Animals

Female NOD/ShiLtJ mice (8- to 9-week-old) were purchased from the Animal Model Research Center of Nanjing University (Nanjing, China). All mice were housed in a specific pathogen-free animal facility at the Tongji Medical College on a 12/12 h light/dark cycle. The Mice were housed in our animal facility for 1-week before the treatment of vehicle or fluvoxamine (20 mg/kg) (HY-B0103A, Medchem Express, Shanghai, China) by peritoneal injection every other day for two weeks. After the mice reached 12 weeks of age, non-fasting blood glucose was measured three times per week using an Accu-Check Advantage glucometer (Roche Diagnostics, Indianapolis, IN, USA) and classed as diabetic once two consecutive blood glucose exceeded 13.8 mmol/l. All experimental procedures were approved by the Tongji Hospital Animal Care and Use Committee in accordance with the National Institutes of Health (NIH) guidelines.

Antibodies and reagents

Anti-mouse CD3e (100340), anti-mouse CD28 (102116), recombinant mouse IL-2 (575406), recombinant mouse IL-6 (575702), recombinant mouse TGF-β (763102), recombinant mouse IL-12 (577004), recombinant mouse IL-23 (589002), Brilliant Violet 421-conjugated anti-mouse CD4 (100443), PerCP anti-mouse CD8a (100731), PE-conjugated anti-mouse/human CD44 (103008), APC-conjugated anti-mouse CD62L (104412), PE/Cyanine7-conjugated anti-mouse IFN-γ (505826), Brilliant Violet 421-conjugated anti-mouse IL-17A (512321), PE-conjugated anti-mouse IL-17A (506904), Alexa Fluor® 647 anti-mouse/rat/human FOXP3 (320014), FITC anti-mouse CD11c (117306), Brilliant Violet 421™ anti-mouse I-A/I-E (107620), PE/Cy7 anti-mouse CD86 (105014), and PE anti-mouse F4/80 (123110) were purchased from Biolegend (San Diego, CA, USA). Anti-phospho-PI3 Kinase p85 (Tyr458)/p55 (Tyr199) (4228S), anti-PI3 Kinase p85 (4292S), anti-phospho-AKT (Ser473) (D9E) XP® Rabbit mAb (4060S), anti-AKT (9272S), anti-PKM2 (4053S) and anti-β-Actin (3700S) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA); anti-PGK1 (A12686), anti-ENO1 (A11448), and anti-LDHA (A1146) antibodies were got from Abclonal (Wuhan, China); anti-HK2 (22029-1-AP) and anti-Glut1 (21829-1-AP) antibodies were obtained from Proteintech (Wuhan, China); and 740 Y-P (HY-P0175) was ordered from Medchem Express (Shanghai, China).

Cell purification and polarization

Naïve CD4+ T cells from 8- to 10-week-old NOD mice were enriched with a Naïve CD4+ T Cell Isolation Kit for mouse (130-104-453; Miltenyi Biotec, Auburn, CA, USA), and CD4+ T cells were enriched with a mouse CD4+ T Cell Isolation Kit (130-104-454; Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer’s instruction. The cells were cultured in RPMI 1640 medium (plus β-mercaptoethanol) supplemented with 10% FBS, 1% GlutaMax, 1% sodium pyruvate, and 1% Pen/Strep (all from Gibco, Shanghai, China) for further studies. Naïve CD4+ T cells were activated with 10 μg/ml plate coated anti-CD3 and anti-CD28. Th1 cocktail (10 ng/ml IL-2 and 10 ng/ml IL-12), Th17 cocktail (10 ng/ml IL-1β, 10 ng/ml IL-6, 10 ng/ml IL-23, and 5 ng/ml TGF-β), and Treg cocktail (10 ng/ml IL-2 and 10 ng/ml TGF-β) were added to drive Th1, Th17 and Treg polarization, respectively.

T cell proliferation assay

CD4+ T cells purified from the spleen of 8- to 10-week-old NOD mice were stained with Cell Tracer Carboxyfluorescein Succinimidylester (CFSE) (565082; Biolegend). The cells (1 × 106 /ml) were plated in the anti-CD3 and anti-CD28 coated 96-well plate in triplicates, followed by FACS analysis after 3 days.

Metabolic assays

In vitro differentiated cells were cultured in the presence of vehicle or fluvoxamine for 72 h, and washed extensively before the assay. ECAR was measured using a Seahorse XFe24 analyzer (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instruction. In brief, the cells were seeded on XFe24 microplates that had been pre-coated with poly-D-lysine (Sigma) to immobilize cells. The basic assay media contained 2.5 μM dextrose and 2 mM glutamine. Injections of 2 μM oligmycin, 2 μM FCCP, 10 mM 2-deoxyglucose, and 0.5 μM rotenone/antimycin A were then performed sequentially. XFe Wave software (Agilent Technologies, Santa Clara, CA, USA) was used to analyze the data.

RNA-sequencing and bioinformatic analysis

CD4+ T cells were stimulated with or without 10 μM fluvoxamine for 24 h and then collected. DESeq2 was used to analyze the differential expression between the two groups, and the P value was corrected using the Benjamini & Hochberg method. The corrected P value and |log2foldchange| are used as the threshold for significant difference of expression levels. The enrichment analysis was performed based on the hypergeometric test. For KEGG, the hypergeometric distribution test was performed with the unit of pathway. GO analysis was conducted based on the GO term. Construction of the cDNA and sequencing were performed in Wuhan Metware Biotechnology Co., Ltd (Wuhan, China).

ELISA

Cytokine levels in the serum and culture supernatants, including TNF-α, IFN-γ, IL-1β, IL-4, IL-17A, TGF-β and IL-10, were measured using the commercial ELISA kits obtained from BD Biosciences (San Diego, CA, USA) and eBioscience (San Diego, CA, USA).

GSIS assay

Pancreatic islets were isolated and GSIS was carried out using established methods (He et al. 2018). All islets obtained from 5- to 6-week-old NOD donors. Analysis of insulin concentration was carried out using an Ultrasensitive mouse insulin immunoassay kit (EZassay, China) following the manufacturer’s instruction. Serum insulin was determined using a Rat/Mouse Insulin ELISA kit (80-PINMS-E01, ALPCO, Salem, NH, USA) as instructed.

Apoptosis assay

Cell apoptosis was measured by flow cytometry using an Annexin V-FITC/PI Apoptosis Detection Kit from Vazyme (A211-01; NanJing, China). Cells were collected and washed with cold PBS twice. The cells were then resuspended in 100 μl of Annexin V binding buffer and incubated with 10 μl FITC-conjugated Annexin V and 5 μl propidium iodide for 15 min in the dark. All samples were examined using a Miltenyi flow cytometer.

Real-time PCR and western blot analysis

Total RNA extraction and cDNA synthesis were performed using the established techniques (Hu et al. 2016). Protein extraction and Western blot analysis were performed as previously described (Chen et al. 2021). The primer sequences for examined genes are listed in Additional file 1: Table S1. The relative expression levels for each target gene were calculated using the 2−ΔΔCt method. Samples were excluded from analyses if mRNA or protein was not detected.

Human samples

Blood samples were collected in Tongji Hospital from participants with newly diagnosed type 1 diabetes. Subjects with active infection, neoplasia, or other comorbidities were excluded from our study. CD4+ T cells were isolated from PBMCs and cultured with or without 20 μM fluvoxamine for 48 h. Consent form was obtained from each study subject, and the study was approved by the Tongji Hospital Human Assurance Committee.

Statistical analysis

Data were represented as mean ± SEM. All in vitro studies were performed at least three times. Statistical analyses of the data, as indicated in each figure legend, were conducted with the GraphPad Prism 5 software (GraphPad Software Inc., San Diego, CA). Two-tailed Student’s t-test was conducted to compare values between two groups. In all statistical cases, differences with p values < 0.05 was considered as statistically significant.