Anergic T cells are metabolically anergic

Abstract

Full T cell activation requires TCR engagement (signal 1) in the context of costimulation (signal 2). Costimulation is required for maximal expression of effector cytokines and prevention of T cell anergy. It has become increasingly clear that another major function of costimulation is to up-regulate the metabolic machinery necessary for T cell function. In this report we demonstrate that anergic T cells are metabolically anergic, in that upon full stimulation (signals 1 plus 2) they fail to up-regulate the machinery necessary to support increased metabolism. These findings suggest that one mechanism responsible for the maintenance of T cell anergy is failure to up-regulate the metabolic machinery. Furthermore, we demonstrate that by blocking leucine, glucose, and energy metabolism, T cell activation is mitigated. Additionally, inhibition of these metabolic pathways during T cell activation leads to anergy in Th1-differentiated cells. Overall, our findings extend the role of T cell metabolism in regulating T cell function.

FIGURE 1

Costimulation is required for full T cell activation and up-regulation of metabolic machinery. A, A.E7 T cells were left un-stimulated or treated with anti-CD3 alone (signal 1) or anti-CD3 plus anti-CD28 (signals 1 plus 2) for 16 h, then interrogated for proliferation by thymidine incorporation. B, Supernatants from A were interrogated for IL-2 by ELISA. C, CD98 cell surface expression of cells treated as in A by FACS. The shaded tracing indicates the iso-type control for stimulated T cells. D, As in C, but for CD71. E, Resting A.E7 T cells were left unstimulated or stimulated through the TCR (anti-CD3) or TCR and costimulation (anti-CD3 plus anti-CD28) for 3 h and interrogated for phospho-S6K1. F, As in E, but for phospho-Akt (S473). Total enzyme (pan) probes are included as loading controls. Data are representative of three independent experiments.

FIGURE 2

Upon rechallenge with signals 1 plus 2, anergic T cells fail to not only produce IL-2 and proliferate but up-regulate metabolic machinery. A, Mock-treated and anergic (signal 1) A.E7 T cells were rechallenged with APCs plus PCC peptide for 48 h. Cells were then interrogated for proliferation by thymidine uptake. B, Mock-treated and anergic (signal 1) T cells were rechallenged with anti-CD3 and anti-CD28 for 16 h and interrogated for IL-2 by ELISA. C, Supernatants from rechallenged T cells were tested for L-lactate production. For these experiments some cells were left unstimulated, and some anergic T cells were rescued with high-dose IL-2. D, CD98 cell surface expression of cells treated as in B by FACS. The shaded tracing indicates the isotype control for the stimulated T cells that were initially mock treated. E, As in D, but for CD71. F, As in D and E, but some anergic cells were rescued with IL-2, G, Mock-treated or anergic A.E7 T cells were left unstimulated or were stimulated for 3 h with anti-CD3 and anti-CD28; lysates were made and probed for phospho-S6K1 by immunoblot. H, As in G, but for phospho-Akt (S473). Total enzyme (pan) probes are included as loading controls. Data are representative of three independent experiments. Statistics were performed using the Student’s t test.**, p < 0.01; ***, p < 0.001; NS, p < 0.05.

FIGURE 3

Metabolic inhibition leads to decreased T cell function. Previously activated (Th1) 5C.C7 T cells were stimulated in varying concentrations of 2DG (mM), NALA (mM), or AICAR (μM). Supernatants were then interrogated for (A) IL-2 or (B) IFN-γ. Data are normalized values from three independent experiments. Error bars indicate SD. C, Metabolic inhibition induces anergy even in the presence of costimulation. A.E7 T cells were stimulated 16 h with anti-CD3 alone or anti-CD3 plus anti-CD28 in the presence of rapamycin (200 nM), NALA (2 mM), AICAR (10 μM), or 2DG (500 μM). The cells were washed and rested without drug for 7 days in unsupplemented media or media containing 1 ng/ml murine IL-2. The cells were then rechallenged for 16 h with anti-CD3, anti-CD28, without any drugs, and interrogated for IL-2 production by ELISA. Error bars indicate SD. Data are representative of three independent experiments.

FIGURE 4

Full T cell stimulation in the presence of metabolic inhibition leads to tolerance induction via mTOR inhibition. A, NALA, 2DG, and AICAR mimic starvation signals in an mTOR-dependent fashion. Previously activated 5C.C7 T cells were left unstimulated or stimulated with anti-CD3 plus anti-CD28 (TCR + Costim) in the presence or absence of NALA (2 mM), 2DG (500 μM), AICAR (10 μM), or rapamycin (200 nM) for 3 h, then lysed and interrogated for phospho-S6K1 and phospho-Akt (S473). Total enzyme (pan) probes are included as loading controls. B, 5C.C7 T cells were left unstimulated or were stimulated with anti-CD3 and anti-CD28 (TCR + Costim) with or without AICAR (10 μM); lysates were made and probed for phospho-AMPK, phospho-S6K1, and pan S6K1 by immunoblot. Immunoblots are representative of at least three independent experiments.

FIGURE 5

Anergic T cells are metabolically anergic. Thompson and colleagues have proposed that when a T cell receives signal 1 alone, it fails to fully up-regulate the metabolic machinery necessary to support T cell activation (left panel) (3). As such, a major function of costimulation is to facilitate metabolic function necessary to fuel the T cell response. We propose that just as anergic T cells fail to produce IL-2 upon rechallenge with full stimulation (signals 1 plus 2), they also fail to up-regulate the metabolic machinery such as the transferrin receptor, amino acid transporters, and glucose transporters necessary to promote a full effector response (right panel). IL-2 has the ability to activate mTOR; however, in the absence of signal 2 (left panel) or when a cell is anergic (right panel), very little IL-2 is produced. In the presence of costimulation (middle panel) AMPK is modestly activated (see Fig. 4B); however, this is not enough to inhibit mTOR in the setting of full activation. Thus, in the absence of energy, glucose, or amino acids mTOR is inhibited, resulting in the induction of anergy. Likewise, a dearth of these nutrients or the inability to import and metabolize these nutrients can directly inhibit T cell function.