Microbiota-derived 3- I AA is involved in cancer treatment

Bioinformatics study of first-line chemotherapy for metastatic adenocarcinoma in a large cohort of patients with benign mPDAC

Patients who had been diagnosed with mPDAC and scheduled for treatment with GnP or FOLFIRINOX were recruited to the study (n = 30). In order to have informed consent from their patients, they had to get approval from the ethics commission in Hamburg, Germany. Antibiotics received in the first few months of treatment did not deliver pre-treatment stool, so they were excluded from the analyses. Stool was collected before the start of chemotherapy using a home sampling kit (OMNIgene, Gut OMR-200) for shotgun metagenomic sequencing. One sample was excluded due to overloading of the sample tube which led to unsuccessful fixation and limited representation of the microbiota. Therefore, the final cohort for microbiota analysis contained 22 patients. The best response to first-line therapy is determined by the size of the primary tumours, the time point before treatment begins and the number of scans that reveal the largest metastases in comparison to the time point that shows the best response. The criteria of a PFS of at least 140 days, or a decline from at least 40% in tumour markers, are used in cases of disease stabilization. Stool for functional experiments was collected from tubes without any Additive and transported directly to the laboratory, where it was washed with 20% glycerol and frozen at 80 C. To preserve most of the donor microbiota for functional experiments, the maximum allowed turnaround time from toilet to freezer was 4 h. Blood was drawn at cycle three or four of chemotherapy treatment, mixed 1 to 1 with phosphate-buffered saline (PBS) and plasma was isolated using gradient centrifugation. Serum material of the Munich cohort was taken before the start of chemotherapy and processed according to local standards. Informed consent was obtained from all patients as approved by the ethics commission Munich (project number: 284-10).

Source: https://www.nature.com/articles/s41586-023-05728-y

Bone marrow chimeras for mice of Isocages based on an experimental study of the neurotoxicity of C57BL/6 mice

All mice used in this study were of a C57BL/6 background. The institutional review board is known as the Behrde fr Soziales, Familie, Gesundheit und Verbraucherschutz. Mice were kept under SPF or germ-free conditions, with an ambient temperature of 20 ± 2 °C, humidity of 55 ± 10% and a dark–light cycle of 12 h. Age- and sex-matched littermates between 4 and 16 weeks old were used for the most part. Mpo–/– bone marrow used to establish bone marrow chimeras was provided by S. Baldus and M. Mollenhauer. Ahr–/– bone marrow used to establish bone marrow chimeras was provided by C. Esser.

The stool was washed with brain heart effusion broth and then put into a container that was washed with stool. The suspension was grated to gnotobiotic mice in Isocages. Two to four weeks later, tumour experiments were initiated.

Sample sizes were calculated on the basis of small pilot experiments and mice were randomized before the beginning of treatment. The person treating the mice didn’t lose sight because they had complex treatment schedules. For models of orthotopic PDAC, 5 × 104–10 × 104 KPC, 2 × 105 Hy19636, 1 × 105 mSt-ATG4B/mSt or 1 × 105 knockdown KPC (AhR, GPX, scramble control) cells were injected orthotopically in a 1:1 mixture of PBS and Matrigel (Corning, 356231). Tumour weight was assessed at day 20 after tumour cell injection unless otherwise indicated. For subcutaneous tumour growth, 2.5 × 105 LLC-GFP or 1 × 106 MC38 cells were subcutaneously injected into the flank. Subcutaneous tumour growth was measured every other day using a caliper. Tumour weight was assessed at day 17 after tumour cell injection. There were limits for the diameter of the tumours on active experimental mice and they were not exceeded.

For bone marrow chimeras, recipient mice were irradiated with 9.6 Gy (BioBEAM 2000) 24 h before bone marrow transfer. One day later, 1 × 106–4 × 106 bone marrow cells isolated from wild-type, Ahr–/– or Mpo–/– mice were transferred by intravenous injection. Cancer cells were injected after three to four weeks after transfer. Quantitative PCR on tumours-infiltrating immune cells was used for validation.

In order to begin treatment with chemotherapy, it is necessary to start it in the abdominal region. at the indicated time point (usually day 11 after cancer cell injection). The five-and-one combination of Oxalipoline, irinotecan, sufentanil, and medac were used forFIRINOX treatment as described in other studies. Folinic acid was not used and gemcitabine (Hexal) 120 mg kg−1 and nab-paclitaxel (Celgene) 30 mg kg−1 were used for GnP treatment. If 200 g anti-CD8 clone 53-6.3 was used alone or in combination with 200 g Anti-CD4 clone GK 1.5, the T cells would be destroyed every third. Control mice were treated with the respective isotype control clone 2A3 (BioXcell, BE0089) at similar concentrations and intervals. One day before and on the day of cancer treatment, two injections of trehalose were administered. Similarly, 60 mg kg−1 chloroquine (Sigma, C6628) dissolved in PBS was injected i.p. On the day of the treatment.

The 3-IAA treatment was applied by oral gavage for five days in a row, two days before and two day after the treatment is finished. Indole-3-propionic acid (3-IPA; Sigma, 57400), GCA (Sigma, G2878), hippuric acid (Sigma, 112003) and DCA (Sigma, 30960) were dissolved in 1 M NaOH in PBS and pH-adjusted to 7.4 using 1 M HCl. The solution was gavaged for five consecutive days orally at a concentration of 500 mg kg−1 (3-IPA, hippuric acid) or 250 mg kg−1 each (GCA and DCA). In the drinking water ad libitum, NAC was applied for five days at a concentration of 1 g l1.

The treatment lasted until one day after the start of theDietary Trypton Modulation. The experiment had 14 days of it being applied. Standard diet (2.3 g kg−1 tryptophan; Altromin, 1320) was changed to either synthetic crystalline AA tryptophan-free (0 g kg−1; SSNIFF, S9336-E701) diet or crystalline AA tryptophan-high (12 g kg−1; SSNIFF, S5714-E711) diet. A change was made to the diet back to the standard diet.

Dac is given through the diet for up to seven days after a cancer cell injection. The standard diet was changed back to it’s previous state.

Metagenomic library preparation, sequencing and bioinformatics of human microbiome41 using NEBNext Ultra II FS DNA libraries

Blood was collected by cardiac puncture in EDTA-coated tubes. After a period of 15 min, the red blood cells were removed from the samples, and the blood was then measured using the recommended method for measuring blood sugar.

For shotgun metagenomics, Illumina library preparation was performed using the NEBNext Ultra II FS DNA Library Prep Kit (New England Biolabs, E7805). The library preparation was performed according to the manufacturer’s instructions. The size selection was performed using AMPure XP beads (Beckman Coulter, A63882) and adaptor enrichment was performed using seven cycles of PCR using the NEBNext Multiplex oligos (New England Biolabs, E7335) from Illumina and then subjected to Illumina NovaSeq (2 × 150 bp) sequencing.

For bioinformatic analysis, raw reads were trimmed for low quality and filtered against the phix174 and human hg19 genome with bbduk (ref. 40). For taxonomic species profiling, all libraries were mapped against the Unified Human Gastrointestinal Genome collection (n = 4,644) using BBMap (refs. 41,42 Mapping rates were normalized into transcripts per million (TPM) and genomes with less than 10% genome coverage (genome breadth) were considered not prevalent in the sample. Data were summarized as metagenomics operational taxonomic units (OTUs) into biom format and analysed with phyloseq and LEfSe (refs. The book is 43,44). Functional profiling was done with H UMAnN3 and the 9.9 million gene integrated reference catalogue of the human microbiome41.

The German Collection of Microorganisms and Cell Cultures (DSMZ) obtained several Bacteroides thetaiotaomicron, B. fragilis and Prevotella coPri. For bacteria isolation, faecal samples frozen in glycerol were thawed and streaked out anaerobically in serial dilutions on BHI blood agar plates (5% defibrinated sheep’s blood & vitamin K3) supplemented with vancomycin. After growing the agar plates inside an incubator at 37 °C for 2 days, single colonies were picked into BHI-S medium in a 96-well plate and were further incubated for 24 h. The resulting cultures were screened using specific primers for B thetaiotaomicron. After passaging PCR-positive wells on agar plates to obtain pure cultures and additional confirmation of identity by Sanger sequencing, resulting strains were maintained in BHI-S until further use. All strains were cultured and kept in BHI soup, which was supplemented with Fetal bovine serum and BHI-S.

The BHI-S medium was inoculated from a fully grown overnight Bacterial Culture that had a 1:200,000 ratio and then incubated until the early exponential phase. The cultures were taken from the chamber and brought to a temperature that was cooler than room temperature. The supernatant was removed and immediately frozen at −20 °C.

A Bayesian analysis of the 16S rRNA gene in tumours using the Ribosomal Database Project (DADA2)

DNA from tumours was extracted using the DNeasy Blood & Tissue Kit (Qiagen, 69504). Approximately 10 mg tissue was digested with Proteinase K in ATL buffer at 56 °C for 1 h. The manufacturer’s protocol is used to process samples. Controls for blank extraction were included in the samples.

Variable regions V1 and V2 of the 16S rRNA gene were amplified using the primer pair 27F-338R in a dual-barcoding approach according to a previous report45. 3.5 l DNA and agarose gel were used for amplification of the tumours. Final PCR products were normalized using the SequalPrep Normalization Plate Kit (Thermo Fisher Scientific, A1051001), pooled equimolarly and sequenced on the Illumina MiSeq v3 2×300bp (Illumina). Demultiplexing after sequencing was based on 0 mismatches in the barcode sequences. The data processing was performed with the DADA 246 workflows, which were adjusted for the V1–V2 region. The Ribosomal Database Project version 16 release provides a framework for the use of the BayesianClassification provided in DADA2. The finest possible classification was used to classify the sequence that was not assignable to genera level.

Detection and quantification of 630 metabolites in plasma, chemiluminescence and serum cultures using MxP Quant 500 Kit (Biocrates)

We obtained data in both positive and negative ionized modes, which allowed for identification and quantification of 630 metabolites. Plasma samples were processed using the MxP Quant 500 Kit (Biocrates) according to the manufacturer’s instructions. In brief, 10 µl of plasma sample, calibration standard and control sample were transferred onto a filter containing internal standards for internal standard calibration. Filters were dried under a stream of nitrogen using a pressure manifold (Waters). The phenyl isocyanate reagent was used to incubated the samples. After drying under nitrogen, analytes were prepared with 5 lmol l 1 Ammonium acetate in methanol, which was further difection for the UPLC–MS/MS analysis. The targeted analysis covered 630 metabolites (https://biocrates.com/mxp-quant-500-kit/) detected by MS/MS after UPLC separation and flow injection analysis (FIA). Two UPLC runs and threeFIA runs are required for each measurement. All analyses were performed on an ACQUITY UPLC I-Class system (Waters) coupled to a Xevo TQ-S mass spectrometer (Waters). Reversed-phase chromatographic separation was accomplished using a C18 LC-column (Biocrates) with 0.2% formic acid in water with 0.2% formic acid in acetonitrile as the eluent system. Methanol is the solvent used in the kit provided by the manufacturer. The UPLC–MS/MS results were analyzed using a seven-point curve and one-point calibration. The lower threshold values were set to zero. The data was analysed using MetaboAnalyst. Concentrations were log-transformed before analysis and raw P values and log2-transformed fold change values are shown in the graphics of Fig. 1.

To quantify 3-IAA or DCA serum concentrations, mouse or human serum was diluted 1 to 10 with PBS and the chemiluminescence immune assay (CLIA) (Abbexa, 3-IAA abx190011; DCA 258844) was performed according to the manufacturer’s protocol. For detection of 3-IAA in cultures, supernatant was processed as described above and directly used for the assay. The conjugate detection was done with 1-s sampling and a gain of 3,400 to 4,000 was adjusted individually for each test. The concentration was determined using supplied standards and interpolated using Prism 8.4.0.

Tumour cell viability and proliferation were assessed using a metric to measure synthesis (matt) or metric to measure synthesis (mathematics test) according to the protocol. The FLUOstar Omega was used to assess absorbance. In other experiments, viability was assessed by flow cytometry using SYTOX (Thermo Fisher Scientific, S34857), PI (Biolegend, 421301) and 2,500–5,000 counting beads (Spherotec, ACBP 100-10) as a reference. The cells were measured using CellROX according to the manufacturer’s protocol and then assessed by flow cytometry.

KPC cells were obtained from Ximbio under catalogue number 153474; Hy19636_GLRM reporter cells and mSt-ATG4B/mSt cells were provided by A. Kimmelman31; MC38 and LLC-GFP (ATCC) cells were provided by A. Giannou; and MIA PaCa-2, BxPC-3 and T3M-4 cells (all ATCC) were provided by C. Güngör. All cells tested negative for mycoplasma contamination. Under standard conditions, Cells were maintained at 37 C and 5% CO2 and regularly inspected. Cells were grown in DMEM GlutaMAX (Thermo Fisher Scientific, 10566016) supplemented with penicillin and streptomycin (Gibco, 15140122) and 10% FBS (Gibco, 10500064). In a 96-well plate, the cancer cells were plated with 5,000 to 10,000 cells per well. Cells were allowed to seed overnight, except in experiments with neutrophil co-culture, treatment of GPX-knockdown cells or co-treatment with MPO. Subsequently, treatment with indicated compounds and treatment duration was initiated. 3-IAA or 3-IPA (3-IPA, Sigma, 57400; 3-IAA, Sigma, I3750) were dissolved in 1 M NaOH in PBS and PH-adjusted to 7.4 using 1 M HCl or DMSO when indicated. NAC was put into PBS at a concentration of 1 mM.

The bone marrow-derived macrophages came from the femurs. Red blood cells were lysed using 10× RBC lysis buffer (BioLegend), and remaining cells were plated on petri dishes at 5 × 106 cells per 10 cm dish in IMDM supplemented with 10% FBS, 1% pen/strep, 50 μM β-mercaptoethanol and 25 ng ml−1 of M-CSF (Peprotech). Five days after differentiation, sweeteners were added for an additional 2 days at final concentration of 0.5 mM. LPS (Sigma E. coli O111:B4 LPS25) was added at day 7 post differentiation at a final concentration of 1 ng ml−1 and BD GolgiStop (1:1340 dilution) for 4 h followed by surface and intracellular cytokine staining for tumour necrosis factor (TNF) (clone MP6-XT22) and IL-1β-pro (clone NJTEN3). Samples were acquired on the BD LSR Fortessa and on the BD FACSymphony. Flow cytometry data was analysed using FlowJo v10 (TreeStar).

Tumour tissue from three different groups of mice were pooled and used to derive some important information. The samples were labelled for 2 h with scioDye 2. The sample was labeled with scioDye 1. The buffer was exchanged with PBS after 2h after the reaction was stopped. The samples were all kept at 20 C until use. The samples were analysed in a dual-colour approach using a reference-based design. The samples were put into a bin and then incubated with a reference sample using a dual-colour approach after the scio Block was blocked. After incubation for 3 h, the slides were thoroughly washed with 1× PBSTT, rinsed with 0.1× PBS as well as with water and subsequently dried with nitrogen. Slide scanning was conducted using a Powerscanner (Tecan) with constant instrument laser power and PMT settings. GenePix Pro 6.0 was used to perform spot segment. Acquired raw data were analysed using the (LIMMA) package of R-Bioconductor after uploading the median signal intensities. A special invariant Lowess method was used. The data from the 3-I AA and theFIRINOX samples were uploaded to the STING database. The standard pipeline was used to perform KEGG enrichment analysis. Only pathways with a false discovery rate (FDR) < 0.05 were considered statistically significant.

Raw reads were quality and adapter trimmed using cutadapt (version 2.10) before alignment50. Both the RSEM 1.3.1 and STAR 2.8.6 were used for the mapping of reads and the count of genes. Normalization of raw count data and differential expression analysis was performed with the DESeq2 package (version 1.30.1)47 within the R programming environment (v4.0.3)45. The pairwise comparisons were sucralose 24 h and tm 24 h; saccharin 24 h and tm 24 h; and sucralose 48 h and tm 48 h. DAVID 53,54 was used to look for pathways and functions.

Total RNA was extracted from cell lines using Trizol Reagent (Invitrogen,15596018) and the total RNA extraction kit (Qiagen, 74004/74104) according to the manufacturer’s protocol. The High-Capacity cDNA Synthesis Kit (Thermo Fisher Scientific, 4368813) was used for cDNA synthesis. Applied Biosystems bought primer and probes. Mouse probes and primer are called Gpx7 (Mm0048) and AHR. The TaqMan Master Mix is used on the StepOne Plus system. Forty to 44 cycles were applied for every test, including technical doublets. If two of the three values were not visible, the expression was non-detectable. Relative expression was normalized to Gapdh (Mm99999915_g1).

The human U6 promoter of the Lentiviral vectors directed against the three mouse species. The production of lentiviral particles has been described in detail elsewhere50, and protocols are available online (http://www.LentiGO-Vectors.de). For the transduction of KPC cells with the HIV-1-derived lentiviral vectors, 2.5 × 104 cells were plated in 0.5 ml medium with 8 μg ml−1 polybrene per well of a 24-well plate. The stable integration of shRNAs and the puromycin resistance genes was lead to by the addition of 10 ls of nonconcentrated lentiviral particles. The plate was 1,000g and 25 C for 1 h to increase its spin-inoculation rate. The selection of successfully transduced cells with 1 μg ml−1 puromycin in the culture medium was started four days after transduction.

Quantification of LC3B and Ki67 Using ImageJ v.2.1.0/1.53c and Tissue-TEK OCT

In a blinded manner, quantification was performed for LC3B and Ki67. Positive cells were determined using ImageJ v.2.1.0/1.53c and the threshold was adjusted according to the staining intensity of the respective antibody and maintained for all tumours analysed with the same staining. The number of positive cells was counted per 250–500 × 500-μm field (10× to 40× magnification) in three to five fields per sample.

The tissues were fixed overnight in 4% PFA solution at 4 C, and were cultured for 24 hours in PBS with 30% sugar and embedded in the Tissue-TEK OCT compound. For further analysis, 7-μm sections were used. The Widefield image was performed using the THUNDER imager 3D Live Cell and 3D Cell Culture with a 40 1.10 NA water impact objective. LED power and exposure time and other system-specific settings were first optimized using positive control tissue and not changed between image acquisitions of the different groups to provide optimal comparability. The tissue section had five areas of interest randomly selected and imaged. ImageJ imaging software was used for file navigation, adjustment of colour balance and image analysis. GFP and RFP were determined using a program called ImageJ. The mean fluorescence intensity for each respective signal was determined per slide in at least five areas per tumour.

Source: https://www.nature.com/articles/s41586-023-05728-y

An animal study of chronic indigestion in caged male C57BL/6J Tcra/ mice (Ad libitum, water, sucralose)

All statistical analyses were done using GraphPad. Normality and log-normality were tested using Shapiro–Wilk or Kolmogorov–Smirnov tests. If normality was not given, non-parametric testing was performed. If not otherwise indicated, tests were performed two-sided and resulted in significant. P values are shown.

At least four mice were used in the experiment. For more complex models, we used more animals to compensate for the increased expected variability. The mice were assigned to different treatment groups. After measuring starting bodyweight, mice were divided into groups according to their weightmatched cohort.

Six- to eight-week-old male C57BL/6J Tcra−/− mice were given either water or sucralose (0.72 mg ml−1 or 0.17 mg ml−1) ad libitum for 2 weeks before T cell transfer and until the end of the experiment. 0.5 × 106 cells congenic CD45.1+CD4+CD45RB+ T cells were injected per mouse. Inflammation at day 21 was assessed by intracellular cytokine staining followed by flow cytometric analysis. The UK Home Office’s project licence authorized a maximum weight loss of 15% and chronic indigestion as humane endpoints. None of these limits were exceeded.

Individually caged male C57BL/6J littermates were used to calculate their body weight, food intake and solution intake. Body weight and food intake were measured weekly. Solution intake was measured twice weekly by measuring the weight of the solutions. Fresh solutions were provided after every measurement every 3–4 days.

After 48 h of acclimatization, we continually measured O2 consumption, CO2 production, respiratory exchange ratio, and energy expenditure in mice housed in metabolic cages of a TSE system. The body composition was measured using a device. This animal study was approved by the Animal Ethics Committee of the government of Upper Bavaria (Germany).

The male C57BL/6J mice were given water or a different sweeteners for more than 12 weeks. Mice were food deprived for 6 h before receiving an oral gavage of 2 mg kg−1 of glucose (Merck, 158968). Blood glucose was measured using a glucometer (Accu-CHEK) before the oral gavage and every 30 min up to 120 min post gavage.

Faecal samples were freshly collected from individually caged littermates C57BL/6J male mice aged 8 weeks exposed to the different drinking solutions as indicated. The samples were collected after 12 months of treatment. At least two faecal pellets were collected for each mouse, snapped frozen in liquid nitrogen and kept at 80 C until they were out of the mouse. Faecal DNA was isolated using QIAamp PowerFecal DNA KIT (Qiagen, 12830-50). The 16S amplicons were prepared using a reduced volume reaction based on the illuminati 16S Metagenomics Sequencing Protocol. 2 l of each sample was given with 3.25 l H2O, 8.25 l 2 NEB Q5 High-fidelity DNA Polymerase Master Mix and 1 l of a unique 10. A final extension of 72 C was added for 5 minutes after the samples had been placed at 95 C for 3 min.

The clean up was done with a bead-based method. The quality of the libraries was assessed by using a D1000 ScreenTape. They were on the Illumina MiSeq platform. 2× 300 paired-end reads were produced using the 600 cycle MiSeq Reagent kit v3. The libraries were loaded at 8 pM.

The fastq files were processed using DADA2 (v1.18)44, truncating the forward (respectively reverse) reads to 280 and 210 bases and trimming them by 17 and 21 bases, respectively, with a maximum of two expected errors. Taxa and species assignment was carried out using v132 of the SILVA database. The count data was then combined with the processed data in a way that would allow it to be analysed. The log fold changes and P values could be estimated using the DESeq2 but they did not account for the effects of the experimental groups.

Source: https://www.nature.com/articles/s41586-023-05801-6

Monitoring of the bacterial infection response of C57BL/6J Rag2/ male and female mice with CFSE or sucralose injections

For 2 weeks, C57BL/6J Rag2/ male and female mice were provided either water or sucralose. The conjugates of C57BL/6J donors were injected with 1 106 cells per mouse, according to manufacturer’s instructions. At day 3 post injection spleens were assessed for CFSE dilution of donor T cells by flow cytometry.

The iRFP fluorescence was measured using the LiCOR Odyssey Pearl Imager and analysed with a database of more than 1 million images.

EL4-OVA cells (1 × 106 cells per mouse) resuspended in PBS were subcutaneously injected into the left flank of C57BL/6J recipient mice either exposed to water or sucralose (0.72 mg ml−1 or 0.17 mg ml−1) 2 weeks before challenge and until the end of the experiment, 10 days post injection. Tumours were digested and analysed for OVA-specific CD8+ T cells and IFNγ production in response to SIINFEKL peptide stimulation, followed by intracellular cytokine staining. Samples were acquired on the BD LSR Fortessa and on the BD FACSymphony. Flow cytometry data were analysed using FlowJo v10 (TreeStar).

The load ofbacteria was measured at 3 days after the infection. Spleen and liver were isolated and weighed, followed by tissue disruption. Spleen and blood vessels were resuspended in PBS. The plates were plated with fifty micro litres of each serial dilution then placed into a antibacterial incubator at 37 C. The number of colony-forming units were counted the following day and normalized to tissue weight.

C57BL/6J mice (one cohort of male mice aged 8–10 weeks and one cohort of female mice aged 8–10 weeks) were given sucralose (0.72 mg ml−1) or water ad libitum for 2 weeks and until the end of the experiment. Every second day, mice were injected with an anti-IL-4 monoclonal antibody, clone 11B11 or PBS. After the second injection, mice were sacrificed. Peritoneal macrophages were collected by injecting 5 ml of PBS into the peritoneal cavity. The exudate was collected and stained for flow cytometry. The samples were collected by the LSR Fortessa. The data was analysed using Flow Jo v10

Source: https://www.nature.com/articles/s41586-023-05801-6

Joint Clinical Research Facility (JCF): Recruitment of staff and student populations within an informed written consent, ethically approved participatory advertising campaign at the university

Staff and student populations at the university were recruited. Potential participants responded to ethics committee approved advertising by contacting the local clinical research facility. The clinical research facility oversaw recruitment through informed written consent in response to an ethically approved participant information sheet that explained the study. The Joint Clinical Research Facility conducted the recruitment with no bias. Informed written consent and ethical approval was obtained from Wales Research Ethics Committee 6 (13/WA/0190).

Source: https://www.nature.com/articles/s41586-023-05801-6

InDO 1AM – A phosphate free medium for cell proliferation studies. The mouse Pdx1-Kras G12D pancreatic cancer cell line

InDO 1AM. Lymph nodes were made into single-cell suspensions and FLT3L-generated dendritic cells were incubated in 50M of INDO-1AM in RPMI with 1% of FBS for 30. Lymphocytes were washed and spun at 1,300rpm for a short period and then stained with the Fixable VIability to identify viable cells. Cell suspensions were spun and used in the media at a rate of 106 cells per liter. The cultures were stained with the CD11c, B220, MHC II, SIRP1 and XCR1 versions. Cells were heated to an Ambient temperature of 37 C before being taken to the LSL Fortessa. Anti-CD3–biotin (5 μg ml−1) (clone 145-2C11; Invitrogen eBioscience) was added during baseline reading for 1–2 min, followed by 20 μg ml−1 of streptavidin (Invitrogen). After the baseline read, the anti-IgM was injected into the tube. The Dendritic cell samples were injected after the baseline reading. Calcium flux was determined by the ratio between 400 nm (bound) to 500 nm (free) readings. Samples were acquired on the BD LSR Fortessa. Flow cytometry data was analysed using FlowJo v10 (TreeStar). To quantify the percentage of responders, cells were gated after the addition of streptavidin for 250 s.

The primary pancreatic tumours from the Pdx1-cre model have been used to make the Mouse Pdx1-KrasG12D pancreatic cancer cell line. Cells were maintained in culture in DMEM, 10% FBS, and 1% penicillin-streptomycin. The EL4-OVA thymoma cell line was maintained in TCM with 0.4 mg ml−1 of G418 (Roche Diagnostic GmbH). All cells were incubated at 37 °C and 5% CO2 humidified incubators.

TCR-independent proliferation was achieved either by supplementing the media with 100 ng ml−1 of IL-2 or with a high dose combination of PMA (10 ng ml−1) and ionomycin (500 ng ml−1) or low dose of PMA (1 ng ml−1) and ionomycin (50 ng ml−1). T cells were allowed to grow for a few days.

CD8+ Teff cells. Naive CD8+ T cells were isolated using the manufacturer’s protocol and activated using 250,000 cells per well of a 96-well plate coated with anti-CD3 (5 μg ml−1) and anti-CD28 (2 μg ml−1). It was added with IL-2 as well. CD8+ T cells were re-stimulated, followed by surface staining and intracellular cytokine staining for IFNγ expression.

FLUO-3AM. Single-cell suspensions were obtained from lymph nodes. Cells that were resuspended in TCM had Fluo 3AM loaded onto them and were to be kept for 30 minutes with 5 M Fluo 3AM in 20% of the cells. Excess Fluo was removed by two washes with PBS. Cells were finally resuspended in calcium-free PBS supplemented with 10 mM glucose, 2 mM glutamine and 50 μM β-mercaptoethanol and kept on ice. Cells were warmed to 37 °C for 5 min before acquisition using the LSR Fortessa. After a baseline reading of 1-8 min, stimulation of the calcium release with either 1 M Thapsigargin or 200 ng ionomycin (Merck) was required. For quantification, Fluo3 intensity was plotted against time using FlowJo (TreeStar) kinetic option with the mean value and Gaussian smoothing. The area of the curve was obtained by calculating a range of the same amount of time for both treatment and basic condition.

Jurkat T cells were cultured with or without 0.5 mM sucralose for 48 h. The cells were washed twice with ice-cold PBS and then centrifugation was performed on the last cell at 13,500g after the release of digitonin. The pellet was resuspended in 100 μl of ice-cold extraction buffer. The metabolites were extracted and processed as described above. For whole cells controls, cells (3.5 × 106) cultured in presence or absence of 0.5 mM sucralose were washed twice with ice-cold PBS and extracted in 1 ml ice-cold extraction buffer.

T cells isolated from C57BL/6J male mice aged between 6 and 10 weeks—were activated with anti-CD3 (2 μg ml−1) and anti-CD28 (1 μg ml−1) for 3 days. Cells were stained for their surface markers and then loaded with a phase sensitive probe in the presence of a small amount of Pluronic F-127. 30 min at 37 C and 15 min at room temperature were used for the loading. Cells were kept at 37 °C and protected from light before acquisition. ANEq fluorescence emission was measured at 570 nm (high order) and 610 nm (low order) using a BD LSR Fortessa. Flow cytometry data was analysed using FlowJo v10 (TreeStar).

Cells were lysed using RIPA buffer (Millipore) supplemented with 1% SDS and phosphatase and protease inhibitor cocktail (La Roche), denatured at 95 °C, and resolved on NuPAGE polyacrylamide pre-cast gels (Invitrogen, Thermo Fisher Scientific). Gels were transferred onto nitrocellulose membranes (GE Healthcare). T cell or Jurkat T cell lysates were probed as indicated in the manuscript. Working dilution of primary and secondary antibodies are listed in the antibodies section. All uncropped and unprocessed scans and images are in the Source Data files.

Blood was collected by cardiac puncture into EDTA-coated tubes. Blood was spun for 15 minutes in the Eppendorf tubes. The supernatant (plasma) was collected and stored at −80 °C.

Source: https://www.nature.com/articles/s41586-023-05801-6

Preparation and Detection of the mRNA HyperPrep Kit for the Advanced Sequencing Facility at the Francis Crick Institute

The Advanced Sequencing Facility at the Francis Crick Institute uses the KAPA mRNA HyperPrep Kit in their library preparation. 50 million 101-bp reads per sample were generated from technical triplicate libraries using the Illumina HiSeq 4000 platform.

Source: https://www.nature.com/articles/s41586-023-05801-6

Isotopic tracing of U-[13C]glucose in IMDM supplemented with 10% dFBS for glucose-free T cells

Stable isotope tracing of U-[13C]glucose (Cambridge Isotopes) was performed in glucose-free IMDM (The Francis Crick Media Services) supplemented with 10% dFBS. CD4+ and CD8+ T cells were activated with anti-CD3 (5 mg ml−1) and anti-CD28 (2 mg ml−1) for 48 h in the presence and absence of 0.5 mM sucralose. The 10 mM were counted, washed, and replaced with T cells. U-[13C]glucose solution at a concentration of 2 × 106 cells ml−1 followed by a 4 h pulse. Cells were washed twice with ice PBS and the remaining part of the cell was taken out using a buffer containing 50% methanol, 30% acetonitrile and 20% water. Jurkat T cells were exposed to 0.7 mM sucralose for 48 h unless otherwise stated and washed 2 times with PBS. Metabolites were extracted as described above using 1 ml of extraction buffer for 3.5 × 106 cells. For sucralose detection in plasma, the extraction procedure was adapted from ref. 55. A ratio 3:1 (v/v) was added to 50 l of plasma as a result of the thaw on ice. The mixture was put into an ice cube tray for 5 minutes. Centrifugation (13,000 rpm, 10 min, 4 °C) was used to pellet the protein. The supernatant was dried in a vacuum. 350 l chloroform:methanol:water was added to make a polar metabolites, which contained at least 0.275mol of [13C]valine. The phases were separated by centrifugation (13,000 rpm, 10 min, 4 °C). The polar phase was transferred into a LC–MS vial equipped with an insert and dried in a rotary vacuum concentrator. Adding 75 l of a mixture of H2o:methanol to the dried extract was the final step. LC–MS analysed the samples. The sucralose calibrate curve was prepared by spiking 50 ls of plasma with various concentrations of the standard. As described above, the sample preparation and the extraction were done.

Primary or Jurkat T cells were treated for 48 h with 0.5 mM deuterium-labelled sucralose (SCBT). Sucralose localization was performed by cryo-OrbiSIMS analysis using a Hybrid-SIMS instrument (IONTOF GmbH, Thermo Fisher Scientific) at the National Physical Laboratory incorporating an Orbitrap Q-Exactive HF analyser and a time of flight (ToF) analyser57, 58. T cells were deposited onto silicon wafers coated with 50 nm gold, excess media removed, and frozen by plunging into liquid nitrogen. The samples were transported under liquid nitrogen and mounted on a custom Leica sample holder, then transferred into the OrbiSIMS onto a stage pre-cooling to 160 C. A 21 eV flood gun was used with a current of 21 A and a pressure of 9.7 107 mbar for charge compensation. All acquisitions were performed in positive polarity with a sample potential of 80 V and a ToF analyser. The 30 keV Bi+ liquidmetal ion gun with a spot size of 500 nm is used as the analysis beam with a current of 1.74 pA. The 3D image was captured using 10 shots per frame and 3 frames per scanning, with the field of view 200 m by 200 m and non-Interlaced sputtering of 400 s between frames. Analysis was performed at a temperature of ~−160 °C throughout. The instrument liquid nitrogen dewars were covered with bags to keep ice out. The m/z scale of the mass spectrum is used to calibrate it. Sucralose peaks were identified compared with analysis of a pure standard and confirmed by isotope cluster distribution. Spectral analysis was performed using the Orbitrap analyser with a cycle time of 200 μs. A 20 keV Ar3500+ GCIB was used as the analysis beam with a current of 172 pA, a duty cycle of 30% and a spot size of ~20 μm. The analysis was done using a field of view of 40 m and a 20 m width with a random mode. The collisional pressure was less than 10 mbar. The injection time was 2000 ms at a mass resolution of 240,000 m/m with a quick time of 512 ms. Data were acquired and analysed using SurfaceLab 7.3 (IONTOF GmbH). A depth profile was shown in the picture. In 2d, relative increasing depth is indicated by successive data points. The GCIB removes material after each point. The grey region in Fig. The region below the ion signal is considered to be noise if the value is calculated from a control sample. The data represents the mean and s.e.m. There are eight cells located within the same field of view. The ion selection was used to pick the cell regions of interest. A marker for cells.