Inflammatory T cell responses rely on amino acid transporter ASCT2 facilitation of glutamine uptake and mTORC1 kinase activation

Abstract

Glutamine has been implicated as an immunomodulatory nutrient, but how glutamine uptake is mediated during T cell activation is poorly understood. We have shown that naive T cell activation is coupled with rapid glutamine uptake, which depended on the amino acid transporter ASCT2. ASCT2 deficiency impaired the induction of T helper 1 (Th1) and Th17 cells and attenuated inflammatory T cell responses in mouse models of immunity and autoimmunity. Mechanistically, ASCT2 was required for T cell receptor (TCR)-stimulated activation of the metabolic kinase mTORC1. We have further shown that TCR-stimulated glutamine uptake and mTORC1 activation also required a TCR signaling complex composed of the scaffold protein CARMA1, the adaptor molecule BCL10, and the paracaspase MALT1. This function was independent of IKK kinase, a major downstream target of the CARMA1 complex. These findings highlight a mechanism of T cell activation involving ASCT2-dependent integration of the TCR signal and a metabolic signaling pathway.

Figure 1. ASCT2 deficiency perturbs T-cell homeostasis

Flow cytometry analyses of T-cell populations in the spleen of age-matched Slc1a5^+/+ (+/+) and Slc1a5^−/− (−/−) mice (5 month old). Data are mean ± SD values of multiple mice (each circle represents a mouse). (A and B) Frequency (upper) and absolute number (lower) of CD4⁺ and CD8⁺ T cells (A) and the memory (CD44^hiCD62L^lo) and naïve (CD44^loCD62L^hi) CD4⁺ T cells (B). (C) Frequency of IFN-γ-producing Th1 and IL-17-producing Th17 cells (gated on CD4⁺CD44^hiCD62L^lo CD4⁺ T cells) as well as Treg cells (CD4⁺CD25⁺Foxp3⁺). Data are representative of four independent experiments. *P < 0.05, **P < 0.01. Also see Figures S1 and S2

Figure 2. ASCT2 is dispensable for T-cell proliferation but is required for naïve CD4⁺ T-cell differentiation

(A and B) Proliferation assay (A) and IL-2 ELISA (B) of naïve CD4⁺ T cells from age-and sex-matched wild-type (+/+) and Slc1a5^−/− (−/−) mice (8 wk old), stimulated for 48 h with the indicated doses of plate-bound anti-CD3 plus 1µg/ml anti-CD28. (C–F) Naïve CD4⁺ T cells from Slc1a5^+/+ and Slc1a5^−/− mice were stimulated under Th1, Th2, Th17, or Treg cell skewing conditions and analyzed, on day 4, for frequency of IFN-γ⁺ Th1, IL-17⁺ Th17, and Foxp3⁺ Treg cells by flow cytometry (C and D), relative mRNA amount of the indicated genes by QPCR (E), and the indicated secreted cytokines by ELISA (F). Data are representative of three (A, B, E and F) or four (C and D) independent experiments with three or more mice per group (mean ± s.d. in A, B, D, E and F). *P < 0.05 and **P < 0.01. Also see Figure S3

Figure 3. ASCT2 regulates CD4⁺ T-cell differentiation in vivo

(A and B) Body weight (BW) changes (percent of initial weight) (A) and frequency of the indicated effector T cells (percent of CD4⁺CD44⁺ cells) in the mesenteric lymph nodes (B) of RAG1-deficient mice (6 wk old) adoptively transferred with wild-type (+/+) or Slc1a5^−/− (−/−) CD45RB^hi CD4⁺ T cells (5 wk after adoptive transfer). (C) Slc1a5^+/+ or Slc1a5^−/− mice were infected with L. monocytogenes for 7 days, and splenocytes were isolated and either not treated (None) or stimulated for 6 h with the LLO_190–201 peptide (LLO) in the presence of momensin, followed by flow cytometry analysis of the frequency of CD4⁺ T cells producing IFN-γ (gated on CD4⁺CD44⁺ cells). (D) RAG1-deficient mice were adoptively transferred with CD4⁺ T cells from Slc1a5^+/+OT-II or Slc1a5^−/− OT-II mice and infected with L. monocytogenes. On day 7 postinfection, splenocytes were isolated and either incubated with medium (None) or stimulated with OVA_323–339 and then subjected to intracellular IFN-γ staining and flow cytometry analysis of CD4⁺ T cells producing IFN-γ (gated on CD4⁺CD44⁺ cells). Data are representative of three (A–C) or two (D) independent experiments with least four mice per group (mean ± s.d. in B–D). *P < 0.05, **P < 0.01, ***P<0.001.

Figure 4. ASCT2 has a T cell-intrinsic role in mediating EAE pathogenesis

(A–E) EAE induction in Slc1a5^+/+ and Slc1a5^−/− mice (8 wk old). (A) Mean clinical scores. (B) H&E and Luxol Fast Blue (LFB) staining of spinal cord sections from MOG_35–55-immunized EAE mice (day 30 post-immunization) for visualizing immune cell infiltration and demyelination, respectively (arrows). Original magnification, ×100. (C–E) Flow cytometry analyses of the CD4⁺ T-cell and the CD11b⁺ myeloid cell numbers in the CNS (C) and the absolute number and frequency of the IL-17⁺ Th17 cells and IFN-γ⁺ Th1 cells in the CNS (D) and draining lymph nodes (E) of day 13 EAE-induced Slc1a5^+/+ and Slc1a5^−/− mice. (F–I) EAE induction in Rag1^−/− mice (8 wk old) adoptively transferred with Slc1a5^+/+ or Slc1a5^−/− CD4⁺ T cells. (F) Mean clinical scores. (G–I) Flow cytometric analyses of the CD4⁺ T-cell and the CD11b⁺ myeloid cell numbers in CNS (G) and the absolute number and frequency of Th17 and Th1 cells in the CNS (H) and draining lymph nodes (I) of day 14 EAE-induced RAG1-deficient mice adoptively transferred with Slc1a5^+/+ or Slc1a5^−/−CD4⁺ T cells. *P < 0.05, **P < 0.01, ***P<0.001.

Figure 5. TCR and CD28-stimulated glutamine uptake requires ASCT2 and regulates CD4⁺ T-cell differentiation

(A) Glutamine uptake analysis of Slc1a5^+/+ or Slc1a5^−/− naïve CD4⁺ T cells, stimulated for 30 min with anti-CD3 and anti-CD28 mixed with a crosslinking anti-hamster IgG (left) or for 20 h with plate-bound anti-CD3 and anti-CD28 (right). (B) Intracellular glutamine analysis of Slc1a5^+/+ or Slc1a5^−/− naïve CD4⁺ T cells, stimulated for 20 h with plate-bound anti-CD3 and anti-CD28. (C and D) Flow cytometry analysis of Th1, Th17, and Treg cells generated thorough in vitro differentiation of Slc1a5^+/+ or Slc1a5^−/− naïve CD4⁺ T cells (for 4 days) under the polarizing conditions described in Figure 2 in glutamine-free medium supplemented with the indicated amount of l-glutamine. Data are representative of three (A and B) or four (C and D) independent experiments with at least three mice per group (mean ± s.d. in A and C). *P < 0.05, **P < 0.01, ***P<0.001. Also see Figure S4.

Figure 6. ASCT2 mediates TCR and CD28-stimulated mTORC1 activation in naïve CD4⁺ T cells

(A and B) IB analyses of the indicated phosphorylated (P-) and total proteins in whole-cell lysates of Slc1a5^+/+ or Slc1a5^−/− naïve CD4⁺ T cells, stimulated with anti-CD3 and anti-CD28 using the crosslinking method in regular RPMI 1640 medium (A) or glutamine-free medium supplemented with the indicated amount of l-glutamine (B). (C) Flow cytometry analysis of Th1, Th17, and Treg cells generated thorough in vitro differentiation of Slc1a5^+/+ or Slc1a5^−/− naïve CD4⁺ T cells (for 4 days) under the Th1 or Th17 polarizing conditions in glutamine-free medium supplemented with the indicated amount of l-glutamine. (D) Leucine uptake analysis of Slc1a5^+/+ or Slc1a5^−/− naïve CD4⁺ T cells, either not treated (NT) or stimulated for 20 h with plate-bound anti-CD3 and anti-CD28. (E) IB analysis of the indicated proteins in whole-cell lysates of Slc1a5^+/+ or Slc1a5^−/− naïve CD4⁺ T cells that were either not (−) or stimulated (+) with anti-CD3 and anti-CD28 for 30 min. (F) 2-deoxyglucose uptake analysis of the cells described in D. (G) Slc1a5^+/+ or Slc1a5^−/− naïve CD4⁺ T cells were either not treated (NT) or stimulated with plate-bound anti-CD3 plus anti-CD28 for 20 h in a 96-well plate. Lactate concentration in the culture medium was measured and calculated as described in the Extended Experimental Procedures. (H) IB analyses of Gult1 and c-Myc in whole-cell lysates of the Slc1a5^+/+ or Slc1a5^−/− naïve CD4⁺ T cells described in D. (I and J) Oxygen consumption in Slc1a5^+/+ or Slc1a5^−/− naïve CD4⁺ T cells, either not treated (NT) or stimulated with plate-bound anti-CD3 plus anti-CD28 for 20 h in a 96-well plate. Fluorescence-probe intensity was measured for the indicated time periods and is presented as relative fluorescence unit (RFU) per 10⁶ cells (I). The increase rate of oxygen consumption was calculated at early phase (0 – 60 min) and late phase (60 – 120 min) (J). Data are representative of three independent experiments with at least three mice per group. *P < 0.05, **P < 0.01, ***P<0.001. Also see Figures S5 and S6.

Figure 7. CBM complex is required for TCR and CD28-stimulated glutamine uptake and mTORC1 activation

(A) Glutamine uptake analysis of naïve CD4⁺ T cells derived from the indicated genetic ablation and their wild-type control mice, either not treated (NT) or stimulated for 30 min with anti-CD3 and anti-CD28 using the crosslinking method. (B) IB analysis of phosphorylated (P-) and total S6 in whole-cell lysates of the indicated genetic ablation and wild-type control naïve CD4⁺ T cells stimulated with anti-CD3 and anti-CD28 as in A. (C) IB analyses of phosphorylated (P-) and total S6 and S6K in whole-cell lysates of parental Jurkat cells and the CARMA1-deficient JPM50.6 cells stimulated with anti-CD3 and anti-CD28. (D) QPCR analysis of the relative mRNA amount of the indicated genes in Card11^+/+ or Carma1^−/− naïve CD4⁺ T cells, stimulated for 20 h with plate-bound anti-CD3 and anti-CD28. (E) HEK293 cells were transfected with Myc-tagged CARMA1 or Myc-tagged Bcl10 in the absence (−) or presence (+) of HA-tagged ASCT2. ASCT2 was isolated by IP using anti-HA, and its associated CARMA1 and Bcl10 were detected by anti-Myc IB (upper). The expression of ASCT2, CARMA1, and Bcl10 was monitored by direct IB (lower). (F) Jurkat cells and JPM50.6 cells were stimulated for 15 min with anti-CD3 and anti-CD28 and were stained with rabbit anti-ASCT2 or mouse anti-TCRβ, followed by incubation with Alexa-labeled goat anti-rabbit or goat anti-mouse secondary antibodies. The subcellular localization of ASCT2 (green), TCRβ (red) and nucleus (blue) were visualized by confocal fluorescent microscopy. Scale bar, 5 µm. Data are representative of three independent experiments with three or more mice per group (mean ± s.d. in A and D). *P < 0.05, **P < 0.01. Also see Figure S7.