Fig 1: Leucine (LEU) sustained CD8+ tumor-infiltrating lymphocytes (TILs) immunity in vivo. (A) The mean fluorescence intensity (MFI) of CD98 on tetramer+CD8+ T cells from TILs or spleen, or on tumor cells in wild-type (WT) mice bearing MC38 tumor (n=6). (B) Human CD8+ TILs isolated from tumor and para-tumor with patient with colon cancer. The CD98 MFI was analyzed by flow cytometry (n=4). (C) Slc3a2 transcripts in tumors and paired adjacent normal tissue samples for several types of tumor from The Cancer Genome Atlas (TCGA). BLCA, bladder urothelial carcinoma; COAD, colon adenocarcinoma; KICH, kidney chromophobe; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma. (D) Effect of supernatants (sup) from short hairpin RNA (shRNA)-Slc3a2-treated MC38 on mammalian target of rapamycin complex 1 (mTORC1) activity of CD8+ T cells. CD8+ T cells from Rragdfl/flCd4cre (Rragd−/−) or Rragd+/+Cd4cre (Rragd+/+) spleen were rested with sup for 90 min, followed by anti-CD3 and anti-CD28 stimulation for 30 min under the indicated conditions. p-4E-BP1 were stained and analyzed by flow cytometry (n=3). (E) Quantification of CD44 and CD62L expression on splenic CD8+ T cells of Rragdfl/flCd4cre (Rragd−/−) or Rragd+/+Cd4cre (Rragd+/+) fed complete L-amino acids (control), or leucine-deficient (LEU-diet) (n=3). (F) OT-I CD8+ T cells were transferred into the Rag1−/− host bearing MC38-OVA tumor, LEU (70 mg/kg) or PBS was given by intratumor. MC38-OVA tumor growth kinetics (n=6). (G–I) Effect of combination of LEU (70 mg/kg) and antiprogrammed cell death protein 1 (αPD-1) treatment on WT mice bearing MC38 tumor. (G) MC38 tumor growth kinetics (n=6). (H) Intracellular cytokine staining for interferon (IFN)-γ and tumor necrosis factor (TNF)-α, on restimulation of phorbol myristate acetate (PMA) and ionomycin for 4 hours, or Adpgk peptides for 6 hours. The quantification of TNF-α+CD8+ TILs, IFN-γ+CD8+ TILs. Data are shown as mean±SD (error bars) (A–E, H–I). Data are shown as mean±SEM (error bars) (F, G). Representative plots, Student’s t-test were used. *P<0.05; **p<0.01; ***p<0.001.
Fig 2: RagD deficiency restricted T-cell receptor (TCR)-induced mammalian target of rapamycin complex 1 (mTORC1) activity in CD8+ T cells. (A) Naïve CD8+ T cells from Rragdfl/flCd4cre (Rragd−/−) or Rragd+/+Cd4cre (Rragd+/+) spleen, stimulated with plate bounded anti-CD3 and anti-CD28 crosslinking. The expression of p-S6 or p-4E-BP1 were analyzed by flow cytometry, normalized to 0 min (n=5). (B) Immunoblot analysis of p-4E-BP1 levels in Rragdfl/flCd4cre (Rragd−/−) or Rragd+/+Cd4cre (Rragd+/+) CD8+ T cells stimulated with plate bounded anti-CD3 and anti-CD28 overnight (n=5). (C) Immunofluorescence staining of mTOR and lysosomal-associated membrane protein 1 (LAMP1) in naïve CD8+ T cells from Rragdfl/flCd4cre (Rragd−/−) or Rragd+/+Cd4cre (Rragd+/+) spleen stimulated with plate bounded anti-CD3 and anti-CD28 overnight (n=5). Scale bars, 5 μm. (D–J) Naïve CD8+ T cells from Rragdfl/flCd4cre (Rragd−/−) or Rragd+/+Cd4cre (Rragd+/+) spleen were sorted and stimulated with plate bounded anti-CD3 and anti-CD28 for 48 hours (n=5). (D) The quantification of Ki67+ cells in CD8+ T cells. (E) The quantification of AnnexinV+ cells in CD8+ T cells. (F) The quantification of Caspase3+ cells in CD8+ T cells. (G–I) The mean fluorescence intensity (MFI) of CD71, CD44 and CD69 on CD8+ T cells. (J) Quantification of MitoTracker and tetramethylrhodamine (TMRM) in CD8+ T cells. Data are shown as mean±SD (error bars). Student’s t-test was used. *P<0.05; **p<0.01; ***p<0.001.
Fig 3: RagD are required for CD8+ tumor-infiltrating lymphocyte (TIL) functions in tumor microenvironment (TME). (A) OT-I CD8+ T cells were sorted and activated with plate bounded anti-CD3 and anti-CD28, followed by gene editing with short hairpin RNA (shRNA) targeting Rraga, Rragb, Rragc and Rragd. CD8+ T cells were transferred into CD45.1 mice bearing B16-OVA tumor. The intracellular cytokine staining for interferon (IFN)-γ on CD8+ TILs, on restimulation of phorbol myristate acetate (PMA) and ionomycin for 4 hours. The quantification of IFN-γ+CD8+ TILs (n=3). (B–C) Human CD8+ TILs isolated from patient’s tumor and para-tumor with colon cancer (n=7) and liver cancer (n=4). The RagD mean fluorescence intensity (MFI) was analyzed by flow cytometry. (D) Flow cytometry analysis for the correlation between RagD expression with CD39+CD103+ or CD39-CD103- on CD8+ TILs from the patient with colon cancer (n=7). (E–F) Flow cytometry analysis for the correlation between frequencies of IFN-γ+ with RagD expression in CD8+ TILs from the patient with colon cancer (n=4). (F)The intracellular cytokine staining for IFN-γ on CD8+ TILs, on restimulation of PMA and ionomycin for 4 hours. Representative flow cytometry plots of RagD expression (E), IFN-γ expression (F), RagDlow (blue), RagDhigh (red), unstimulated control (gray). (G) Flow cytometry analysis for the correlation between RagD expression with KLRG1+CD39+ or TIM3+CD39+ in H-2Db Adpgk Neoepitope Tetramer+ CD8+ TILs from mice MC38 tumor (n=5). Data are shown as mean±SD (error bars). Student’s t-test was used. *P<0.05; **p<0.01; ***p<0.001.
Fig 4: Tumor cells limited T-cell access to leucine (LEU) and impaired mammalian target of rapamycin complex 1 (mTORC1) activity. (A–B) Expression of p-S6 or p-4E-BP1 in naïve CD8+ T cells from Rragdfl/flCd4cre (Rragd−/−) or Rragd+/+Cd4cre (Rragd+/+) spleen (A), and healthy human donor (B), pretreated with or without amino acids, followed by anti-CD3 and anti-CD28 crosslinking in the presence or absence of amino acids (n=5). (C) CD8+ tumor-infiltrating lymphocytes (TILs) from MC38 tumor were stimulated with anti-CD3 and anti-CD28 in amino acid-deficient (AA−), amino acid-sufficient (AA+) or different single amino acid-deficient medium in vitro, p-4E-BP1 on CD8+ TILs was stained and analyzed by flow cytometry (n=5). (D) Naïve CD8+ T cells rested in AA− for 90 min and then stimulated in LEU, arginine (ARG) or alanine (ALA) medium for 30 min. p-4E-BP1 on CD8+ T cells was stained and analyzed by flow cytometry (n=5). (E) Naïve CD8+ T cells rested in AA− for 90 min and then stimulated in LEU medium for 30 min, in the presence or absence of RagD inhibitor Bi-Li-0186 (10 μM). p-4E-BP1 on CD8+ T cells was stained and analyzed by flow cytometry (n=5). (F–G) Effect of tumor cell culture supernatants on mTORC1 activity of CD8+ T cells. CD8+ T cells were rested with supernatants (sup) from cultured MC38 (F) or B16F10 (G) cells with varying concentrations of LEU, AA− or completed amino acid-sufficient fresh medium for 90 min and then stimulated with plate-bounded anti-CD3 and anti-CD28 for 30 min under the indicated conditions. p-4E-BP1 on CD8+ T cells was stained and analyzed by flow cytometry (n=5). (H) CD8+ T cells from Rragdfl/flCd4cre (Rragd−/−) or Rragd+/+Cd4cre (Rragd+/+) spleen were rested with sup from cultured MC38 with or without LEU (100 μM) for 90 min, followed by anti-CD3 and anti-CD28 stimulation for 30 min under the indicated conditions. p-4E-BP1 on CD8+ T cells were stained and analyzed by flow cytometry (n=3). (I) MC38 tumor cells and CD8+ T cells were cultured at different ratios for 48 hours in a Transwell system with 20 μM or 100 μM LEU. CD8+ T cells stimulated with anti-CD3 and anti-CD28 for 30 min, p-4E-BP1 on CD8+ T cells was stained and analyzed by flow cytometry (n=3). Data are shown as mean±SD (error bars). Student’s t-test was used. *P<0.05; **p<0.01; ***p<0.001.
Fig 5: RagD-deficient CD8+ T cells show dysfunctional phenotypes. (A–D) MC38 mice tumor model on Rragd+/+Cd4cre (Rragd+/+) or Rragdfl/flCd4cre (Rragd−/−) mice. MC38 tumor growth kinetics (n=5) (A), tumor weights at end point (n=5) (B), quantification of interferon (IFN)-γ+ in CD8+ tumor-infiltrating lymphocytes (TILs) on restimulation of Adpgk peptides (n=3) (C), quantification of PD1+TIM3+ in CD8+ TILs (n=3) (D). (E–L) Rragdfl/flCd4cre OT-I (Rragd−/−) CD8+ T cells or Rragd+/+Cd4cre OT-I (Rragd+/+) CD8+ T cells were transferred into Rag1−/− mice bearing MC38-OVA tumor. (E) Graphic of tumor model. (F) MC38-OVA tumor growth kinetics (n=10). (G) Mice survival (n=10). (H) Tumor weights of MC38-OVA tumor at 14 days after adoptive transfer (n=15). (I–J) At 14 days after adoptive transfer, intracellular cytokine staining for IFN-γ, tumor necrosis factor (TNF)-α and granzyme B (GZMB) on restimulation of phorbol myristate acetate (PMA) and ionomycin for 4 hours, or OVA257-264 for 6 hours (n=5). (K) At 14 days after adoptive transfer, quantification of CD39+TIM3+ in CD8+ TILs (n=7). (L) At 14 days after adoptive transfer, quantification of PD1+TIM3+ in CD8+ TILs (n=4). Data are shown as mean±SD (error bars) (B–D, G–L). Data are shown as mean±SEM (error bars) (A, F). Student’s t-test was used. **P<0.01; ***p<0.001.
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