Journal: Science advances

Article Title: CD38-mediated metabolic reprogramming promotes the stability and suppressive function of regulatory T cells in tumor.

doi: 10.1126/sciadv.adt2117

Figure Lengend Snippet: Fig. 1. Intratumoral Tregs display elevated CD38 expression. (A to E) Frequency of FoxP3+CD38+ T cells within the Tregs compartment (CD4+FoxP3+ T cells) was assessed in [(A) and (B)] tumor tissue and paired blood samples from patients bearing (A) breast cancer (Ca) and (B) muscle-invasive bladder cancer (MIBC); [(C) to (E)] nonlymphoid tissues, lymphoid tissues, and tumor tissue from B6 mice bearing 15 days subcutaneously established (C) B16-F10 melanoma, (D) YUMM1.7 melanoma, and (E) EL-4 thy- moma. Adjacent bar plots represent cumulative data from 9 (A), 12 (B), 4 (C), 4 (D), and 4 (E) samples. (F to H) The fluorescence intensity of CD38 was assessed in Tregs from (F) B16-F10, (G) YUMM1.7, and (H) EL-4 thymoma bearing mice. The bar diagrams represent cumulative data from four [(F) to (H)] independent experiments. (I) t–Distributed stochastic neighbor embedding (t-SNE) representation highlights the five identified Tregs (CD3+CD4+FOXP3+) subclusters derived from the single-cell RNA sequencing (scRNA-seq) clusters of CD3G+ T cells. (J) t-SNE plot showing single-cell transcription levels of key genes, with colors indicating their expression: blue for CD3G and CD4, green for FOXP3, and red for CD38. (K) Pie chart depicting the relative abundance of five identified Tregs clusters across the five breast cancer samples. (L) The heatmap displays the top 100 differentially expressed genes, ranked by log2 fold change, across all identified Tregs clusters. *P < 0.05; ****P < 0.0001. FMO, fluorescence minus one.

Article Snippet: HEK293T cells were transfected with lentiviral plasmid DNA encoding CD38 shRNA (Origene) or scrambled negative shRNA (Origene) along with structural plasmid ps- PAX2 and p- MD2G using CaCl2 and HBS method.

Techniques: Expressing, Fluorescence, Derivative Assay, RNA Sequencing

Journal: Science advances

Article Title: CD38-mediated metabolic reprogramming promotes the stability and suppressive function of regulatory T cells in tumor.

doi: 10.1126/sciadv.adt2117

Figure Lengend Snippet: Fig. 2. CD38 endows Tregs with superior suppressive function. (A) Naïve CD4+ T cells from B6 mice were differentiated into Teff or Tregs, and CD38 expression was assessed. The bar diagram shows cumulative data from 11 experiments. (B) Quantitative polymerase chain reaction (qPCR) analysis of various genes in CD38Hi and CD38Lo iTregs. Data are representative of five independent experiments. (C) CellTrace Violet (CTV)–labeled naïve T cells were activated with anti-CD3/CD28 (1 μg/ml each) in the presence of fluorescence-activated cell sorting (FACS)–sorted CD38Hi and CD38Lo iTregs for 3 days. CD8+ T cell proliferation was assessed via CTV dilution. Bar diagram shows data from four experiments. (D) Supernatant from (C) was used to measure interferon-γ (IFN-γ) levels. (E) CD4+FoxP3+ T cell frequency on day 3 of iTregs differentiation (left) and after sorting into CD38Hi and CD38Lo subsets, followed by culturing in interleukin-2 (IL-2) for 2 days (right). The bar plot represents data from four experiments. (F) FoxP3 expression in CD38Hi and CD38Lo iTregs after culturing in IL-2 for 2 days. The bar plot shows data from four independent experiments. (G) CTV-labeled naïve T cells were activated with anti-CD3/CD28 in the presence of wild-type (WT) and CD38−/− iTregs for 3 days. CD8+ T cell proliferation was assessed via CTV dilution. Adjacent bar diagram represents data from five independent experiments. n.s., not significant. (H) Supernatant from (G) was used to assess IFN-γ levels. (I) Human iTregs transduced with either control shRNA or CD38 shRNA were cocultured with CTV-labeled naïve T cells in the presence of anti-CD3/CD28 for 3 days. CD8+ T cell proliferation was assessed via CTV dilution. The adjacent bar diagrams represent cumulative data from five independent experiments. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.0001.

Article Snippet: HEK293T cells were transfected with lentiviral plasmid DNA encoding CD38 shRNA (Origene) or scrambled negative shRNA (Origene) along with structural plasmid ps- PAX2 and p- MD2G using CaCl2 and HBS method.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Labeling, Fluorescence, FACS, Transduction, Control, shRNA

Journal: Science advances

Article Title: CD38-mediated metabolic reprogramming promotes the stability and suppressive function of regulatory T cells in tumor.

doi: 10.1126/sciadv.adt2117

Figure Lengend Snippet: Fig. 3. CD38 expression restricts the mitochondrial metabolism in Treg. (A to G) iTregs were sorted into CD38Hi and CD38Lo subsets and were used to determine: (A) extracellular acidification rate (ECAR) in response to glucose, oligomycin, and 2DG; (B) glycolysis rate after glucose addition; (C) glycolytic capacity; (D) oxygen consump- tion rate (OCR) under basal condition and in response to different inhibitors as indicated; (E) basal respiratory capacity; (F) maximal respiratory capacity; and (G) spare respiratory capacity. Data are representative of four [(A) to (G)] independent experiments. (H to K) WT and CD38−/− iTregs were assessed for (H) OCR under basal condition and in response to the indicated inhibitors, (I) basal respiratory capacity, (J) maximal respiratory capacity, and (K) spare respiratory capacity. Data are representative of four [(H) to (K)] independent experiments. (L and M) iTregs were differentiated in the presence or absence of rotenone (Rote; 1 μM, added at 48 hours) and antimycin A (Anti; 1 μM, added at 48 hours). After washing, they were cocultured with CTV-labeled T cells in the presence of a T cell activation cocktail (anti-CD3/CD28) for 3 days. (L) CD8+ T cell proliferation was assessed via CTV dilution. The adjacent bar diagram shows data from four independent experiments. (M) Supernatant from (L) was used to measure IFN-γ levels. (N to R) Mass spectrometry (MS)–based determination of indicated intracellular metabolites in iTregs differentiated in the presence or absence of rotenone (Rote; 1 μM, added at 48 hours). Data are representative of four independent experiments. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.0001.

Article Snippet: HEK293T cells were transfected with lentiviral plasmid DNA encoding CD38 shRNA (Origene) or scrambled negative shRNA (Origene) along with structural plasmid ps- PAX2 and p- MD2G using CaCl2 and HBS method.

Techniques: Expressing, Labeling, Activation Assay, Mass Spectrometry

Journal: Science advances

Article Title: CD38-mediated metabolic reprogramming promotes the stability and suppressive function of regulatory T cells in tumor.

doi: 10.1126/sciadv.adt2117

Figure Lengend Snippet: Fig. 4. CD38-NAD+ axis regulates the mitochondrial metabolism and suppressive function in Tregs. (A and B) iTregs sorted into CD38Hi and CD38Lo subsets were used to determine: (A) intracellular NAD+ levels and (B) indicated intracellular metabolites using MS. (C to G) FACS-sorted CD38Hi iTregs were cultured overnight in the presence or absence of NMN (500 μM) and were used for (C) determining the indicated intracellular metabolites using MS, (D) OCR under basal condition and in response to the indicated inhibitors, (E) basal respiratory capacity, (F) maximal respiratory capacity, and (G) spare respiratory capacity. (H to I) CD38Hi iTregs cultured overnight in the pres- ence or absence of NMN were cocultured with CTV-labeled naïve T cells in the presence of a T cell activation cocktail (anti-CD3/CD28) for 3 days and assessed for (H) re- sponder CD8+ T cell proliferation via CTV dilution and (I) measuring IFN-γ levels from the supernatant of the experiment shown in (H). (J) qPCR-based analysis of CpG methylation at CNS regions of the Foxp3 locus was performed on CD38Hi and CD38Lo iTregs. (K and L) CD38Hi iTregs cultured overnight in the presence or absence of cell- permeable α-KG (100 μM) were used to assess (K) CpG methylation at CNS regions of the Foxp3 locus and (L) suppressive potential by measuring responder CD8+ T cell proliferation after 3 days of coculture with naïve T cells under T cell activation conditions. (M) qPCR-based analysis of CpG methylation at CNS regions of the Foxp3 locus was performed in CD38Hi iTregs supplemented with NMN. The data are representative of three (A), three (B), six (C), four [(D) to (I)], five [(J) and (K)], and three [(L) and (M)] independent experiments. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.0001.

Article Snippet: HEK293T cells were transfected with lentiviral plasmid DNA encoding CD38 shRNA (Origene) or scrambled negative shRNA (Origene) along with structural plasmid ps- PAX2 and p- MD2G using CaCl2 and HBS method.

Techniques: Cell Culture, Labeling, Activation Assay, CpG Methylation Assay

Journal: Science advances

Article Title: CD38-mediated metabolic reprogramming promotes the stability and suppressive function of regulatory T cells in tumor.

doi: 10.1126/sciadv.adt2117

Figure Lengend Snippet: Fig. 5. CD38 is crucial for the metabolic adaptability of Tregs to use lactate and maintain suppressive function. (A to C) Isotopologue distribution for (A) PEP, (B) acetyl CoA, and (C) pyruvate in CD38Hi and CD38Lo iTregs overnight cultured in glucose-free medium containing 13C3 l-lactate. (D and E) iTregs were differentiated in glucose-free, l-lactate–containing medium and sorted into the CD38Hi population. These were then treated overnight with the indicated inhibitors in the same l-lactate– containing medium and were used for coculturing with CTV-labeled naïve T cells in the presence of a T cell activation cocktail (anti-CD3/CD28) for 3 days and assessed for (D) CD8+ T cell proliferation via CTV dilution and (E) measuring IFN-γ levels from the supernatant of the experiment shown in (D). (F) T cell proliferation was assessed via CTV dilution after 3 days of coculturing naïve T cells with CD38Hi iTregs that were differentiated in glucose-free, l-lactate–containing medium, following the indicated RNA interference–mediated knockdown. (G to J) iTregs were differentiated in glucose-free, l-lactate–containing medium and sorted into the CD38Hi population before being treated overnight with NMN and indicated inhibitors. Cells were then assessed for (G) OCR under basal condition and in response to the indicated inhibitors, (H) CpG methylation at CNS1, (I) CpG methylation at CNS2, and (J) CpG methylation at CNS3 regions of the Foxp3 locus. (K and L) NMN-treated and untreated CD38Hi iTregs differ- entiated in glucose-free, l-lactate–containing medium were cocultured with CTV-labeled naïve T cells for 3 days in the presence of anti-CD3/CD28. (K) Proliferation of CD8+ T cells was assessed by CTV dilution, and (L) supernatant from (K) was used to measure IFN-γ levels. The data are representative of three [(A) to (C)], three [(F) to (J)], four [(D) and (E)], and four [(K) and (L)] independent experiments. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.0001.

Article Snippet: HEK293T cells were transfected with lentiviral plasmid DNA encoding CD38 shRNA (Origene) or scrambled negative shRNA (Origene) along with structural plasmid ps- PAX2 and p- MD2G using CaCl2 and HBS method.

Techniques: Cell Culture, Labeling, Activation Assay, Knockdown, CpG Methylation Assay

Journal: Science advances

Article Title: CD38-mediated metabolic reprogramming promotes the stability and suppressive function of regulatory T cells in tumor.

doi: 10.1126/sciadv.adt2117

Figure Lengend Snippet: Fig. 6. Small-molecule targeting of CD38 impairs intratumoral Tregs and elicits antitumor response. (A) Frequency of adoptively transferred Pmel (CD8+vβ13+) T cells from mice bearing 10 days subcutaneously established B16F10 melanoma. The adjacent bar represents data from n = 4 mice. (B) The bar diagrams depict the frequency of donor Tregs population (Foxp3+ CTV+) from the tumor-bearing mice shown in (A). (C and D) iTregs were differentiated either in complete medium (CM) or in 30% TDS in the presence or absence of CD38i and were assessed for (C) evaluating suppressive potential by checking the proliferation of CTV-labeled CD8+ T cells from the coculture and (D) CpG methylation at CNS1, CNS2, and CNS3 regions of the Foxp3 locus. The data are representative of four (C) and three (D) independent experiments. (E to J) C57BL/6 mice (n = 5 mice per group) with subcutaneously established YUMM1.7 melanoma tumor were treated either with vehicle control or CD38i (15 mg/kg, intra- peritoneal, once a week) and evaluated for (E) tumor growth; (F) Tregs frequency at the spleen, DLN, and tumor site; (G) assessment of Teff (CD8+ T cells) to Tregs ratio at the tumor site; and [(H) to (J)] the ability of CD8+ T cells from the tumor site to coproduce different effector cytokines upon restimulation in vitro. TNF-α, tumor necrosis factor–α. (K and L) BrdU incorporation in (K) CD38Lo and CD38Hi intratumoral Tregs and (L) intratumoral Tregs from Ctrl and CD38i-treated mice. The representative data are from five [(E) to (L)] independent samples. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.0001. FSC-A, forward scatter area.

Article Snippet: HEK293T cells were transfected with lentiviral plasmid DNA encoding CD38 shRNA (Origene) or scrambled negative shRNA (Origene) along with structural plasmid ps- PAX2 and p- MD2G using CaCl2 and HBS method.

Techniques: Labeling, CpG Methylation Assay, Control, In Vitro, BrdU Incorporation Assay