Fig 2: Enforced expression of CXXC4 leads to suppression of gastric cancer cells proliferation and the activation of T cells by inhibiting the expression of CDK18. A, The expression of CDK18 in gastric cancer and adjacent normal tissues measured by RT‐qPCR, n = 37, *P < .05 tumour tissues vs adjacent normal tissues. B, The expression of CDK18 in GES‐1 and the gastric cancer cell line SGC7901 assessed by RT‐qPCR, n = 3, *P < .05 SGC7091 cell line vs GES‐1 cell line. C, The expression of CDK18 in gastric cancer cell line SGC7901 normalized to β‐actin measured by Western blot analysis, n = 3, *P < .05 oe‐MIR100HG vs oe‐NC. D, The expression of CDK18 in gastric cancer cell line SGC7901 normalized to β‐actin assessed by Western blot analysis, n = 3. E, The expression of CXXC4, MIR100HG and CDK18 measured by RT‐qPCR, n = 3. F, The proliferation of SGC7901 cells detected by CCK‐8 assay, n = 3. G, The proliferation ability and the proportion of activated IFN‐γ+ T cells of CD3+ detected by flow cytometry. H, The cytokine IFN‐γ secreted by T cells measured by ELISA, n = 3, *P < .05 oe‐CXXC4 + oe‐NC vs oe‐NC, # P < .05 oe‐CXXC4 + oe‐CDK18 vs oe‐CXXC4 + oe‐NC. Measurement data were expressed as mean ± SD. Comparisons between tumour and adjacent normal tissues in panel A were analysed using paired t test. The data conforming to normal distribution and homogeneous variance between two groups in panels B and C were analysed by unpaired t test. Comparisons among multiple groups in panels D, E, G and H were analysed using the one‐way ANOVA, followed by Tukey's post hoc test. The data at different time points in panel F were analysed by the repeated measures ANOVA followed by Bonferroni's post hoc test. The experiment was repeated three times independently

Fig 3: Mito-MGN induces melanoma regression and modulates the melanoma immune landscape(A) Melanoma engraftment and treatment schedule. Tumors were harvested and dissociated into single cell suspension for flow cytometric analyses using the strategy shown in Figure S2.(B) Tumor volumes measured ex vivo from mice treated with Mito-MGN or vehicle control.(C) Percent viable CD45+ leukocytes from vehicle or Mito-MGN treated melanoma tumors measured by flow cytometry.(D) Percent viable CD3+ T cells infiltrating vehicle control or Mito-MGN-treated melanoma tumors.(E–G) Effects of Mito-MGN on the melanoma infiltrating T cells. Ratio of CD8+-to-CD4+ T cells (E), percent of CD3+CD8+ T cells expressing granzyme B (F), and levels of CD4+CD25+FoxP3+ regulatory T cells (Treg) (G).(H and I) Enumeration of melanoma infiltrating myeloid cells. Percent CD11b+ myeloid cells (H), CD45+CD3−CD11b+CD11c−Ly6C−F4/80hi tumor-associated macrophages (TAM) (I), suppressive CD206+ TAM (J), monocytic MDSC (M-MDSC) (K), and granulocytic MDSC (PMN-MDSC) (L).(M–O) Impact on peripheral immune populations measured. Ratio of splenic CD8+ to CD4+ T cells (M), CD11b+CD11c−Ly6ChiLy6G− M-MDSC (N), and CD11b+CD11c−Ly6CintLy6Ghi PMN-MDSC (O) as a percent of total CD45+ leukocytes in the spleen. Values in B-O are mean ± SD from 7-10 individual mice from 2 replicate cohorts. ∗, p< 0.05; ∗∗, p< 0.01; ∗∗∗, p< 0.001; ∗∗∗∗, p < 0.0001; ns, not significant as measured using Student's t-test.

Fig 4: 6PGD-/- ablation results in enhanced non-oxidative PPP and accumulation of glycogen(A–C) Naive CD8+ T cells (CD3+CD8+CD44lowCD62Lhigh) were isolated from 6PGD-/- and 6PGDfl/fl mice and stimulated with aCD3 + aCD28 mAbs and IL-2 (20 IU/mL) in the presence of D7-glucose plus 13C5, 15N2-glutamine in (A) or 13C6-glucose in (B) and (C). Isotope-labeling patterns of metabolites of cell extracts were analyzed by IC-UHRMS as fractional enrichment (A and C) or 1H NMR (B). (A) Shows the conversion of D7-glucose and 13C5, 15N2-glutamine into the metabolites of PPP and gluconeogenesis (GNG) via the Krebs cycle. (B and C) Tracks 13C6-glucose incorporation, respectively, into glycogen as well as glucose-1-phosphate (G1P) and UDP-glucose (UDPG) as intermediates of glycogen biosynthesis.(D) Accumulation of dark glycogen particles was evident in electron microscopy (EM) analysis of naive 6PGD-/- CD8+ T cells compared with 6PGDfl/fl after 4 days of stimulation. Measurements of scale bars are given in the figures.(E) Schematic diagram indicating the site of GPI action.(F) Accumulation of glycogen in 6PGD-/- Cd8+ T cells after 48 h of culture evaluated in colorimetric method.(G) 6PGDfl/fl CD8+ T cells were stimulated for 48 h with aCD3 + aCD28 mAbs in the presence of either 6-AN, 6-AN + glycogen phosphorylase inhibitor (GPI), or DMSO and then glycogen content was assessed via colorimetric method. Results were generated from two independent experiments. Legend in x axis: 0 = unlabeled; Dx = sum of D1 to Dx or Glc-derived species; C*Dx = sum of 13C-labeled species with 0-x number of D; N*Dx = sum of 15N-labeled species with 0-x number of D; C3NxDx = sum of 13C3 with 0-x number of D and 15N, which could reflect incorporation of GNG product into the ribose unit of NADPH (cf. atom tracing diagram Figure S9). C*NxDx = sum of 13C-labeled species with 0-x number of 15N and D. G6P, glucose-6-phosphate; 6PG, 6-phosphogluconate; R5P, ribose-5-phosphate; S7P, sedoheptulose-7-phosphate; Gly3P, glyceraldehyde-3-phosphate; X5P, xylulose-5-phosphate; E4P, erythrose-4-phosphate; F6P, fructose-6-phosphate; OAA, oxaloacetate; PEP, phosphoenolpyruvate; TK, transketolase; TA, transaldolase. Error bars represent ± SEM. **p < 0.01; ***p < 0.001.

Fig 5: Blocking 6-phosphogluconate dehydrogenase (6PGD) in regulatory T cells (Tregs) induces reprogramming of glycolysis, mitochondrial respiration, and non-oxidative pentose phosphate pathway (PPP).(A–B) Extracellular acidification and oxygen consumption analysis on YFP+ sorted cells from Pgdfl/fl Foxp3Cre and wild-type (WT) mice was performed using Seahorse XF24 metabolic analyzer as described in supplemental information. The extracellular acidification rate (ECAR) (A) and oxygen consumption rate (OCR) (B) are shown (N = 4). (C) YFP+ cells glucose (Glc) uptake capacity was determined by 2-NBDG uptake, mitochondrial potential (??m) by TMRE, mitochondrial mass by MitoTracker Deep Red FM and mitochondrial ROS by MitoSox Red. Results are representative of three independent experiments with N = 4 per group. (D) YFP+ cells were sorted from WT and Pgdfl/flFoxp3Cre mice and cultured in the presence of IL-2 (700 IU/ml) and anti-CD3/anti-CD28 coated beads (Treg:beads ratio 1:3) plus D7-Glc and 13C5,15N2-glutamine (Gln) for 48 hr. Isotope labeling patterns of metabolites of cell extracts were analyzed by IC-UHRMS. Data shown demonstrates D7-Glc and 13C5,15N2-Gln incorporation into the PPP metabolites via non-oxidative PPP and gluconeogenesis. Results were generated with N = 2. Legend in X-axis D* = sum of D1 to Dx or Glc-derived species; C* = sum of 13C1 to 13Cx or Gln-derived species; C*D* = sum of dual 13C1 to 13Cx and D1 to Dx or Glc and Gln-derived species; G6P, glucose-6-phosphate; 6PG, 6-phosphogluconate; R5P, ribose-5-phosphate; S7P, sedoheptulose-7-phosphate; Gly3P, glyceraldehyde-3-phosphate; X5P, xylulose-5-phosphate; E4P, erythrose-4-phosphate; F6P, fructose-6-phosphate; OAA, oxaloacetate; PEP, phosphoenolpyruvate.