Glutamine uptake and metabolism are coordinately regulated by ERK/MAPK during T lymphocyte activation

Abstract

Activation of a naive T cell is a highly energetic event, which requires a substantial increase in nutrient metabolism. Upon stimulation, T cells increase in size, rapidly proliferate, and differentiate, all of which lead to a high demand for energetic and biosynthetic precursors. Although amino acids are the basic building blocks of protein biosynthesis and contribute to many other metabolic processes, the role of amino acid metabolism in T cell activation has not been well characterized. We have found that glutamine in particular is required for T cell function. Depletion of glutamine blocks proliferation and cytokine production, and this cannot be rescued by supplying biosynthetic precursors of glutamine. Correlating with the absolute requirement for glutamine, T cell activation induces a large increase in glutamine import, but not glutamate import, and this increase is CD28-dependent. Activation coordinately enhances expression of glutamine transporters and activities of enzymes required to allow the use of glutamine as a Krebs cycle substrate in T cells. The induction of glutamine uptake and metabolism requires ERK function, providing a link to TCR signaling. Together, these data indicate that regulation of glutamine use is an important component of T cell activation. Thus, a better understanding of glutamine sensing and use in T cells may reveal novel targets for immunomodulation.

Figure 1

T cell activation requires high levels of glutamine. (A) C57BL/6J splenocytes were stimulated with titrated amounts of anti-CD3 antibody in medium containing the indicated concentration of glutamine. 2 mM is the standard glutamine concentration in RPMI1640 medium. Proliferation was measured after 3 days of stimulation. Means at a given antibody concentration without a common letter differ from each other and from 0 mM glutamine, P<0.05. Means with no letter do not differ significantly from 0 mM glutamine. (B, C) C57BL/6J splenocytes were stimulated for 36 hours with 1 μg/mL anti-CD3 antibody in complete (2 mM glutamine) or glutamine-free RPMI1640 medium, and IL-2 (B) and IFN-γ (C) levels were measured in culture supernatants. For resting samples, cells were cultured in complete medium in the absence of anti-CD3 antibody. *, P<0.01, ***, P<0.0001. All results are representative of at least 3 independent experiments.

Figure 2

Biosynthetic precursors cannot substitute for glutamine in T cell activation. C57BL/6J splenocytes were stimulated as in Figure 1A, in complete medium or glutamine-free medium, or in glutamine-free medium supplemented with 2 mM glutamate (A), asparagine (B), or proline (B). Proliferation was measured after 3 days of stimulation. ***Different from glutamine-free, P<0.001. *Different from glutamine-free; P<0.05. All results are representative of at least 3 independent experiments.

Figure 3

Glutamine is not required for induction of early T cell activation markers. Purified C57BL/6J T cells were stimulated for 24 hours with immobilized anti-CD3 and anti-CD28 antibodies in complete or glutamine-free medium. Cells were stained with PE-conjugated anti-CD69 (A), anti-CD25 (B), or anti-CD98 (C) antibodies and analyzed by flow cytometry. For resting samples, cells were cultured in complete medium in the presence of immobilized control hamster antibodies. (D) Forward scatter (FSC) plots of the T cells analyzed in A–C. Results are representative of 3 independent experiments.

Figure 4

T cells selectively enhance glutamine uptake during activation. (A) Purified T cells were cultured for 24 hours with either control hamster IgG (rest) or anti-CD3 plus anti-CD28 antibodies (24 hr stim), and glutamine uptake was measured. ***Different from rest, P<0.001. (B) Purified T cells were stimulated with either control hamster IgG (rest), anti-CD3 antibodies alone (CD3), or anti-CD3 plus anti-CD28 antibodies (CD3/28), and glutamine uptake was measured. (C) Purified T cells were stimulated as in A, and rates of uptake of glutamine and glutamate were measured. ***Different, P<0.001. (D) Constitutive glutamine uptake in EL-4 lymphoma cells was compared to uptake in resting and stimulated T cells. Means without a common letter differ, P<0.01. (E) Rates of constitutive glutamine and glutamate uptake were measured in EL-4 lymphoma cells. **Different from glutamate, P<0.01. All results are representative of at least 3 independent experiments, except for (B), which is representative of 2 independent experiments.

Figure 5

T cell activation induces expression of SNAT transporters. (A) Purified C57BL/6J T cells were stimulated with immobilized anti-CD3 and anti-CD28 antibodies for the indicated times. SNAT1 and SNAT2 mRNA levels were determined by quantitative real-time PCR. (B) Purified T cells were stimulated for the indicated times, and surface proteins were biotinylated prior to lysis. SNAT2 protein was precipitated from lysates with anti-SNAT2 antibody, resolved by SDS-PAGE, and detected with avidin-HRP. The predicted mobility of SNAT2 is indicated to the left. Results are representative of 3 (A) or 2 (B) independent experiments.

Figure 6

Glutamine uptake in activated T cells is regulated by ERK signaling. Purified T cells were stimulated in the presence or absence of the ERK inhibitor PD98059 (40 μM), and glutamine uptake was measured as in Figure 4. **Different, P<0.01. Results are representative of 3 independent experiments.

Figure 7

T cell activation induces enzymes involved in glutamine metabolism. (A) Purified T cells were cultured for 24 hours with immobilized control (rest) or anti-CD3 plus anti-CD28 (stim) antibodies, in the presence or absence of 40 μM PD98059 (stim + PD), and enzymatic activity of glutaminase was measured. Glutaminase activity in continuously growing EL-4 cells was also measured. (B) T cells were cultured with control (rest) or anti-CD3 plus anti-CD28 antibodies for the indicated times, in the presence or absence of 40 μM PD98059 (stim + PD), and glutaminase mRNA levels were determined by quantitative real-time PCR. (C) Purified T cells were cultured as in (A), and enzymatic activities of GDH, GOT, and GPT were measured. (D) Activities of GDH, GOT, and GPT in continuously growing EL-4 cells were determined. Means without a common letter differ, P<0.05. All results are representative of at least 3 independent experiments.