustains expression of the glucose transporter GLUT1 (37). Thus, transgenic expression of Bcl-2 can only partially rescue the T cell defect in IL-7Rα-deficient mice (38, 39). Consistent with this, crossing of Vps34f/fLck-cremice to a Bcl-2 transgenic line only partially rescued the defective T cell compartment. Interestingly, deletion of Bim did not have any rescuing effect on the T cell compartment in Vps34f/fLck-cremice. This is in sharp contrast to a near normal T cell compartment in IL-7Rα−/−Bim−/− mice (40). This result strongly suggests that Vps34f/fLck-creT cells have additional defect that is not caused by the lowered surface expression of IL-7Rα. This is in agreement with the profound effect seen on DP cells, which do not express IL-7Rα, yet still succumb to apoptosis. Although the deletion efficiency in more mature populations was over 80% (Supplemental Figure 1), the remaining DP cells still had 75% of WT Vps34 protein levels. This is consistent with a long half-life of Vps34 protein and suggests that as Vps34 is gradually deleted in developing Vps34f/fLck-cre thymocytes, more mature T lineage cells die from a progressive Vps34 loss. Since the developing DP thymocytes were not synchronized, it remains possible that the loss of Vps34 in DP thymocytes was not synchronous, and that thymocytes which lost Vps34 earlier in development were the first victims of apoptosis. However, those DP thymocytes which deleted Vps34 later in development could have produced or scavenged PI(3)P at levels sufficient to maintain cell viability.

Our results have also suggested a clear route for normal IL-7Rα intracellular trafficking. Under steady-state conditions, IL-7Rα that has not been bound to ligand is internalized, and returned to the surface through homeostatic recycling (8). IL-7R internalized by the cell is sorted via the early endosome into the multivesicular body. The internalized receptor proteins are shuttled through the network of MVB compartments, and our data shows ESCRT-0 and ESCRTII seem to be distinctly located within the network. Some of the receptor under optimally signaled conditions will be destined for the late endosome, and ultimately, the lysosome (7). However, during the MVB to late-endosomal transition, Vps34 activity can save the IL-7Rα chain from destruction for a retrograde endosomal to Golgi translocation for surface reexpression. It is likely that this MVB to late-endosomal transition is the rate-limiting step in this whole process, since we observed a majority of the internalized IL-7Rα within the Vps36 compartment, but not the HRS-bearing compartment, after taking the cells from an IL-7 rich environment in vivo and starving them.

This process appears to be disrupted in the absence of Vps34 at several stages. First, Vps34 has a profound effect on the localization and maturation of HRS-containing multivesicular bodies. This early ESCRT protein has been shown to regulate the fate of Na+ channel by binding to the ubiquitinated form and either targeting it for degradation or shuttling it into a later cell surface recycling pattern (24). The HRS-containing ESCRT-0 complex is generally thought to cluster ubiquitinated cargoes for further intralumenal vesicle formation (41), and we observe a striking aggregation of this complex in the MVB of T cells lacking Vps34.

Second, Vps34 deficiency disrupts the localization of Vps36, which contains a GLUE domain. This protein functions as part of the ESCRT-II complex, which has been shown to be important for membrane invagination and budding of ubiquitin positive-containing intralumenal vesicles (41). The consequence of this mislocalization likely accounts for not only the inability of IL-7Rα to enter the late-endosomal to retromer pathway, but also accounts for the inability for internalized IL-7 to be properly targeted to lysosome. In the absence of Vps34, internalized IL-7Rα might be either mislocalized within abnormal Eea1 negative early endosomes (42), or in HRS negative early MVB compartments, that are unable to further mature without PI(3)P and become aggregated intracellularly. The alternative intracellular sorting pathway in the absence of Vps34 kinase activity remains to be determined.

Finally, it has been shown that Vps34 has a profound effect on the localization of Eea1, which contains the eponymous FYVE domain, in the early endosome, synergizing with Rab5 (42). This interaction has been proposed to be necessary for both endosomal membrane fusion within the early endosomal compartment, as well as providing an inward directionality to vesicle 25 transport (42). However, internalization of IL-7Rα is intact in the absence of Vps34, and the surface levels of many other receptors, which undoubtedly require unique sorting mechanisms, are normal in Vps34f/fLck-cre T cells.

Ideally in a clinical setting, pharmacological inhibition or enhancement of Vps34 activity could be used to “tune” the amount of pro-survival receptors in various pathogenic settings. If IL- 7Rα surface expression can be modulated, allowing for maximal numbers of T lymphocytes to be sustained off highly limited amounts of growth factors, then the repertoire of polyclonal naïve CD4+ T cells can be maintained or diversified into old age. Additionally, the amounts and clonalities of memory responses might be modulated in this manner, as IL-7Rα has been implicated as a marker for memory cell precursors. This would suggest that Vps34 is a highly attractive candidate for therapeutic intervention in preventing or modulating various lymphopenic conditions.

Supplementary Material

1

Acknowledgments

We thank Dr. Jeff Rathmell for his thoughtful reading and advice on the manuscript.

This work is supported through NIH grants AI074944 and AI074754 to Y. He

Footnotes

Online Supplemental Information

Supplementary information is associated with this manuscript

Competing Interests

The authors declare that they have no competing financial interestsraises

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