Reagents

Purified anti-human CD3 (OKT3) and purified anti-human CD28 (CD28.2) were from eBioscience. Anti-human Bcl6 (EP529Y) was from abcam (Cambridge, MA, USA). Anti-CBP (D6C5) and anti-P300 (E6D1T) were from Cell Signal Technology (USA). Anti-β-actin (AB21800) and HRP-conjugated anti-rabbit and anti-mouse IgG (ABL3012 and ABL3032) were from Absci (Baltimore, MD, USA). Anti-β-hydroxybutyryllysine antibody (PTM-1204) was from PTMbio (China). Protein A/G-PLUS-agarose beads (37478) were from Cell Signaling Technology (Danvers, USA). Fluorochrome-conjugated anti-CD4 (RPA-T4), anti-human CD185 (RF8B2), anti-human CD279 (MIH4), and anti-human CD278 (DX29) were from BD Bioscience. Recombinant human IL-12, human TGFβ, human IL-23, and human IL-6 were from R&D Systems.

Cell culture and transfection

HEK 293T cells were purchased from the Chinese Academy of Science (Shanghai, China) and cultured in DMEM containing 10% FBS and penicillin/streptomycin at 37 °C under 5% CO2. For plasmid transfection, the cells were transfected with Flag-Bcl6, Flag-Bcl6-KKYR, Flag-Bcl6-KRYK, Flag-Bcl6-RKYK, and Flag-Bcl6-3KR recombination plasmid or pcDNA3.1 using JetPRIME (Polyplus Transfection, USA) according to the manufacturer’s instructions; transfection efficiency is verified by fluorescence intensity. After transfection, cells were treated with BHB according to different groups. Then, the cells were harvested for Western blot detection.

Isolation of PBMCs and Tfh cell differentiation assays

Blood was acquired and diluted with PBS in a ratio of 1:1. The diluted blood was laid carefully above lymphocyte separation solution (Cedarlane Laboratories, Hornby, Ontario) and then centrifuged at 400×g for 30 min; the peripheral blood mononuclear cells (PBMCs) were collected from the interphase. Then, wash once with PBS and centrifuge at 300×g for 10 min.

Naïve CD4+ T cells were enriched from PBMCs by a magnetic cell isolation kit (Miltenyi Biotec, Germany). Subsequently, the cells were activated by plate-bound anti-CD3(2 μg/mL) and soluble anti-CD28 (1 μg/mL) and cultured with anti-IL-12/23 p40 antibody (10 ng/mL), rhIL-12 (1 ng/mL), rhTGFβ (5 ng/mL), rhIL-23 (10 ng/mL), and rhIL-6 (10 ng/mL) in RPMI 1640 medium containing 10% FBS (Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin. The cells were cultured at 37 °C, in a 5% CO2 incubator for 6 days for further analysis.

Western blotting

The cells were harvested and washed with PBS twice and then homogenized with RIPA lysis buffer (Merck Millipore, Billerica, MA, USA) containing protease inhibitor (1:100, I3786, Sigma-Aldrich, St. Louis, MO, USA) and PMSF (1 mM). The cell lysate supernatants were examined by Western blot according to standard protocols.

Immunoprecipitation (IP)

The transfected 293T cells were harvested and lysed in RIPA lysis buffer. The cell lysates were first incubated with 2-mL anti-Flag antibody for 1 h at 4 °C. Then, 20-μL protein A/G-PLUS-agarose beads (for Flag-Bcl6) were incubated in the mixture at 4 °C overnight. Collect immunoprecipitates by centrifugation at 2500 rpm for 5min at 4 °C. Then, carefully aspirate and discard the supernatant. Wash the beads four times with 1-mL RIPA buffer, each time repeating the centrifugation step above. After the final wash, discard the supernatant and resuspend beads in 40 μL of 1× electrophoresis sample buffer. The following steps are the same as Western blotting.

CCK8 assay

HEK 293T and PBMCs were inoculated 5000 cells/well in 96-well plate and then stimulated with different concentrations of BHB at 37 °C under 5% CO2 for 24 h. Then, add 10-μL CCK8 solution to each well and continue to culture for 1–4 h. The absorbance was read at 450 nm. Cell viability was calculated by the following formula: cell viability (%) = [(As-Ab)/(Ac-Ab)] × 100 and inhibition (%) = [(Ac-As)/(Ac-Ab)] × 100 (As, absorbance of experimental well (contains cells, medium, CCK-8, and compound); Ab, absorbance of blank well (contains medium and CCK-8); Ac, absorbance of control well (contains cells, medium, and CCK-8)).

Flow cytometry analysis

The stimulated PBMCs were stained with anti-CD4 at 4 °C for 30 min, and these cells were then fixed and permeabilized using a fixation/permeabilization buffer (BD Biosciences, San Jose, CA, USA). Intracellular staining was performed using anti-human CD185, anti-human CD279, and anti-human CD278. All samples were acquired on a BD Accuri C6 flow cytometer (BD Biosciences), and the data were analyzed using FlowJo software (TreeStar, America).

RNA extraction and quantitative real-time PCR

To determine the mRNA expression levels of the transcription factors T-bet, total RNA was extracted from the PBMCs with an RNAprep Pure Cell/Bacteria kit (TIANGEN, Beijing, China). Then, cDNA was synthesized with a FastQuant RT Super Mix (TIANGEN, Beijing, China) according to the manufacturer’s protocol. qPCR was performed in technical triplicate using a ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) with a LightCycler® 96 analysis system (Roche, Basel, Switzerland)). The thermocycler program was as follows: 30 s at 95 °C for denaturation and then 40 cycles (10 s at 95 °C and 30 s at 60 °C) for PCR amplification, followed by amplicon melting analysis to evaluate the specificity of the reaction and identify the presence of primer dimers. All primers were purchased from GenScript Biotech Corp. The primers were as follows: hβ-actin, 5′-TGG ACT TCG AGC AAG AGA TG-3′ and 5′-GAA GGA AGG CTG GAA GAG TG-3’; hBcl6, 5′-ACA CAT CTC GGC TCA ATT TGC-3′ and 5′-AGT GTC CAC AAC ATG CTC CAT-3′; and hPrdm1: 5′-TAA AGC AAC CGA GCA CTG AGA-3′ and 5′-ACG GTA GAG GTC CTT TCC TTTG-3′. The RNA expression level of each gene of interest was normalized to that of β-actin, and the results were expressed using the 2–ΔΔCt method.

Mass spectrometric analysis

The band of interest was excised from the gel and rinsed three times with Milli-Q water. The band was then cut into approximately 1-mm2 pieces and dried. The gel slices were reduced with 30 μL of 10-mM DTT at 56 °C for 60 min and cooled down to RT. Sixty microliters of 100-mM iodoacetamide was added and the protein was alkylated for 45 min at RT in the dark. Subsequently, the gel slices were completely dried and added to a trypsin solution to incubate at 37 °C overnight. After the reaction was cooled down to RT, the supernatant was removed and saved. The gel was subsequently extracted with 100 μL 0.1% and 5% TFA in 50% ACN by gently mixing and incubated at RT for 15 min, respectively. Each wash was combined with the saved supernatant, and the resulting solution was lyophilizated for further MALDI-TOF/TOF (MALDI-7090, Shimadzu Kratos) analysis. The peptide mass fingerprints and peptide ion MS/MS spectra were acquired on MALDI-7090. The total MS/MS data was searched against SwissProt database using the following parameters: trypsin digestion allowing up to 2 missed cleavages, fixed modifications of cysteine (carbamidomethylation), variable modifications of methionine (oxidation) and lysine (β-hydroxybutyrylation), precursor peptide tolerance of 0.05 Da, and MS/MS tolerance of 0.2 Da. Search results with e values less than 0.01 were judged as positive identifications.

Interacting protein assays

Bcl6 protein was isolated by SDS-PAGE after IP enrichment. The targeted gel was obtained for in-gel tryptic digestion. Gel pieces were destained in 50-mM NH4HCO3 in 50% acetonitrile (v/v) until clear. Gel pieces were dehydrated with 100 μL of 100% acetonitrile for 5 min, the liquid was removed, and the gel pieces were rehydrated in 10-mM dithiothreitol and incubated at 56 °C for 60 min. Gel pieces were again dehydrated in 100% acetonitrile, liquid was removed, and gel pieces were rehydrated with 55-mM iodoacetamide. Samples were incubated at room temperature, in the dark for 45 min. Gel pieces were washed with 50-mM NH4HCO3 and dehydrated with 100% acetonitrile. Gel pieces were rehydrated with 10-ng/μL trypsin resuspended in 50-mM NH4HCO3 on ice for 1 h. Excess liquid was removed and gel pieces were digested with trypsin at 37 °C overnight. Peptides were extracted with 50% acetonitrile/5% formic acid, followed by 100% acetonitrile. Peptides were dried to completion and resuspended in 2% acetonitrile/0.1% formic acid. The follow-up analysis was performed by LC-MS/MS.

Enzyme-linked immunosorbent assay

According to the agreement of the manufacturer, the levels of IL-21 were determined by using a specific ELISA kit (MULTISCIENCES, Hangzhou, China). All cytokines were quantified using the specific standard curve of recombinant cytokines provided by the corresponding enzyme-linked immunosorbent assay kit.

Statistical analysis

Measurement data are represented by mean ± SEM. Statistical comparisons between groups were evaluated by the Student’s unpaired (2-tailed) t-tests. The Spearman test was used to analyze the correlation between two continuous variables. The resulting MS/MS data were processed using Proteome Discoverer 1.3. All statistical analyses were performed using GraphPad Prism software version 7.0 (GraphPad Software, Inc., La Jolla, CA, USA). A p value less than 0.05 (two-tailed) was considered statistically significant and labeled with *. p values less than 0.01 were labeled with **, and p values less than 0.001 were labeled with ***, respectively.