A Role for Autophagic Protein Beclin 1 Early in Lymphocyte Development

Autophagy is essential for maintaining cellular energy homeostasis during nutritional stress, and it also appears to play important roles in development and differentiation. During nutrient starvation-induced autophagy, portions of the cytoplasmic material are sequestered within double-membrane vesicles and then delivered to the lysosome for degradation. Beclin 1, the mammalian ortholog of yeast Atg6, is a critical component of a complex containing class III PI3K and other proteins, including UVRAG, Ambra-1, Bif-1, and Atg14L, that stimulates autophagy by initiating the isolation membrane formation (1, 2). Beclin 1 also interacts with antiapoptotic Bcl-2 family members, Bcl-2 and Bcl-x_L (3, 4), and it is believed that this interaction could represent an important link between autophagy and apoptosis. Several studies have demonstrated that Beclin 1 can affect the apoptotic pathway in different types of eukaryotic cells (5–7). In addition, Bcl-2 has been shown to inhibit Beclin 1-mediated autophagic death induced by nutrient starvation in cultured cells (4).

The analyses of several genetically modified mouse strains and early embryonic stem cell (ESC)-derived mouse embryos deficient in proteins essential for autophagy, including Beclin 1, indicate that autophagy is critical for maintaining cellular energy homeostasis and normal development (8–12). Beclin 1-deficient mice die during early embryonic development (8, 13), which prevents the analysis of Beclin 1 deficiency in specific tissues. In our previous report using Beclin 1-GFP transgenic mice, we provided evidence that Beclin 1 could be involved in T cell development in the thymus (14). In this study, we used a recombination activating gene 1 (Rag1) blastocyst complementation approach (15) and in vitro ESC differentiation to T cells to analyze the role of Beclin 1 in T and B cells in more detail. Our studies indicate that Beclin 1 plays an essential role in maintaining normal thymic cellularity and early B cells in the bone marrow. However, it appears to be largely dispensable for initial generation and proliferation of peripheral T and B cells. The absence of Beclin 1 does not appear to cause any specific block in the development of T or B lineage cells. Rather, Beclin 1 deficiency results in impaired maintenance of lymphoid progenitors in vivo and in vitro. Our results thus reveal a selectively early role for Beclin 1 in lymphocyte development distinct from that observed for other autophagic proteins, such as Atg5 and Atg7.

Rag1^−/− mice of both sexes were purchased from The Jackson Laboratory. Rag1^−/− blastocysts harvested from pregnant Rag1^−/− female mice (3.5 post-coitum) were injected with 8–15 of either Beclin 1^−/− or Beclin 1^+/− ESCs and implanted into pseudopregnant foster mothers. Both ESC clones used in this study were described previously (8). All chimeric mice were maintained under specific pathogen-free conditions at either The Mount Sinai Animal Care Facility or The Rockefeller University Animal Care Facility and used at 4–8 wk of age. All studies and procedures were approved by Animal Care and Use Committees at both institutions.

Fluorescently labeled mAbs against mouse CD4 (clone GK1.5), CD8 (clone 53-6.7), CD43 (clone IM7), IgM (R6-60.2), CD43 (clone S7), B220 (clone RA3-6B2), CD5 (53-7.3), CD229.1 (Ly9.1, 30C7), CD21 (7G6), CD23 (B3B4), and CD117 (c-Kit, 2B8), and allophycocyanin-conjugated lineage mixture were purchased from BD Biosciences (San Jose, CA). PE-conjugated anti-mouse IgD (clone 11-26) was from Southern Biotech (Birmingham, AL). Abs against mouse CD69 (clone H1.2F3), CD25 (PC61.5), CD44 (IM7), CD127 (IL-7Rα, clone A7R34), and Ly-6A/E (Sca-1, clone D7) were purchased from eBioscience (San Diego, CA). Single-cell suspensions were generated from the spleen, bone marrow, and thymus, and stained for 30 min on ice using saturating concentrations of directly conjugated mAbs. Cells were washed three times with PBS containing 2% FBS, and data were acquired using either FACScan or LSR II flow cytometers (BD Biosciences, San Jose, CA). Flow cytometry data were analyzed using FlowJo software (Tree Star, Ashland, OR).

In vitro differentiation of ESCs to T cells was done as previously described (16). In brief, both ESC clones, Beclin 1^−/− and Beclin 1^+/−, were maintained in an undifferentiated state on mitomycin C arrested mouse embryonic fibroblasts (Millipore/Chemicon, Billerica, MA) in ESC media (16) with addition of 10³ U/ml LIF (ESGRO from Millipore/Chemicon). Coculture experiments were initiated by seeding 5 × 10⁴ ESCs on top of a ∼80% confluent monolayer of the OP9-R stromal cell line in OP9 media (16). Mesoderm colonies were trypsinized on day 5 of coculture, and 5 × 10⁵ cells were plated on a new OP9-R monolayer with additional supplement of Flt-3 ligand (R&D Systems, Minneapolis, MN) to a final concentration of 5 ng/ml in the OP9 media. The formation of hematopoietic stem cells (HSCs) was observed by day 8 of coculture, and HSCs were transferred to an OP9-DL1 monolayer of cells that were virally transduced to express the Notch ligand Delta-like 1 (DL1) (17). From this point, the OP9 medium was supplemented with Flt-3L and IL-7 (PeproTech, Rocky Hill, NJ) to a final concentration of cytokines of 5 and 1 ng/ml, respectively. Cocultures were subjected to alternating media change and no-trypsin passages in 2-d intervals. Differentiating lymphocytes were analyzed on days 12 and 19/20 by flow cytometry.

Live cells were gated by excluding dead cell discriminator-positive cells (Invitrogen, Carlsbad, CA). OP9 feeders were excluded by staining with rat anti-mouse CD45 Ab conjugated to PE (Caltag/Invitrogen, Carlsbad, CA) and gating on the CD45⁺ population of cells. For day 12 analyses, the differentiated lymphocytes were further stained with rat anti-mouse CD44 conjugated to fluorescein (FITC) and rat-anti mouse CD25 conjugated to allophycocyanin (both from Caltag/Invitrogen Laboratories). On day 19/20, differentiated lymphocytes were analyzed with rat anti-mouse CD4 conjugated to allophycocyanin (Caltag/Invitrogen Laboratories) and rat anti-mouse CD8α conjugated to FITC (BD Pharmingen, Franklin Lakes, NJ).

For T cell stimulation, flat-bottom 96-well plates were precoated with 10 μg/ml 2C11 Ab in PBS (BD Biosciences) for 4 h in a tissue culture incubator at 37°C or for 12 h at 4°C. Plates were washed three times with ice-cold PBS, and splenocytes (2 × 10⁵ cells/well) were incubated in RPMI 1640 media supplemented with 10% FBS. For B cell proliferation assays, naive splenic B cells were purified by depletion-type magnetic separation using CD43 (Ly-48) MACS beads (Miltenyi Biotec, Auburn, CA) and purified B cells (2 × 10⁵ cells/well) cultured in RPMI 1640 media supplemented with 10% FBS in the presence of 5 ng/ml IL-4 (Sigma-Aldrich, St. Louis, MO) and 25 μg/ml LPS (Sigma-Aldrich).

Cultures were incubated in a tissue culture incubator at 37°C in a humidified atmosphere containing 5% CO₂ for 24 or 48 h. Proliferation assays were performed using BrdU-based calorimetric ELISA kit (Roche, Nutley, NJ). In brief, cells were stimulated in a tissue culture incubator for 48 h, pulsed overnight with BrdU (10 μM), followed by incubation with peroxidase-labeled anti-BrdU Ab. Development of color after substrate (tetramethyl-benzidine) addition was monitored using an ELISA reader at 370 nm.

For CFSE-based T cell proliferation assays, spleen cells were labeled with 0.5 μM CFSE in PBS (Invitrogen) for 8 min at room temperature (RT). To quench the reaction, we added FCS immediately to the final concentration of 20%, and washed cells three times in PBS and resuspended them in RPMI 1640 media supplemented with 10% FBS. The CFSE-labeled spleen cells were then incubated with plate-bound 2C11 Ab (10 μg/ml) in a tissue culture incubator at 37°C in a humidified atmosphere containing 5% CO₂ for 72 h and analyzed using FACScan flow cytometer.

Spleen T cells were isolated using Dynal mouse T cell-negative isolation kit (Invitrogen) and stimulated in 24-well plates in the presence of T cell-activating magnetic beads coupled with anti-CD3 and anti-CD28 Abs (Invitrogen) and IL-2 (0.5 ng/ml) in a CO₂ incubator for 7 d. After incubation, stimulating beads were removed with a magnet, and cytospins were prepared using Shandon cytospin (Thermo Fisher Scientific, Waltham, MA) with ∼1 × 10⁵ T cell on each microscope slide. T cells were subsequently fixed for 20 min in 4% paraformaldehyde in PBS on ice, washed twice with PBS/1% BSA, and permeabilized 5 min at RT with 0.5% saponin/0.03 M sucrose/1% BSA in PBS. The cells were washed once with PBS/1% BSA and blocked using 5% normal goat serum in PBS/1% BSA for 15 min at RT. After a single wash with PBS/1% BSA, T cells were incubated with polyclonal anti-LC3 Ab PM036 (MBL International Corporation, Woburn, MA) for 60 min at RT. After this incubation, T cells were three times and then blocked with 5% normal goat serum PBS/1% BSA. After subsequent wash with PBS/1% BSA, T cells were incubated with FITC-conjugated goat anti-rabbit Ab for 30 min at RT. T cells were finally washed three times in PBS/1% BSA, nuclei counterstained with DRAQ5 (Biostatus Limited, Leicestershire, U.K.) DNA stain for 5 min at RT, and mounted using SlowFade Gold antifade reagent (Molecular Probes, Eugene, OR). The cells were visualized using an Olympus Fluoview300 confocal microscope (Olympus, Center Valley, PA) at a magnification of ×600.

Statistical analysis was performed using two-tailed Student t test. A p value <0.05 was considered statistically significant.

Targeted disruption of Beclin 1 in mice results in early embryonic lethality before generation of a lymphoid system, thus preventing the analysis of in situ lymphoid development (8). To circumvent this obstacle, we generated Beclin 1^−/−→Rag1^−/− chimeric mice and control Beclin 1^+/−→Rag1^−/− chimeras using Beclin 1^−/− and Beclin 1^+/− ESC clones, respectively. Because Rag1^−/− mice cannot produce mature T and B cells because of deficient V(D)J recombination (18), any mature lymphocytes in chimeric mice must be derived from microinjected ESCs, indicating that they have the potential to complement Rag1 deficiency in lymphopoiesis. The injection of Beclin 1^−/− and Beclin 1^+/− ESC clones into Rag1^−/− blastocyst mostly produced mice with extensive agouti-black coat-color chimerism (≥80%), which confirmed the developmental potential of Beclin 1^−/− and Beclin 1^+/− cells. Because ESC clones used in this study are of 129/SvJ origin, any lymphocytes derived from these cells can be easily distinguished from Rag1^−/−-derived cells, which are of C57BL/6 origin, based on the expression of the Ly9.1 alloantigen. This alloantigen, present on ES but absent on Rag1^−/−-derived cells, is a surface glycoprotein expressed by most thymocytes, peripheral B and T cells, bone marrow lymphoid cells, and hematopoietic progenitors.

To determine T cell development in Beclin 1-deficient chimeric mice, we analyzed thymocytes using flow cytometry. Virtually all double positive (DP) and single positive thymocytes in chimeric mice expressed the Ly9.1 surface marker, indicating that they developed from injected ESCs (data not shown). In 20% of Beclin 1^−/−→Rag1^−/− chimeras, a complete reconstitution of the major thymocyte populations was observed compared with control Beclin 1^+/−→Rag1^−/− chimeras (Fig. 1A). However, in about 80% of chimeras, we have detected various degrees of loss of Beclin 1-deficient DP thymocytes (Fig. 1B, 1C). This loss ranged from 20% to almost complete depletion of DP thymocytes in some Beclin 1^−/−→Rag1^−/− chimeras (Fig. 1C) but was never observed in control animals. Enumeration of thymocytes and spleen cells by trypan blue exclusion also revealed a dramatic reduction of thymocyte numbers in Beclin 1^−/−→Rag1^−/− chimeras (Fig. 1D). Different levels of chimerism in individual mice could potentially explain the high variability of thymocyte numbers observed in control chimeras (Fig. 1D). Nevertheless, the differences between Beclin 1-deficient and control chimeras are significant (17.7 × 10⁶ versus 72.8 × 10⁶; p = 0.0002).

The dramatic loss of thymocytes in Beclin 1^−/−→Rag1^−/− chimeras could potentially arise from a specific block during double negative (DN) thymocyte development. To address this possibility, DN thymocytes expressing the Ly9.1 marker were stained with CD44 and CD25 Abs, and analyzed by flow cytometry (Fig. 1E). Compared with control mice, Beclin 1-deficient chimeras displayed perturbed DN thymocyte populations with accumulation of both DN1 cells (CD44⁺CD25⁻) and the intermediate DN1-DN2 (CD44⁺CD25^lo) cells. Our results indicate that Beclin 1 deficiency results in the loss of thymocytes and apparent abnormalities in the DN thymocyte compartment. However, from these data alone, it is not possible to discern whether the abnormalities observed in Beclin 1-deficient DN thymocytes represent a true developmental block or are a result of competition between Beclin 1^−/− and Rag1^−/−-derived DN cells.

To further investigate the thymocyte abnormalities discovered in Beclin 1^−/− chimeric animals, we used in vitro ESC differentiation system with OP9 stroma cells expressing the DL1 protein (17) to generate T cells in the absence of Rag1^−/− cell competition. After 12 d of coculture, both Beclin 1^−/− and control ESCs were capable of generating normal DN thymocyte subsets, as indicated by similar CD25 versus CD44 profiles, as well as similar numbers of c-Kit^hi DN1 (CD25⁻CD44⁺) cells containing early T-lineage progenitors up to day 16 (Fig. 2A and data not shown). We also observed that the rate of DN2/3 cell generation from CD45⁺ precursors (as indicated by the ratio of CD25⁺/CD45⁺ cells) was equivalent in both knockout and control cocultures (Fig. 2B). This would rule out an intrinsic developmental block at this stage of thymocyte development caused by Beclin 1 deficiency. Remarkably, however, day 19/20 cocultures derived from Beclin 1^−/− ESCs displayed sharp declines in DN thymocyte populations compared with control Beclin 1^+/− ESC cocultures (10.44 ± 3.1 versus 1.66 ± 0.52; p < 0.05; Fig. 2C, 2D). We also observed impaired output of more mature CD8⁺ T cells that is likely secondary to the loss of their progenitors in the DN compartment (Fig. 2C, 2D). The coculture-derived Beclin 1-deficient DP thymocytes exhibited normal sensitivity to apoptosis and normal TCR β chain expression (data not shown). Because earlier coculture time points, including days 12 (Fig. 2A) and 16, indicated no difference in the ability of Beclin 1^−/− and control ESCs to initially generate T cells in vitro (unpublished observation), our results from Rag1^−/− chimeras, as well as in this ESC differentiation system, strongly suggest that the failure of Beclin 1^−/− ESC coculture to sustain normal T cell generation in vitro at later time points is caused by impaired maintenance of thymocyte precursors at, or prior to, the DN stage.

Despite the dramatic loss of thymocytes in Beclin 1^−/−→Rag1^−/− chimeras, we did not observe any change in peripheral, spleen CD4⁺ and CD8⁺ T cells numbers (Fig. 1A–C, right dot plot panels, 3A), suggesting that Beclin 1 is not essential for maintaining normal numbers of T cells in the periphery. The analysis of peripheral T cell subsets did not indicate any imbalance among naive CD44^loCD62L^hi, effector CD44^hiCD62L^lo, and memory CD44^hiCD62L^hi T cells in Beclin 1-deficient chimeras (Fig. 3B). Surprisingly, peripheral T cells exhibited normal proliferative response in vitro compared with control T cells (Fig. 3C, 3D), as determined by both BrdU incorporation (Fig. 3C) and CFSE (Fig. 3D) assays, and also displayed normal induction of activation markers, CD69 and CD25 (Fig. 3E). Because previous studies have suggested that autophagy is required for peripheral T cell proliferation (19, 20), we analyzed whether Beclin 1-deficient T cells contained autophagosomes by anti-LC3 staining. The confocal imaging of LC3-stained, activated T cells revealed a reduced number of fluorescent puncta in Beclin 1-deficient versus control T cells (Fig. 4). Beclin 1^−/− T cells contained, on average, 0.72 ± 0.09 fluorescent puncta per cell compared with 1.70 ± 0.12 puncta in control Beclin 1^+/− T cells (p = 0.002). Because the presence of LC3 puncta has been considered a universal marker of autophagosomes (21), our results suggest that Beclin 1-deficient T cells have reduced autophagic activity but normal proliferation.

To determine whether Beclin 1 plays a more general role in lymphocyte development, we used flow cytometry to analyze whether Beclin 1-deficient ESCs could effectively contribute to the bone marrow, as well as to the bone marrow-derived B cell compartment in chimeric mice. This analysis revealed ∼2-fold reduction in the frequency of Ly9.1⁺ bone marrow cells in Beclin 1^−/−→Rag1^−/− chimeras, indicating a somewhat reduced contribution potential of Beclin 1^−/− ESCs (Fig. 5A, 5B, middle panel). In addition, we also observed severely reduced numbers of B220⁺ IgM⁺ cells (Fig. 5B, top panel). The analysis of Ly9.1 marker expression during early stages of B cell development as defined by B220 and CD43 expressions indicates that, as opposed to control chimeras, pro-B cells (B220^lowCD43⁺) from Beclin 1^−/−→Rag1^−/− chimeras contained only a small number of ESC-derived Ly9.1⁺ cells (Fig. 5B, bottom panel). These results indicate that Beclin 1^−/− ESCs fail to efficiently contribute to the early B cell compartment of Rag1^−/− bone marrow.

To analyze the peripheral B cell compartment in chimeric mice, we stained spleen cells using fluorescently labeled Abs and analyzed them by flow cytometry. We found that Beclin 1^−/−→Rag1^−/− chimeras had slightly reduced populations of IgM⁺IgD⁺ B cells (12.6 ± 4.5 versus 19.1 ± 5.3; p < 0.05; n = 7; Fig. 6A, top panel). The frequencies of CD21^hiCD23⁺IgM⁺ T2, as well as CD21⁺CD23⁻IgM⁺ marginal zone B cells, were generally unchanged (Fig. 6A, middle and bottom panels). However, the most developmentally early population of CD21⁻CD23⁻IgM⁺ (T1) peripheral B cells (representing recent bone marrow emigrants) was significantly diminished in Beclin 1^−/−→Rag1^−/− chimeras (5.6 ± 1.7 versus 19.9 ± 3.6; p < 0.05; n = 3; Fig. 6A, bottom panel). Importantly, the analysis of peritoneal B1 cells using CD5 and IgM marker expression revealed normal development of the B1 cell population in Beclin 1^−/−→Rag1^−/− chimeric mice (Fig. 6B). To determine whether peripheral Beclin 1-deficient B cells were functional, we analyzed the proliferative response of purified spleen B cells stimulated in vitro with LPS and IL-4. Our results indicate that in vitro proliferative response of Beclin 1^−/− B cells is not significantly different from the response of control Beclin 1^+/− B cells (Fig. 6C). In summary, we find that Beclin 1 deficiency has far more dramatic effects at early stages of B cell development than at later stages. Thus, similar to T cells, Beclin 1 appears to be largely dispensable for normal numbers and proliferation of B cell populations in the periphery.

The early-stage defects in T and B cell lineage cells of Beclin 1^−/−-deficient chimeras prompted an analysis of early lymphoid precursors in the bone marrow. We analyzed Beclin 1^−/−→Rag1^−/− chimeric mice for potential alterations in common lymphoid progenitor (CLP) and HSC populations, as previously described (22). We observed that Ly9.1⁺ CLPs were substantially reduced in Beclin 1^−/−→Rag1^−/− chimeras compared with control mice, indicating that Beclin 1-deficient ESCs poorly contributed to the CLP pool in chimeric mice (17.54 ± 11 versus 55.2 ± 3.3, p = 0.03; Fig. 7A). In addition to the CLP pool reduction, the CD127⁻ Lin⁻c-Kit⁺ Sca-1⁻ fraction containing common myeloid progenitors (23, 24) was virtually absent in Beclin 1^−/−→Rag1^−/− chimeras even as numbers of Ly9.1⁺ splenic macrophages (CD11b⁺) appeared normal in these mice (Supplemental Fig. 1). These data suggested an upstream defect in earlier hematopoietic progenitors. We therefore further analyzed the cell compartment upstream of the oligolineage progenitors for the presence of HSCs. Beclin 1-deficient ESCs did generate Ly9.1⁺ HSCs, albeit at much reduced frequency (2.3 ± 3.2 versus 42.4 ± 2.8; p < 0.01; Fig. 7B). These results suggest that Beclin 1 is required for normal and/or sustained contribution to the pools of progenitors of lymphocytes in the competitive environment of chimeric mouse bone marrow. This may help explain the early-stage defects observed in Beclin 1-deficient T and B lineage cells.

Several studies have recently addressed the role of autophagy in lymphocytes. First, autophagy has been found to play a role in CD4⁺ T cell death induced by HIV (25), indicating that this process can be triggered in T cells by extracellular cues different from starvation. In addition, T cell activation in vitro induces increased autophagic activity in CD4⁺ T cells (26). These reports, in combination with our previous analyses of Beclin 1 gene regulation in thymocytes (14), prompted the current study of the role of the Beclin 1 autophagy gene in T cell development.

Mice with T cell-specific inactivation of Atg5 or Atg7 genes exhibited a variety of intrinsic defects in thymocytes and peripheral T cells. In the case of thymocytes, these defects involve reduction of thymic cellularity in the absence of a specific developmental block or impaired survival. As for peripheral T cells, reduced numbers of CD4⁺ and CD8⁺ T cells exhibiting impaired survival and proliferation defects, as well as alterations of activation/memory/homeostatic expansion, were observed in the absence of Atg5 or Atg7 (19, 20, 27). These defects are likely caused by impaired autophagy-dependent removal of damaged or aging mitochondria in peripheral T cells (20, 27). It is therefore clear that Atg5 and Atg7 autophagy genes are required at both early and late stages of T cell development.

We found that Rag1 blastocyst chimeras lacking Beclin 1, similar to Atg5 and Atg7-deficient mice, show reduced thymocyte numbers (Fig. 1). We also detected an altered distribution of DN thymocytes in these chimeras with accumulation of cells in the DN1 population (Fig. 1E). This, however, likely does not reflect a genuine developmental block. Rather, it would seem that Beclin 1-deficient DN thymocytes are outcompeted by Rag1^−/−-derived DN cells. This is because, using an in vitro ESC differentiation system for generating T cells, we observed a normal rate of initial DN subset generation and did not detect significant DN subset aberrations at early time points in the coculture (Fig. 2A, 2B). However, we did observe a dramatic reduction in the DN compartment at later time points (Fig. 2C, 2D). Importantly, we observed this reduction in the absence of competition from Beclin 1-sufficient cells that exist in the chimeric mice. Thus, the defects in thymocyte progenitor maintenance seen in vitro (and likely the related progenitor maintenance defects seen in vivo) are intrinsic to Beclin 1 deficiency. These in vitro data help illuminate the likely basis for the reduction of total thymocyte numbers observed in Beclin 1-deficient chimeras. This could be the result of a decreased frequency (over time) of hematopoietic progenitors seeding the thymus. Collectively, these results point to the impaired maintenance of thymocyte progenitors in the absence of Beclin 1.

In sharp contrast with the peripheral T cell defects previously reported for Atg5- and Atg7-deficient T cells (19), we could not detect a significant impact of Beclin 1 deficiency on peripheral T cell numbers and their proliferative response in vitro (Fig. 3). Considering the essential roles of Atg5 and Atg7 in autophagy, their apparent requirement for T cell proliferation is commonly regarded as a general requirement for autophagy in peripheral T cells. However, we were able to detect LC3 puncta in Beclin 1-deficient T cells (Fig. 4), suggesting that a noncanonical, Beclin 1-independent autophagy appears to be sufficient to sustain normal peripheral T cell proliferation. Similar examples of Beclin 1-independent autophagy have been observed in other cell types under certain conditions (28–31). The fact that Beclin 1-deficient T cells still retain some autophagic activity could thus potentially explain different outcomes of Beclin 1 and Atg5/Atg7 deficiencies in peripheral T cell phenotype and proliferation. In addition, we did not observe any significant accumulation of mitochondria in Beclin 1^−/− T cells (unpublished observation), as seen in Atg5^−/− T cells (20, 27). In summary, our data indicate that the Beclin 1 autophagy gene plays an essential, but selectively early, role during T cell development. This is because, unlike Atg5 and Atg7, Beclin 1 function seems largely dispensable in peripheral T cells.

Although previous studies indicate an important role for autophagic protein Atg5 in both early- and late-stage T cells, the data describing the impact of Atg5 deficiency in B cells are less clear. Atg5-deficient fetal liver chimeric mice display defects that suggest reduced lymphoid progenitor activity (19) including a significant reduction in the number of peripheral B cells. In contrast, a subsequent study in which the Atg5 gene was conditionally targeted in B cells (using CD19-cre) showed partial deficiencies at the pro-B to pre-B cell transition in the bone marrow, but a largely normal peripheral B cell compartment (29). The differences observed in the severity of the Atg5-deficient B cell phenotype undoubtedly stem from the disparate experimental systems used in these two studies. Nevertheless, these prior data suggest at least an early-stage role for Atg5 in the B cell compartment, even if its role in peripheral B cells remains nebulous.

In concordance with the Atg5 knockout fetal liver chimera data, we detect deficiencies in lymphoid progenitors in the absence of Beclin 1. Our data also show that Beclin 1 deficiency causes significant defects at early stages of B cell development in the bone marrow, and a lesser deficit in the earliest peripheral B cell subset representing recent bone marrow emigrants. In contrast, Beclin 1-deficient B cells at later stages of development appear largely normal. Taken together, the data indicate that both Beclin 1 and Atg5 proteins are required in lymphoid progenitors and early-stage T and B cell development. Although Atg5 may be required in peripheral B cells (19), our data suggest that, as in peripheral T cells, Beclin 1 activity is not essential for most peripheral B cells. Although peripheral lymphocytes seem largely normal in our Beclin 1^−/− chimeric mice, we cannot rule out the possible emergence of long-term deficits over time. It is conceivable that Beclin 1 activity will prove significant in preventing age-related decline in lymphocyte homeostasis and function. Long-term monitoring of Beclin 1^−/− chimeric and control mice will be required to test this hypothesis.

Our findings of an essential role for Beclin 1 at early stages of both T and B cell development suggest that the reduction of early T and B cell numbers in Beclin 1-deficient chimeras could result from a reduced pool of lymphoid progenitors in the bone marrow. We examined this possibility by analyzing CLPs and HSC in both control and Beclin 1-deficient chimeras (32). Our results indicate that Beclin 1^−/−→Rag1^−/− chimeras have significantly reduced Ly9.1⁺CLPs and also a severely reduced population of Ly9.1⁺HSCs compared with control chimeras (Fig. 7). The impaired maintenance of lymphoid progenitors in the bone marrow may therefore explain the defects seen in the early T and B cell compartments in the absence of Beclin 1. It is not yet clear whether the problem in progenitor maintenance is selectively at a single stage (such as the HSC stage) or is a more general defect in maintenance of multiple types of undifferentiated progenitors. Nevertheless, using an in vitro ESC differentiation system to generate T cells, we found a depletion of the DN thymocyte compartment over time resulting in reduced maintenance of T cell generation in the cocultures at later time points. Because this depletion occurs in the absence of competition from Beclin 1-sufficient cells in the chimeras, this defect would be consistent with impaired progenitor activity/maintenance issues intrinsic to cells lacking Beclin 1. In future studies, this in vitro system may enable a comprehensive assessment of the generation, maintenance, and function of thymocyte progenitors at multiple stages to determine which populations are dependent on Beclin 1 function and why.

In summary, Beclin 1 deficiency results in severe depletion of T and B cell precursors, but has limited impact on peripheral T and B cells. Furthermore, loss of Beclin 1 function has no discernible effect on peripheral T cell proliferation despite reduced autophagy. Together with other recent reports, our results thus indicate distinct roles for various known autophagic proteins in T cells. Although recent studies in Atg5- and Atg7-deficient mice indicate a clear role for autophagy, in general, throughout lymphocyte development, we propose that Beclin 1-dependent processes are selectively required in progenitor cells and early stages of lymphocyte development. It remains to be seen, however, whether these processes reflect the role of Beclin 1 in autophagy or could involve other autophagy-independent function(s) of this protein in cell survival/differentiation. Importantly, our results reveal that noncanonical, Beclin 1-independent autophagy is sufficient to support later-stage peripheral T and B cell development and proliferation.

We thank Kevin Kelley (Mount Sinai School of Medicine) and Rada Norinsky (Rockefeller University) for expert Rag1^−/− chimeric mice generation, Chingwen Yang (Rockefeller University) for ESC work, Tatyana Leonova (Rockefeller University) for help with B cell assays, Joon Kim (Hunter College) for flow cytometry, and Derek Sant’Angelo (Memorial Sloan-Kettering Cancer Center) for helpful comments on the manuscript. We are indebted to Roxanne Holmes and Juan-Carlos Zúñiga-Pflücker for invaluable discussions and the ongoing advice and reagents that enabled us to establish in vitro T cell differentiation at City University of New York.

The authors have no financial conflicts of interest.