Glutamine promotes human CD8+ T cells and counteracts imiquimod-induced T cell hyporesponsiveness

Abstract

T cells protect tissues from cancer. Although investigations in mice showed that amino acids (AA) critically regulate T cell immunity, this remains poorly understood in humans. Here, we describe the AA composition of interstitial fluids in keratinocyte-derived skin cancers (KDSCs) and study the effect of AA on T cells using models of primary human cells and tissues. Gln contributed to ∼15% of interstitial AAs and promoted interferon gamma (IFN-γ), but not granzyme B (GzB) expression, in CD8⁺ T cells. Furthermore, the Toll-like receptor 7 agonist imiquimod (IMQ), a common treatment for KDSCs, down-regulated the metabolic gatekeepers c-MYC and mTORC1, as well as the AA transporter ASCT2 and intracellular Gln, Asn, Ala, and Asp in T cells. Reduced proliferation and IFN-γ expression, yet increased GzB, paralleled IMQ effects on AA. Finally, Gln was sufficient to promote IFN-γ-production in IMQ-treated T cells. Our findings indicate that Gln metabolism can be harnessed for treating KDSCs.

Keywords: Dermatology; Immunology.

Graphical abstract

Figure 1

Gln supports IFN-γ production by human CD8⁺ T cells Proteinogenic AA in interstitial tissue fluids of freshly excised biopsies from (A–B) actinically damaged skin and (C–D) KDSCs (each n = 6) were quantified by mass spectrometry. (A and C) Heatmap plot showing 20 endogenous AA in six individual patients. (B and D) Pie chart showing the relative contribution of individual AA to the complete pool of 20 AA detected (mean of the n = 6). (E) CD8⁺ IL-2 T cells were incubated with different concentrations of GPNA for 21 h. IFN-γ expression was measured by flow cytometry after restimulation with PMA/ionomycin for 5 h. (F) IFN-γ expression was assessed by flow cytometry in CD8⁺ IL-2 T cells cultured in Gln-free medium supplemented with 4 mM Gln or not for 21 h after restimulation with PMA/ionomycin for 5 h. (G) IFN-γ expression after restimulation with PMA/ionomycin for 5 h and (H) GzB expression in CD8⁺ IL-2 T cells cultured in Gln-free medium supplemented with 4 mM Gln or not for 21 h were measured by flow cytometry. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001. All data are shown as mean ± SEM. AA, amino acids; CLA, cutaneous lymphocyte antigen; GPNA, L-γ-glutamyl-p-nitroanilide; GzB, granzyme B; KDSC, keratinocyte-derived skin cancer.

Figure 2

IMQ perturbs AA metabolism in CD8⁺ IL-2 T cells CD8⁺ IL-2 T cells were treated with IMQ for 21 h. (A) Quantification of intracellular AA by mass spectrometry (n = 5). (B and C) Proteomics analyses (n = 6). (B) Red dots in volcano plot indicate significantly downregulated, green dots significantly upregulated, and black dots indicate all other proteins. (C) Relative protein expression of ASCT2. (D) ASCT2 expression measured by flow cytometry (n = 6). (E) c-MYC mRNA expression evaluated by qPCR (n = 6). (F) p-S6 expression measured by flow cytometry (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001. All data are shown as mean ± SEM. See also Figures S1–S3, and Table S1. AA, amino acids; ASCT, alanine serine cysteine transporter; IMQ, imiquimod; MFI, mean fluorescence intensity; p-S6, phospho-S6 ribosomal protein.

Figure 3

IMQ represses ASCT2 expression in CD8⁺ T-TILs (A) TLR7 expression in CD8⁺ T-TIL lines assessed by flow cytometry (n = 4). (B–D) CD8⁺ T-TIL were treated with or without IMQ. (B) ASCT2 expression measured by flow cytometry after 21 h (n = 4). (C) c-MYC mRNA expression evaluated by qPCR after 4 h (n = 3). (D) p-S6 expression measured by flow cytometry after 21 h (n = 4). (E and F) Full-thickness biopsies from KDSCs were cultured and left untreated or treated on day 1, 3, and 5 with IMQ. (E) ASCT2 and (F) TLR7 expression in CD8⁺ T cells were determined by flow cytometry analyses on day 7. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗p < 0.0001. All data are shown as mean ± SEM. ASCT, alanine serine cysteine transporter; IMQ, imiquimod; p-S6, phospho-S6 ribosomal protein; TLR7, toll-like receptor 7; unst., unstained.

Figure 4

IMQ reduces IFN-γ expression and proliferation of CD8⁺ T cells T cells were treated with IMQ or not for 21 h (A–E and G–J) or for 5 days (F) and analyzed by flow cytometry (A–D and F–J) and proteomics (E). IFN-γ expression in (A) CD8⁺ IL-2 T cells and (B) T-TILs reactivated with PMA/ionomycin and (C) in T-TILs reactivated with CD3/CD28/CD2 tetramers during IMQ treatment. (D and E) GzB expression in CD8⁺ IL-2 T cells. (F) CD8⁺ IL-2 T cells were analyzed for cell divisions using the CFSE dilution method. Representative histogram plot of one out of five donors. (G) Ki-67 expression in CD8⁺ IL-2 T cells cultured with IL-2 only (n = 7) or (H) reactivated with tetramers and IL-2 (n = 6). (I) Ki-67 expression in CD8⁺ T-TILs cultured with IL-2 only (n = 4) or (J) reactivated with tetramers and IL-2 (n = 5). ∗p < 0.05, ∗∗p < 0.01. All data are shown as mean ± SEM. See also Figures S4 and S5. CFSE, 5(6)-carboxyfluorescein-diacetate-N-succinimidyl ester; CLA, cutaneous lymphocyte antigen; GzB, granzyme B; IMQ, imiquimod; IFN-γ, Interferon-gamma; PMA, phorbol-12-myristat-13-acetat.

Figure 5

Gln is sufficient to promote IFN-γ expression in IMQ-treated CD8⁺ T cells (A) Control and IMQ-treated CD8⁺ IL-2 T cells cultured in AA-free medium supplemented or not with Gln, Asn, Asp, or Ala alone or a pool of all four AA (each 2 mM) (n = 4). IFN-γ expression was assessed by flow cytometry. (B) T-TILs were treated with IMQ and cultured in AA-free medium supplemented or not with Gln alone or a pool of all four AA over 21 h (n = 3). IFN-γ expression was assessed by flow cytometry. (C and D) IMQ-treated CD8⁺ IL-2 T cells were cultured in Gln-free medium supplemented with 4 mM Gln or not for 21 h. (C) IFN-γ expression and (D) GzB expression were measured by flow cytometry. (E) Full-thickness biopsies from KDSCs were treated on day 1, 3, and 5 with IMQ together with or without Gln. After stimulation with activation tetramers on day 6 for 21 h, IFN-γ expression in CD8⁺ T cells was determined by flow cytometry analyses on day 7 (n = 4).∗p < 0.05, ∗∗p < 0.01 (in (E) values were log transformed to align data points to normal distribution). All data are shown as mean ± SEM. AA, amino acids; CLA, cutaneous lymphocyte antigen; T-TILs, tumor-infiltrating T lymphocytes.