Minimal fetal calf serum requirement for IL-7 dependent survival of CD8⁺ T cells.

<p>CD8⁺ T cells were enriched from C57Bl6/J donors and cultured for the times indicated, either alone (open symbols, dashed lines), or in the presence of 50 ng/ml of IL-7 (filled symbols, solid lines) and additionally either in presence (circles) or absence (squares) of foetal calf serum (FCS). Cells were stained with 7AAD and viability amongst CD44^loCD8⁺ cells determined by measuring the frequency of 7AAD⁻ live cells by flow cytometry. Percentage of surviving cells was normalized to the percentage of live cells on day 0. Results are pool of three independent experiments. Error bars indicate SD of biological replicates.</p

Neither amino acids nor glucose are required for IL-7 dependent survival of naïve CD8 T cells.

<p>CD8⁺ T cells were enriched from C57Bl/6J donors and cultured for the indicated time points, either alone (open symbols, dashed lines), or in the presence of 50 ng/ml of IL-7 (filled symbols, solid lines). Cultured cells were stained with 7AAD and frequency of viable 7AAD⁻ cells amongst total CD44^lo CD8⁺ T cells determined by flow cytometry. Cells were cultured in RPMI medium containing standard nutrients (circles) or in RPMI specifically lacking (A) amino acids (aa−), (B) glucose (Glu−) or (C) glucose and amino acids (Glu− aa−, squares throughout). (D) CD8⁺ T cells from C57Bl6/J donors were cultured for 24 h in the presence of 50 ng/ml of IL-7, in RPMI either containing standard nutrients (+Glu +aa) or in the absence of glucose and amino acids (−Glu −aa). Cells were stained for Bcl-2 and expression compared with <i>ex vivo</i> CD44^loCD8⁺ T cells. Histograms show Bcl2 expression by CD44^lo CD8⁺ T cells cultured in the presence (blue solid line) and absence (red solid line) of glu and aa, as compared with <i>ex vivo</i> CD44^lo CD8⁺ T cells (black solid line). Broken lines indicate isotype negative control stainings for each population. (A–C) Error bars indicate SD of technical replicates. Results are representative of three experiments.</p

Regulation of amino acid transporter expression by IL-7 signaling.

<p>Microarray analysis was performed to compare gene expression in purified CD8⁺ populations of F5 T cells from F5 <i>Rag1^−/−</i> mice <i>ex vivo</i> (n = 4) or following culture with 50 ng/ml IL-7 for 24 h (n = 4) (Pearson et, in press). (A) Graphs show relative expression level of indicated genes in F5 T cells <i>ex vivo</i>. (B) Graphs indicate fold change in expression of those amino acid and glucose transporters expressed in F5 T cells, following culture with IL-7, as compared with expression in <i>ex vivo</i> F5 T cells. Error bars indicate SD of technical replicates. * p<0.05.</p

IL-7 induced T cell growth is strictly dependent on exogenous amino acids but not glucose.

<p>CD8⁺ T cells were enriched from C57B6/J donors and cultured for 3 days in RPMI containing standard nutrients, either alone (open symbols), or in the presence of 50 ng/ml IL-7 (filled symbols). Cultured cells were stained with 7AAD and size of viable CD44^lo CD8⁺ T 7AAD⁻ cells determined by measuring FSc by FACS. (A) Where indicated, the inhibitors LY294002 (Ly, squares) (10 µM), rapamycin (Rap, triangles) (20 nM) or the vehicle control (DMSO, diamonds) were added to cultures. (B) Cells were cultured in RPMI medium containing standard nutrients (circles) or in RPMI specifically lacking amino acids (squares), glucose (triangles) or lacking both glucose and amino acids (diamonds). Cells were cultured in the presence (filled symbols) or absence (empty symbols) of exogenous IL-7 (50 ng/ml). Results are the pool of three or more independent experiments. Each point plotted is the mean of triplicate cultures for a single independent experiment. Error bars indicate SD of biological replicates. * p<0.001; ns, not significant.</p

Exogenous Amino Acids Are Essential for Interleukin-7 Induced CD8 T Cell Gowth

<div><p>IL-7 signalling is important in regulating both survival and cellular size (growth) of T cells. While glucose metabolism has previously been implicated in the mechanism of IL-7 induced survival and growth, the role of amino acids has not before been reported. Here, we show IL-7 dependent T cell survival does not require either exogenous glucose or amino acids. In contrast, maintenance of cell size and IL-7 induced growth were specifically dependent on amino acids. Furthermore, cellular amino acid uptake was implicated in the mechanism of IL-7 induced growth. Analysis of IL-7 regulated gene expression revealed that neutral and cationic amino acid transporters were specific transcriptional targets of IL-7 signalling. In contrast, none of the four glucose transporters expressed in T cells were modulated. Taken together, these data reveal for the first time the central importance of amino acid homeostasis for IL-7 regulated T cell growth.</p> </div

IL-7 regulates CD98 expression levels in vitro and in vivo.

<p>CD8⁺ T cells from C57Bl6/J mice were either cultured <i>in vitro</i> in the presence or absence of IL-7 for 24 h, or transferred to either <i>Rag1</i>^−/− or <i>Il7</i>^−/−<i>Rag1</i>^−/− hosts. Upper histograms show CD98 expression by CD44^loCD8⁺ cells cultured for 24 h either alone (red line) or in the presence of 50 ng/ml IL-7 (black line) as compared with <i>ex vivo</i> control CD44^loCD8⁺ T cells (grey fill). Lower histograms show CD98 expression in CD44^loCD8⁺ cells transferred to <i>Rag1^−/−</i> (black line) or <i>Il7^−/− Rag1^−/−</i> hosts (red line) for 3 days, as compared with <i>ex vivo</i> control CD44^loCD8⁺ T cells (grey fill). Results are representative of three independent experiments.</p

IL-7 mediated growth and survival is dose dependent in vitro.

<p>CD8 T cells were enriched from C57Bl6/J donors and cultured with a range of IL-7 concentrations. After 72 h, cells were stained for CD8 expression and with 7AAD and viability and cell size determined by flow cytometry. (A) Graph shows viability of CD8⁺ cells in cultures with different IL-7 concentrations. Viable CD8 T cells were identified as large cells that excluded 7AAD dye. Viability in IL-7 free cultures was <1% at 72 h (data not shown). (B) Graph shows cell size in cultures with different concentrations of IL-7. Data are expressed as size relative to ex vivo CD8 T cell controls. Error bars indicate SD of technical replicates. Results are representative of two or more experiments.</p

Viability of naïve CD8 T cells is not affected by blockade of PI3K or mTOR.

<p>Cells were cultured in RPMI containing standard Glu and aa nutrients and, where indicated, the inhibitors LY294002 (Ly) (10 µM) or rapamycin (Rap) (20 nM), or the vehicle control (DMSO) were added. Percentage of surviving CD44^lo CD8⁺ T cells was normalized to the percentage of live CD44^lo CD8⁺ T cells on day 0. Error bars indicate SD of technical replicates. Results are representative of three independent experiments.</p

Characteristics of study population.

Characteristics of study population.</p

Enrollment, follow-up, and exclusion criteria flow diagram for persons in analysis.

Enrollment, follow-up, and exclusion criteria flow diagram for persons in analysis.</p