Animals

Mice were kept in accordance with guidelines of the Johns Hopkins University Institutional Animal Care and Use Committee. 5c.c7 Rag^–/—and OT-II mice were purchased from Taconic. C57BL/6 mice, CD90.1 mice, CD4 Cre recombinase mice, Foxp3^GFP+ mice, and mice with loxP-flanked Raptor alleles were obtained from Jackson Laboratories. Mice with loxP-flanked Tsc2 alleles were generated by the laboratory of M. Gambello [24]. 5c.c7 Rag^–/—mice were derived from a B10.A background, while all other WT and OT II+ mice described in this manuscript originate from the C57BL/6 strain.

Media and cell culture

Primary cell culture was performed in complete media consisting of 45% RPMI + 45% Clicks media supplemented with 10% FCS, L-glutamine, ßME and antibiotics. Metabolic flux analysis was performed in complete Seahorse media (Seahorse Bioscience, 102365) supplemented with 25mM D-Glucose, 1mM Na-Pyruvate and 2mM L-glutamine. 5c.c7 Rag^–/—splenocytes were stimulated with 0.5ug/ml Pigeon CytochromeC (PCC) peptide (JHU CORE synthesis). C57BL/6 WT splenocytes were stimulated with 0.1–1ug/ml anti-CD3 depending on the experiment. Splenocytes derived from transgenic OT-II+ mice were stimulated with 5ug/ml OVA^323–339 peptide (Anaspec).

Proliferation assay

Sorted cells were cultured for 24hrs in complete media supplemented with 1ng/ml IL-2, then equivalent cell numbers were loaded in a 96 well plate and treated with 1μCi ³H-thymidine for 18hrs. ³H incorporation was measured using a PerkinElmer 1450 Microbeta counter.

Cell cycle analysis

For the determination of intracellular DNA content, cells were washed with PBS and fixed for 30min at 4°C in ice-cold 70% EtOH. Cells were then washed with PBS, and treated with 50U of RNase 1F (New England Biolabs M0243) for 10minutes at room temperature, and suspended in a 50ug/ml solution of propidium iodide. Cell cycle progression was determined by flow cytometry.

Flow cytometry and cell sorting

All flow cytometry experiments were performed on a BD FACS Calibur or LSR II, and analyzed using FlowJo software. Cell sorting was performed on a BD FACS Aria II.

The following antibodies for flow cytometry of murine samples were purchased from BD Biosciences: anti-CD25 (PC61), anti-CD4 (RM4-5), anti-CD69 (HI.2F3), anti-CD90.1 (OX-7), anti-CTLA-4 (UC10-4F10-11), and anti-CD71 (C2). The following antibodies for flow cytometry were purchased from eBioscience: anti-Foxp3 (FJK-16s), anti-CD11a (M17l4), anti-CD62L (MEL-14), anti-CD39 (24DMS1), and anti-GITR (DTA-1). Anti-ribosomal S6 Ser235/236 (2211), anti-ribosomal S6 Ser240/244 (5364), and anti-p70 S6K T389 (9205) were purchased from Cell Signaling Technologies. Anti-Bcl-2 (BCL/10C4) and anti-CXCR3 (CXCR3-173) were purchased from Biolegend. As for antibodies against human: anti-HLA-DR, anti-CD38 (HB7), and anti-Foxp3 (259D/C7) were purchased from BD Biosciences. Anti-CD4 (OKT4), anti-CD127 (A019D5) and anti-CD25 (BC96) were purchased from Biolegend. Intracellular Foxp3 and Bcl-2 staining was performed using the Fixation/Permeabilization kit (eBioscience). For assessment of intracellular phospho-proteins, cells were permeabilized with methanol and fixed with 2% formalin prior to staining. However, for assessment of mTORC1 activity in the activated human samples and in activated cells cultured in rapamycin or TGF-ß, phosphorylated S6 expression was assessed after fixation/permeabilzation in the eBioscience kit. Determination of mitochondrial content was determined by Mitotracker Green staining according to the manufacturer’s instructions (Invitrogen). Cell proliferation analysis was performed using CellTrace CFSE (Invitrogen) or eFluor670 proliferation dye (eBioscience) according to the manufacture’s instructions.

Real Time PCR

RNA from stimulated cells was harvested using Trizol and cDNA generated using M-Mulv reverse transcriptase (NEB). Gene expression analysis was performed using ABI TaqMan 2X Universal Master Mix II (4440040) and rtPCR probes for IFNg (Mm99999071_m1), IL-17a (Mm00439619_m1), IL-17f (Mm00521423_m1), Bcl-2 (Mm00477631_m1), Foxp3 (Mm00475162), and IL2ra (Mm01340213_m1). ΔΔC_t values were normalized to levels of house keeping gene 18S ribosomal RNA (Life technologies). Analysis was performed on an ABI OneStepPlus 96 well instrument.

Extracellular flux analysis

Cellular metabolic parameters were measured using a Seahorse Bioscience XF96 Extracellular Flux Analyzer. Activated T cells were adhered to Poly-D-lysine coated 96 well plates and briefly cultured in complete Seahorse media. Cellular metabolic parameters were assayed by sequential addition of Oligomycin (final concentration 1uM) and FCCP (final concentration 1.5uM).

Immunoblot analysis

Immunoblot analysis was performed as previously described [25]. Antibodies used: anti-Bcl-2 (3498) (Cell Signaling Technologies), and anti-actin (8456) (Sigma).

Suppression Assays

Splenocytes from a WT C57BL/6 mouse were stimulated for 16 hours with 0.1ug/ml anti-CD3 then sorted into mTOR^hi and mTOR^lo populations and cultured with 1ng/ml IL-2 for 72 hrs. Cells from the mTOR^hi and mTOR^lo cultures were mixed at a 1:2 ratio with naïve WT CD90.1+ CD4+ T cells stained with CFSE and stimulated with 1ug/ml anti-CD3 and irradiated APCs. The proliferation of the naïve CD4+ CD90.1+ T cells was measured by CFSE dilution by flow cytometry. For the transwell assays, mTOR^hi or mTOR^lo cells were segregated from the CFSE labeled naïve WT CD90.1+ CD4+ T cells by a Transwell permeable support 0.4uM polycarbonate membrane (Corning). Both the segregated suppressor and responder populations were co-cultured with 1ug/ml anti-CD3 and irradiated APCs.

Human CD4+ T cell experiments

Human samples were obtained through an institutional review board approved protocol. Blood was drawn from a total of 8 healthy volunteers in 3 independent experiments. PBMCs were extracted after Ficoll separation during centrifugation (Ficoll-Paque PLUS, GE Healthcare). CD4+ T cells were purified by magnetic bead separation according to the manufacture’s instructions (Human CD4+ T cell enrichment kit, Stem Cell Technologies). Purified CD4+ T cells were stimulated 20 hrs with dynabeads human T- activator anti-CD3/anti-CD28 beads (Gibco). The 15% biggest ‘mTOR^hi’ and smallest ‘mTOR^lo’ CD38+, CD4+ T cells were sorted on a BD FACS Aria II. Sorted cells were cultured in media supplemented with 3,000units/ml human IL-2 (provided by the NCI) for 72 hrs. Phenotype of sorted cells was determined by flow cytometry.

Statistical Analysis

Data are presented as means and standard deviation. Statistical results were generated using Graphpad Prism. Comparisons were calculated using unpaired T tests, and results with a p-value < 0.05 were considered statistically significant: *p<0.05, **p<0.01, ***p<0.001.

Cell size indicates mTOR activity in recently activated CD4+ T cells

Following TCR stimulation and appropriate co-stimulation, naïve CD4+ T cells dramatically increase in size after 24 hours (Fig. 1A). The cellular processes that regulate cell size are in part controlled by mTOR activity [10,26]. To determine if mTOR regulates the size of activated T cells, we stimulated T cells in the presence of mTOR kinase inhibitor, PP242 [27]. TCR-induced blastogenesis is significantly inhibited by the addition PP242 (Fig. 1A). Furthermore, T cell activation induces mTORC1 activity as indicated by phosphorylation of ribosomal-S6 at S235/236 and S6 Kinase at T389 (Fig. 1B). As expected, phosphorylation of these proteins is diminished with addition of PP242. We consistently find that mTORC1 activity is markedly enriched in the T cells that have increased size (Fig. 1B). The increase in size and mTORC1 activity is best observed after 15 hours of stimulation (S1 Fig.). Thus, there is a direct correlation of cellular size with mTORC1 activity upon activation of a clonal population of naïve T cells.

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Fig 1. CD4+ T cells coordinately increase in size and mTORC1 activity following TCR stimulation.

a-b) 5c.c7 Rag^–/—CD4+ T cells were cultured for 24hrs in media, or media + Pigeon Cytochrome C (PCC) peptide +/- 500nM PP242, a mTOR kinase inhibitor. a) Peptide stimulated cells exhibit a significant increase in cell size relative to unstimulated controls. Stimulation-induced cell growth can be inhibited by treatment with PP242. Forward (FSC) and Side scatter (SSC) MFI are shown in top right corner of each panel. b) Peptide stimulated CD4+ T cells upregulate mTORC1 activity following TCR stimulation, as indicated by phosphorylation of downstream targets S6 and S6K. This increase in mTOR activity is diminished upon PP242 addition. c-d) WT and T-Raptor^–/—OT-II+ splenocytes were stimulated with OVA^323–339 for 24hrs. c) Gates were set on stimulated CD4+ T cells based upon predominant size of stimulated T-Raptor^–/—cells and mTORC1 activity was measured from the smallest (Small) and largest (Big) populations of WT stimulated cells. pS6 MFI shown in upper right corner of histogram FACs plot. d) Gates were set based upon phosphorylated S6 (pS6) expression detected in T-Raptor^–/—cells and size (FSC) was assessed based from pS6—and pS6+ populations of WT stimulated cells. FSC MFI shown in upper right corner of histogram FACs plot. e) 5c.c7 Rag^–/—CD4+ T cells were stimulated with PCC for 24hrs and mTORC1 activity (as indicated by pS6 expression) was measured from the 45%, 20%, and 10% smallest (Small) and largest (Big) populations of cells. f) WT, T-Raptor^–/—and T-TSC2^–/—OT-II+ splenocytes were stimulated with OVA^323–339 for 24hrs. Plots show percentage of smallest and largest cells in each genotype based on the 10% gate generated from WT stimulated cells. The data are representative of at least 3 independent experiments.

https://doi.org/10.1371/journal.pone.0121710.g001

To further examine the relationship of mTORC1 activity with cellular size, we examined TCR-induced blastogenesis in Raptor deficient T cells (T-Raptor^–/–), which have dramatically reduced mTORC1 activity. After 24 hours of stimulation, the vast majority (80%) of the T-Raptor^–/—T cells remain small in size compared with ~30% of the WT T cells (Fig. 1C). Consistent with the role of mTORC1 in regulating T cell size, the largest cells (labeled ‘Big’) detected after stimulation of WT T cells display the highest mTORC1 activity, while the smallest cells (labeled ‘Small’) demonstrate the lowest mTORC1 activity (Fig. 1C). Likewise, WT cells with the highest mTORC1 activity (labeled ‘pS6+’) are the largest in size (Fig. 1D). Examination of cell size at the 45^th, 25^th and 10^th percentiles reveals a graded effect of mTORC1 activity on cell size, whereby regardless of size percentile, mTORC1 activity is consistently highest in the largest cells (Fig. 1E). The largest difference in mTORC1 activity is most evident in the 10% smallest and largest size percentiles (Fig. 1E). Thus, we chose to utilize the 10% size cut off in all following experiments to demonstrate the most robust differences between activated cellular populations. This idea was validated by assessing size of WT, T-Raptor^–/–, and T-TSC2^–/–(T-TSC2^–/—T cells have hyperactive mTORC1 activity) OT-II+ splenocytes after stimulation with OVA^323–339 peptide. Again, we observed that over half of the activated T-Raptor^–/—CD4+ T cells are detected in the gate set from the 10% smallest activated WT cells, while less than 3% of T-TSC2^–/—are detected in the same gating strategy (Fig. 1F). Based on these results, we propose that cell size can be used as a surrogate indicator of mTORC1 activity in recently stimulated T cells, allowing for easy identification and tracking of both mTOR^hi and mTOR^lo cells from an initially monoclonal culture of naïve T cells.

We propose that for an individual cell, antigen recognition (Signal 1) will occur in the context of differential mTORC1 activation depending upon accessory signals (Signal 2) from the environment. However, it was critical to demonstrate that the differential activation of mTORC1 was not simply due to lack of antigen recognition. That is, we wanted to ensure that even the cells with decreased mTOR activity still received full antigen receptor engagement.

To this end, WT, T-Raptor^–/—and T-TSC2^–/—OT-II⁺ splenocytes were stimulated with OVA^323–339 peptide for 24 hours. As expected, this led to differential activation of mTORC1, whereby T-TSC2^–/—T cells have increased activity while T-Raptor^–/—T cells have diminished activity compared to WT cells (Fig. 2A). Notably, there was robust and roughly equivalent upregulation of CD69 and downregulation of CD62L in T cells from all 3 genotypes (Fig. 2B). That is, diminished or enhanced mTORC1 activity did not affect antigen-induced activation. Next, we wanted to demonstrate that such was also the case for the small (mTOR^lo) and big (mTOR^hi) populations of WT cells. Splenocytes from 5c.c7 Rag^–/—TCR transgenic mice were stimulated with Pigeon Cytochrome C (PCC) peptide. Upon stimulation, there is robust activation as indicated by CD69 upregulation (Fig. 2C). Starting from this clonotypic population of naïve T cells, we observe that there emerges a heterogeneous population of antigen experienced cells that differ dramatically in size. Importantly, this difference in cell size was not due to a lack of activation as both large and small cells express high levels of the early T cell activation marker CD69, and demonstrate robust downregulation of CD62L relative to naïve T cells (Fig. 2C). We did note, however, that the levels of CD69 and CD62L were slightly decreased in the T-Raptor^–/—and mTOR^lo cells when compared to WT and mTOR^hi T cells (Fig. 2B and 2C). The significance of this finding is unclear.

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Fig 2. mTORC1 activity is not required for antigenic recognition.

a-b) WT, T-Raptor^–/—and T-TSC2^–/—OT-II+ splenocytes were stimulated with OVA^323–339 for 24hrs. a) mTORC1 activity, as assessed by pS6^S240/244 expression was measured from the CD4+ population of each genotype. The bar graph below displays the MFI ± standard deviation (S.D.) of pS6 per genotype represented in above histogram plot. b) Expression of activation marker CD69, and homing marker, CD62L, are shown from the CD4+ population of each genotype. Below bar graphs indicate MFI of each molecule of interest from 3 independent experiments. c) 5c.c7 Rag^–/—CD4+ T cells were stimulated with PCC for 24hrs and expression of CD69 and CD62L was assessed from the 10% smallest and largest populations of activated CD4+ cells. Expression was compared against non-activated 5c.c7 Rag^–/—CD4+ T cells (CD4+ CD69–). Bar graphs below represent MFI ± S.D. of each molecule of interest from 3 independent experiments. The data are representative of (a,c) and/or are a composite of 3 independent experiments (b,c).

https://doi.org/10.1371/journal.pone.0121710.g002

mTOR^hi and mTOR^lo CD4+ T cells display differential proliferative, metabolic and survival fates

In light of the relationship we observed between cell size and mTOR activity in recently activated CD4+ T cells, we sought to track the fate of T cells with differing levels of mTOR activity, as indicated by cell size, following antigen recognition. To this end, naïve 5c.c7 TCR transgenic Rag^–/—splenocytes were stimulated with PCC peptide for 24 hours. To ensure that only stimulated CD4+ T cell were included in future analysis, cells were stained for CD4 and the mTOR independent early activation marker CD69, then sorted into CD4+CD69+ FSC/SSC “big” mTOR^hi and CD4+CD69+ FSC/SSC “small” mTOR^lo populations (Fig. 3A). To further confirm the relationship of mTORC1 activity with cell size, sorted cells were immediately assessed for mTORC1 activity. As expected, the largest population (labeled ‘Big’) cells demonstrate the highest phosphorylated S6 (pS6) expression (Fig. 3B). At the time of sorting, the process of cellular division had not yet been initiated, with ~90% of both the mTOR^hi and mTOR^lo populations still in G₀ stage of the cell cycle as indicated by propidium iodide staining (S2A Fig.). After the sort, cells were cultured in media supplemented with 1ng/ml IL-2. Sorted mTOR^hi cells rapidly form large homotypic aggregates in culture after 4 hours post sorting, which did not occur in sorted mTOR^lo cultures (Fig. 3C). Twenty four hours after culture, sorted mTOR^hi and mTOR^lo cells were treated with tritiated (³H-) thymidine to monitor proliferation and cultured for an additional 16 hours. Sorted mTOR^hi CD4+ T cells exhibit a much higher rate of proliferation than sorted mTOR^lo cells, as indicated by ³H-thymidine uptake (Fig. 3D). Additionally, CD4+ mTOR^hi cells display robust cell cycle progression relative to sorted mTOR^lo cells, which arrest in G_0/1 following the sort (Fig. 3E). CFSE labeling of 5c.c7 splenocytes prior to stimulation confirmed that neither mTOR^hi and mTOR^lo sorted cellular populations have initiated division at the time of sorting, but mTOR^hi cells have enhanced proliferation as indicated by CFSE dilution after 3 days of culture in IL-2 supplemented media (S2B Fig.).

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Fig 3. Cell size can be used to isolate recently stimulated CD4+ T cells populations with distinct proliferative, metabolic and survival profiles.

a) Splenocytes from 5c.c7 Rag^–/—mice were stimulated with 0.5ug/ml PCC peptide for 24hrs then sorted into CD4+CD69+ populations with FSC/SSC “big” mTOR^hi or FSC/SSC “small” mTOR^lo profiles. b) mTORC1 activity was assessed from “Big” and “Small” cells immediately after sorting. pS6 MFI is shown in the upper corner of the plot. c) Sorted mTOR^hi cells form large homotypic aggregates when cultured in media containing IL-2 for 4hrs after sorting. d-h) Sorted cells were cultured in media containing IL-2 for 24hrs. d) mTOR^hi cells exhibit a higher proliferative rate—as measured by ³H-thymidine incorporation—than their mTOR^lo counterparts. e) Sorted mTOR^hi cells show robust cell cycle progression 24hrs post sorting, while sorted mTOR^lo cells arrest in the G_0/1 stage. f) mTOR^hi cells display a significantly higher Extracellular Acidification Rate (ECAR) and Oxygen Consumption Rate (OCR) than their sorted mTOR^lo counterparts. The increased metabolic activity observed in the sorted mTOR^hi cells is accompanied by a higher mitochondrial load than sorted mTOR^lo cells as indicated by mitotracker green staining (g). Mitotracker green MFI shown in upper corner of FACs plot. h) Sorted mTOR^lo CD4+ T cells exhibit a significantly higher Spare Respiratory Capacity (SRC) than sorted mTOR^hi cells. i) Sorted mTOR^hi cells exhibit a higher rate of cell death when cultured for 5 days in media supplemented with IL-2 than sorted mTOR^lo cells. The bar graph to the right depicts % AnnexinV+7-AAD+ cells ± S.D. from 3 independent experiments. Three days after culture in media + IL-2, sorted mTOR^lo cells show significantly higher levels of (j) Bcl-2 mRNA and (k) protein expression than sorted mTOR^hi cells. mRNA transcript expression shows mean relative expression ± S.D. from 3 independent experiments, with the mTOR^hi values set to 1. Bcl-2 protein expression was normalized to Actin and fold difference in expression is shown above the protein band. The data are representative of at least 3 independent experiments.

https://doi.org/10.1371/journal.pone.0121710.g003

mTOR plays an important role in the regulation of cellular metabolism [13,28]. As such, we next assessed the metabolic profile of the cells. Sorted mTOR^hi cells are highly metabolically active, exhibiting a significantly higher Extracellular Acidification Rate (ECAR)—an indirect indicator of glycolysis—than their mTOR^lo counterparts (Fig. 3F). In addition, the mTOR^hi cells initially demonstrate a higher Oxygen Consumption rate (OCR) compared to mTOR^lo cells (Fig. 3F). This higher rate of metabolic activity observed in the sorted mTOR^hi cells is accompanied by an increase in mitochondria content (Fig. 3G). The higher ECAR observed in sorted mTOR^hi CD4+ T cells is consistent with the role of mTOR in promoting glycolysis [28]. Further, the decreased ECAR observed in the small, mTOR^lo cells phenocopies the metabolic profile of rapamycin treated CD4+ T cells (S3A Fig.). Thus, within a clonal population of T cells stimulated with their cognate antigen, those cells with high levels of mTORC1 activity demonstrate increased proliferation and metabolic activity when compared to those cells with relatively low mTORC1 activity.

We have shown that sorted mTOR^lo CD4+ T cells exhibit a markedly decreased glycolytic metabolic profile than sorted mTOR^hi cells. However, sorted mTOR^lo cells possess increased Spare Respiratory Capacity (SRC) when compared to mTOR^hi cells from the same culture (Fig. 3H). A similar finding was also observed for CD4+ T cell stimulated in the presence of rapamycin (S3B Fig.). SRC is calculated as the differential between a cell’s basal rate of oxygen consumption and the maximal rate of oxygen consumption. SRC is a metric indicating how close a cell is currently functioning to its maximal metabolic potential. Rapidly proliferating cells, such as terminally differentiated effector T cells and tumor cells, display a very low SRC [29,30]. Conversely, long-lived cells, such as stem cells and memory T cells have been shown to have a large SRC [29]. This correlation between longevity and SRC is thought to be due to the need for long-lived cells to survive periods of metabolic stress, which would prove fatal to more terminally differentiated cell types. In light of the previously described association between SRC and longevity, we assayed the in vitro survival of sorted mTOR^hi and mTOR^lo CD4+ T cells. From a clonal population of activated T cells, the mTOR^hi cells undergo increased apoptosis compared to sorted mTOR^lo counterparts when cultured in media supplemented with IL-2 for 5 days after sorting (Fig. 3I). The increased survival observed in the mTOR^lo cells was accompanied by significantly higher expression of the anti-apoptotic protein Bcl-2 at both a transcriptional (Fig. 3J) and protein (Fig. 3K) level.

mTOR^lo cells express increased levels of CD25

While mTORC1 activity is reduced in the sorted small cells compared to big cell counterparts, Bcl-2 levels are highest in small, mTOR^lo cells. Many factors have been shown to control Bcl-2 expression in CD4+ T cells, including cytokine receptor signaling [31]. To this end, we tested for expression of the high affinity IL-2 receptor CD25 in our sorted populations. Interestingly, CD25 expression at the transcript and protein level was higher on the mTOR^lo cells (Fig. 4A and 4B). To determine if this phenotype was graded across a spectrum of cell size, we analyzed CD69+ T cells from 4 distinctly sized populations: the 10% smallest (Q1: mTOR^lo), 10% largest (Q4: mTOR^hi) populations as previously assessed, in addition to the next 25% smallest (Q2) or largest (Q3) groups of cells (Fig. 4C). These studies revealed that CD25 protein and transcript levels, CD62L protein expression, and Bcl-2 transcript levels are inversely related to cell size and hence mTORC1 activity (Figs. 4D and 4E and S4A). Thus, from a metabolic and survival perspective, antigen recognition in the setting of strong mTORC1 activation generates cells with increased glycolytic flux that are destined to be short-lived. Alternatively, antigen recognition in the context of decreased mTORC1 activation leads to the generation of long-lived cells with increased SRC, increased Bcl-2 and increased CD25 (IL-2Ra) expression.

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Fig 4. Divergent fates of activated CD4+ T cells can be tracked with gradients of cellular size following activation.

a-b) Splenocytes from 5c.c7 Rag^–/—mice were stimulated with 0.5ug/ml PCC peptide for 24hrs and then sorted into CD4+CD69+ populations with FSC/SSC “big” mTOR^hi or FSC/SSC “small” mTOR^lo profiles. Cells were cultured in media supplemented with IL-2 for 3 days. a) 3 days after sort, mTOR^lo cells express higher mRNA transcript, and b) surface protein expression of CD25 than sorted mTOR^hi cells. The bar graph to the right depicts the fold increase in CD25 MFI expression in mTOR^lo cells compared to mTOR^hi cells from 6 replicate experiments, with expression of mTOR^hi cells set to a value of 1. Error bars represent ± S.D, n = 3 (a) or n = 6 (b). c-d) 5c.c7 Rag^–/—splenocytes were stimulated with PCC for 24hrs and activated (CD69+) CD4+ cells were sorted based on 4 size profiles. Cells were sorted into 10% smallest (Q1) and largest (Q4) populations in addition to the next 25% smallest (Q2) and largest (Q3) populations. c) Representation of sorting scheme utilized. d) After the sort, cells were cultured separately in IL-2 supplemented media for 3 days. On day 3, expression of CD25 and CD62L was assessed by flow cytometry. An illustration of cell size after sort is depicted above flow plots for clarification of sorted quartile populations. e) Purified CD4+ T cells from 5c.c7 Rag^–/—mice were stimulated by plate bound anti-CD3 and anti-CD28 for 24hrs. Cells were sorted as indicated in c, and mRNA transcript expression of CD25 and Bcl-2 was determined from sorted populations after a 3 day culture in IL-2 supplemented media. Bar graphs show relative expression ± S.D. from 3 independent experiments. The data are representative of 3 independent experiments or show cumulative results.

https://doi.org/10.1371/journal.pone.0121710.g004

Short-lived, metabolically active, mTOR^hi cells demonstrate an effector phenotype

Our data thus far demonstrate that for a population of clonal T cells, metabolic and survival potential is dictated by their level of mTOR activity after activation. Within this context, we hypothesized that the short-lived mTOR^hi cells represent the activated cells destined to become effector cells. Consistent with this hypothesis, recently stimulated and sorted mTOR^hi CD4+ T cells express significantly higher levels of IFN-gamma, IL-17a and IL-17f transcript than sorted mTOR^lo cells (Fig. 5A). Additionally, sorted mTOR^hi cells express much higher cell surface levels of the chemokine receptor CXCR3 (associated with pro-inflammatory effector cells) [32] than sorted mTOR^lo cells (Fig. 5B). As a negative control, no difference was observed in the expression of the LFA-1 subunit CD11a.

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Fig 5. Cell size can be used to isolate recently stimulated CD4+ T cell populations with distinct immunological fates.

a) Relative expression of IFN-gamma, IL-17a and IL-17f mRNA transcripts were determined from mTOR^hi and mTOR^lo populations immediately after sorting stimulated 5c.c7 Rag^–/—splenocytes. Error bars show ± S.D. from 3 independent experiments, and mTOR^hi values were set to 1. b-d) Sorted populations of cells were cultured in media supplemented with IL-2 for 3 days prior to assessment. b) Expression of chemokine receptor, CXCR3, and LFA-1 subunit CD11a was determined by flow cytometry. MFI per population is shown at the top of each FACs plot. c) Relative expression of Foxp3 mRNA transcript ± S.D. was determined from the mTOR^lo population compared to the mTOR^hi cells. The graph was generated from 3 independent experiments, and mTOR^hi values were set to 1. d) Foxp3 protein expression was determined from sorted mTOR^lo and mTOR^hi populations 3 days after culture in IL-2 alone or IL-2 + TGF-ß. Bar graphs to the right depict % Foxp3+ cells ± S.D. generated in each population from 3 independent experiments. e) mTOR^lo Foxp3+ regulatory T cells possess the ability to suppress the proliferation of naïve CD4+ T cells. Sorted mTOR^hi or mTOR^lo populations were cultured in media supplemented with IL-2 for 3 days prior to use in the suppression assay. The percentage of non-divided responders was determined by CFSE dilution 72hrs after culture of 1:2 suppressors: responders in direct contact or in transwell suppression assays. The graphs to the right depict the percentage of non-divided responders after culture with mTOR^hi or mTOR^lo ‘suppressors’ from 3 independent experiments. Connecting lines show values from sorted populations from the same experiment. The data are representative of at least 3 independent experiments or show cumulative results.

https://doi.org/10.1371/journal.pone.0121710.g005

Upon antigen recognition, mTOR^lo cells demonstrate metabolic programs consistent with long-lived cells. However, we have previously demonstrated that TCR engagement in the absence of mTOR induces the generation of Foxp3+ regulatory T cells [14]. Furthermore, others have shown that T cell activation in the presence of the mTOR inhibitor rapamycin promotes the generation of Foxp3+ regulatory T cells [15–18]. Therefore, we wondered if a sub-population of the mTOR^lo cells might be Foxp3+ regulatory T cells. Consistent with this hypothesis, we observed that the mTOR^lo population was enriched for Foxp3+ T cells (Fig. 5C and 5D). That is, under normally activating culture conditions (antigen activation followed by IL-2 addition), 9.4% of the mTOR^lo cells express Foxp3, while only 0.16% of the mTOR^hi cells expressed Foxp3 (Fig. 5D). This difference was also observed and was amplified when TGF-ß was added to the cultures (22.8% versus 4.3%). Foxp3 expression is upregulated in every division of activated mTOR^lo cells after culture in IL-2, and a graded decrease in Foxp3 expression is observed in each population of increased cell size/mTORC1 activity (S4B Fig.). The preferential induction of Foxp3 expression in sorted mTOR^lo cells is not limited to cultures derived from homogeneous Rag^–/—TCR transgenic splenocytes, but can also be observed in cultures generated from anti-CD3 stimulated polyclonal WT CD4+ T cells (S5A Fig.). Not unexpectedly, the mTOR^lo CD4+ T cells cultured in IL-2 alone were better able to suppress the proliferation of activated CD4+ T cells compared to their cultured mTOR^hi counterparts (Fig. 5E). This enhanced suppression was most likely due to the increase of Foxp3+ T cells generated from the mTOR^lo sorted population compared to the mTOR^hi cells (S5B Fig.). The ability of the mTOR^lo T cells to suppress responder T cells was markedly diminished when the responders and suppressors were separated in a transwell, indicating that the suppression is primarily contact dependent (Fig. 5E).

A question remained if Foxp3+ T cells were generated de novo from the activated mTOR^lo cells, or if the enhanced generation of Foxp3+ T cells observed in the sorted mTOR^lo populations was simply a consequence of sorting on a high frequency of already established natural Tregs. In order to answer this question, we utilized mice which express green fluorescent protein (GFP) under the control of the Foxp3 promoter (Foxp3^GFP+) [33]. Splenocytes from WT Foxp3^GFP+ mice were stimulated with 0.1ug/ml anti-CD3 for 20 hrs. Activated (CD69+) CD4+ GFP—cells were sorted from the 10% biggest ‘mTOR^hi’ and smallest ‘mTOR^lo’ populations (S6A Fig.). This approach ensured that all GFP+ natural Tregs would be removed from our sorted populations. Phenotypic assessment of sorted mTOR^hi and mTOR^lo populations 72 hrs after culture in IL-2 revealed that suppressive Foxp3+ T cells were generated de novo from the in vitro stimulated splenocytes (S6B–C Fig.). These observations were consistent with our previous results demonstrating that mTOR^lo CD4+ T cells have a greater propensity to differentiate into Foxp3+ T cells (Fig. 5C–5E).

Our data thus far suggests that under normally activating conditions, the recognition of antigen in the context of low mTOR activation promotes the de novo generation of regulatory T cells. Nonetheless, we could still detect a small percentage of Foxp3+ T cells which displayed high mTOR activity. Therefore, we sought to further phenotype the Foxp3+ T cells generated from the sorted mTOR^lo and mTOR^hi populations. First, Foxp3+ T cells derived from either mTOR^lo and mTOR^hi populations have higher expression of the known T-regulatory cell markers, CD25, CTLA-4, and GITR when compared to Foxp3 negative counterparts (Fig. 6A and 6B). Interestingly, Foxp3+ mTOR^lo T cells have enhanced expression of CD25 and GITR compared to mTOR^hi derived Foxp3+ cells (Fig. 6B). Alternatively, Foxp3+ mTOR^hi T cells have enhanced expression of CTLA-4 compared to Foxp3+ cells derived from the mTOR^lo population (Figs. 6B and S5). Thus, while the generation of Foxp3+ T cells is clearly enriched in the mTOR^lo population of antigen-activated T cells, there also exists a small but distinct population of Foxp3+ CTLA-4^hi T cells which emerges from the mTOR^hi population of antigen activated cells.

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Fig 6. Foxp3+ CD4+ T cells generated from mTOR^hi and mTOR^lo sorted populations have distinct phenotypes.

Splenocytes from 5c.c7 Rag^–/—TCR transgenic mice were stimulated with 0.5ug/ml PCC peptide for 24hrs, and then sorted into CD4+CD69+ populations with FSC/SSC “big” mTOR^hi or FSC/SSC “small” mTOR^lo profiles. Cells were cultured in media supplemented with IL-2 for 96hrs, and analyzed by flow cytometry. a) The percentage of CD4+ Foxp3+ cells was determined from each population. b) Histograms show shifts in MFI of markers associated with a T-reg phenotype. Populations were gated from the Foxp3+ or Foxp3- cells of the mTOR^hi or mTOR^lo sorted samples. The MFI of each protein is shown in the top corner of each FACs plot. The data are representative of at least 3 independent experiments.

https://doi.org/10.1371/journal.pone.0121710.g006

Divergent phenotypic properties of mTOR^hi and mTOR^lo Foxp3+ regulatory T cells

Our data are consistent with previous findings that diminished mTOR activity in T cells promotes the generation of regulatory T cells [14–18]. However, we also find a small, but distinct population of Foxp3+ T cells generated from mTOR^hi cells. Interestingly, one of the most common techniques to generate regulatory T cells in vitro is to induce robust activation in the presence of high concentrations of TGF-ß [34]. In fact, stimulation under these conditions results in robust mTOR activation (Figs. 7A and S7). That is, while strong TCR engagement in the presence of TGF-ß promotes the generation of Foxp3+ T cells, it also results in robust mTORC1 activation. Given the highly divergent metabolic profiles and survival potentials of sorted mTOR^lo and mTOR^hi conventional CD4+ T cells, we wanted to determine if Foxp3+ CD4+ T cells generated in the presence of high or low mTOR activity during TCR stimulation demonstrate similar metabolic and survival potentials. To this end, WT splenocytes were stimulated with 1ug/ml anti-CD3 and 1ng/ml IL-2 in the presence or absence of either 10ng/ml TGF-ß or 500nM rapamycin. Anti-CD3 was washed out at 24 hrs, and the cultures were expanded in media supplemented with IL-2 alone, IL-2 + TGF-ß, or IL-2 + rapamycin for 72 hrs before harvest for flow-cytometric analysis. As expected, the Foxp3+ T cells from the rapamycin treated cultures had reduced mTORC1 activity (mTOR^lo) compared to the Foxp3+ T cells generated in the presence of TGF-ß (Fig. 7A). As was the case for the mTOR^hi CD4+ sorted T cell populations, CD4+ T cells cultured with TGF-ß demonstrate enhanced ECAR compared to the cells activated in the rapamycin treated conditions (Fig. 7B), but reduced expression of Bcl-2 (Fig. 7C). These data suggest that culture in TGF-ß promotes an ‘effector’ phenotype with increased glycolysis but diminished survival. Activation of T cells in the presence of rapamycin or TGF-ß enhanced Foxp3+ T reg generation compared to untreated, activated cells (Fig. 7D), and we further assessed the phenotype of Foxp3+ cells generated from these culture conditions (Fig. 7E). Notably, phenotypic analysis of the Foxp3+ cells generated from the rapamycin versus TGF-ß culture conditions recapitulated the results observed from the sorted mTOR^lo and mTOR^hi Foxp3+ populations. Foxp3+ T cells generated in the presence of rapamycin have enhanced expression of CD25, GITR, CD71, and Bcl-2 compared to Foxp3+ T cells derived from TGF-ß cultures (Fig. 7E). Alternatively, Foxp3+ cells generated in the presence of TGF-ß have enhanced CTLA4 and CD39 expression compared to those generated in rapamycin (Fig. 7E). These data suggest that regulatory T cells generated in the presence of robust mTOR activity (by culture in high concentrations of TGF-ß) have differential survival potential and are metabolically distinct from those generated in the setting of low mTOR activity.

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Fig 7. Foxp3+ CD4+ regulatory T cells exhibit divergent phenotypic profiles, dependent on their method of induction.

Splenocytes from 5c.c7 Rag^–/—TCR transgenic mice (a) or WT C57BL/6 mice (b-e) were stimulated with 1ug/ml soluble anti-CD3 and 1ng/ml IL-2 in the presence or absence of either 10ng/ml TGF-ß or 500nM rapamycin for 24hrs. a) mTORC1 activity was measured by flow cytometric analysis of phosphorylated-S6 expression 24hrs after stimulation. Histograms were gated from activated CD4+CD69+ populations within each culture condition. b-e) After 24hrs of stimulation, cells were expanded in media supplemented with IL-2 alone, IL-2 + TGF-ß, or IL-2 + rapamycin for an additional 72hrs before phenotypic assessment. b) Cells were run on an extracellular flux analyzer and the extra cellular acidification rate, ECAR, was determined over time. c) Bcl-2 expression and d) Foxp3 expression levels were assessed from CD4+ T cells of each culture condition. e) Histograms show shifts in MFI of markers associated with a T-reg phenotype. Populations were gated from Foxp3+ CD4+ T cells. The MFI of each population is shown in the upper corners of the FACS plots depicted in a,c,and e. The data are representative of 3 independent experiments.

https://doi.org/10.1371/journal.pone.0121710.g007

Differential fates of mTOR^hi and mTOR^lo human CD4+ T cells

Next, we wanted to determine if we could use cell size/mTOR activity to track the fate of TCR-activated human T cells. CD4+ T cells from fresh PBMCs were isolated and the T cells were activated with human anti-CD3/anti-CD28 activator beads. Similar to our observations employing murine T cells, mTORC1 activation correlates with cellular size in activated human CD4+ T cells (Fig. 8A). Upon sorting and expanding activated mTOR^hi and mTOR^lo T cells for 72 hrs in IL-2, mTOR^lo T cells have an increase in percentage of CD127^loCD25^hi CD4+ T cells compared to mTOR^hi counterparts (Fig. 8B). Furthermore, from the CD127^loCD25^hi population, there is an increase in Foxp3+ CTLA4+ T cells detected in the mTOR^lo T cell cultures (Fig. 8C). Thus, similar to murine T cells, TCR-stimulation of human CD4+ T cells in the presence of low mTORC1 activity enhances T reg cell differentiation.

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Fig 8. Cellular size (indicative of mTORC1 activity) influences the phenotype of human CD4+ T cells similar to mouse.

CD4+ T cells were purified from the PBMCs of 8 human volunteers. Cells were stimulated with human anti-CD3 and anti-CD28 activator beads overnight. a) 24hrs after stimulation, mTORC1 activity was assessed by the pS6 expression of the 15% largest and smallest activated (HLA-DR+) CD4+ T cells. Histograms in the lower FACs plot were determined from the ‘big’ and ‘small’ gates shown in the upper plot. The pS6 MFI is shown in the upper corner of the FACs plot. b-c) 24hrs after stimulation, the cells were sorted from the 15% largest and smallest activated (CD38+) CD4+ T cells. Sorted populations were cultured in media supplemented with human IL-2 for 72 hrs. After 72 hrs, the phenotype of the large ‘mTOR^hi’ and small ‘mTOR^lo’ populations was assessed by flow cytometry. b) Human T-reg populations were determined from the CD127^lo CD25^hi gating strategy. c) FACs plots show percentage of Foxp3+ CTLA-4+ double positive cells gated from the ‘T-reg’ gate in b. Statistics are shown to the right. The connecting lines demonstrate the changes in percentages between the mTOR^hi and mTOR^lo sorted populations from the same individual. The data are representative of 8 distinct human samples.

https://doi.org/10.1371/journal.pone.0121710.g008