Animal experiments

Animal protocols were performed in accordance with approved guidelines by the KAIST Animal Care and Use Committee (KAIST, KA2018-52). All mice were bred and housed under specific pathogen-free conditions in a 12-hour light–dark cycle. They received food and water ad libitum. CD4-Cre mice and Ipmk-floxed mice (IPMKf/f), who exhibit C57BL/6 backgrounds, were used to generate CD4 helper T cell-specific IPMK knockout mice (IPMKΔCD4) [34]. Male and female mice aged 8–10 weeks old were used for the experiments. Each mice-related experiment used littermates.

Induction of ConA-induced hepatitis

To induce acute hepatitis with T cell activation, PBS or concanavalin A (Sigma-Aldrich, St.Louis, MO) was administered intravenously (IV) via the lateral tail veins to mice weighing 24–26 g at a dose of 12 mg/kg. Serum and livers were obtained from the mice 12 h after the Con A injection. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using VetTest® Chemistry Analyzer and Catalyst One Chemistry Analyser (IDEXX Laboratory Inc., Westbrook, ME, USA). Liver mononuclear cells (MNCs) were isolated from liver tissues by grinding on a 70 mm strainer and centrifuging twice at 50 x g for 5 min. After the supernatant was pelleted (650 x g for 10 min), they were purified using 40% Percoll gradient medium (GE Healthcare; 1,800 x g for 20 minnutes), and RBS lysis buffer (BioLegend).

Histological analysis

Mouse livers were fixed with 10% neutral buffered formalin (Sigma-Aldrich) and embedded in paraffin for histological analysis. Following deparaffinization and rehydration, the sectioned tissues (4 μm thickness) were stained with hematoxylin and eosin (H&E). For apoptotic cell staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed in liver tissues, in accordance with the data protocol provided with the In Situ Cell Death Detection Kit, Fluorescein (Roche).

Cell culture and transfection

Mouse embryonic fibroblasts (MEFs), HEK293T, and NIH3T3 cells were maintained in the following reagents: high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Biowest); 10% fetal bovine serum (FBS) (Atlas Biologicals); 100 mg/mL penicillin/streptomycin (Welgene). MEFs were obtained from IPMKf/fUBCCre−ERT2 mice and they were immortalized by transfecting an SV40 large T-antigen plasmid. To deplete IPMK, 1 µM 4-hydroxytamoxifen was treated with MEFs for 48 h. Thereafter, 10 ng/mL PDGF was administered into cells for each reagent-suitable time and condition (serum starvation for 12 h), when required.

For transient transfection, we used several reagents: jetPRIME reagent (Polyplus) was used for DNA plasmid overexpression in HEK293T cells, following the manufacturer’s protocol. Lipofectamine LTX (Invitrogen) was used for DNA plasmid overexpression in NIH3T3 cells, following the manufacturer’s protocol. NIH3T3 cells were transfected siRNA using Lipofectamine RNAiMAX (Invitrogen). The list of siRNAs used for knockdown is provided as follows (Bioneer): IPMK (Sense: 5′-CAGAGAGGUCCUAGUUAAUUUCA-3′, Antisense: 5′-AGUGAAAUUAACUAGGACCUCUCUGUU-3′).

In vitro CD4+ helper T cell differentiation, proliferation, and TCR stimulation

Naïve CD4+ T cells were isolated from the mouse spleen and inguinal/mesenteric lymph nodes by MACS naïve CD4+ T cell isolation kit, mouse (Miltenyi Biotec) or MagniSort mouse CD4 naïve T cell enrichment kit (Invitrogen), following the manufacturer’s protocols. Isolated CD4+ T cells were plated on plates coated with 10 µg/mL anti-CD3 (16-0031-85, eBioscience) and 10 µg/mL anti-CD28 (16-0281-86, eBioscience) and 55 µM β-mercaptoethanol (Gibco) was also added to the media. The cells were incubated under Th0 cells using 5 µg/mL anti-IFN-γ (16-7311-85) and 5 µg/mL anti-IL-4 (16-7041-85) (eBioscience) in RPMI 1640 medium (Welgene). To trace cell proliferation, the CellTrace™ Carboxyfluorescein succinimidyl ester (CFSE) proliferation kit was used following the manufacturer’s instructions (C34554, Invitrogen).

For TCR signaling stimulation, cells were first stabilized in RPMI 1640 medium at 37 °C for 10 min. Naïve CD4+ T cells were incubated with 5 µg/mL anti-CD3 antibody and 2 µg/mL anti-CD28 antibodies in cold PBS at 4 °C for 20 min. Subsequently, 20 µg/mL anti-Armenian hamster IgG secondary antibody (Jackson ImmunoResearch Laboratory) was added to the RPMI 1640 medium and the cells were treated for either 3–10 min in a 37 °C heat block, after which the medium was rapidly replaced with cold PBS. Additionally, 1 µM PTD and PTD_93–126 (synthesized at Peptron, Daejeon, Korea) were transduced into stabilized naïve CD4+ T cells prior to TCR stimulation in RPMI 1640 at 37 °C for one hour. For ConA-induced TCR signaling stimulation, 5 µg/mL ConA was used in place of antibody-induced TCR stimulation and applied for either 15–30 min.

Plasmid construction

The full-length Sam68 pCMV-GST and fragments were constructed from pcDNA3.0 HA-Sam68 WT DNA plasmid (Addgene) with a pCMV-GST vector. Full-length IPMK pCMV-GST and fragments were constructed as described previously [3, 11]. A MIGR1-Flag-IPMK-DN DNA plasmid was constructed in a MIGR1 vector, which was a gift from Dr. Rho Hyun Seong. LentiCRISPRv2 puro sgIPMK DNA plasmids were also constructed following a previous protocol from Zhang’s Laboratory (GeCKO, Addgene) using Ipmk or Khdrbs1 guided RNA, recommended from CHOPCHOP (https://chopchop.cbu.uib.no) (sgIPMK_1: 5’-AGGCCGTCCGCATCCGTCAG-3’; sgIPMK_2: 5’-GGACCAGACGCCGTAGTATT-3’; sgSam68_1: 5’-TACGCAGAACAAAGTTACGA-3’; sgSam68_2: 5’-AGACGGCGTCTGACGCACCG-3’).

Virus production and transduction of cells

Viruses were generated in HEK293T cells by transfecting the lentiviral DNA plasmid (lentiCRISPRv2 puro vector) with pMD2G and psPAX2.0, or the retroviral DNA plasmid with pCL-Eco viral packaging vector. Viral supernatants were collected 48 h after transfection and used to transduce target cells with 8 µg/mL polybrene for 24 h, after which the medium was replaced with fresh medium. The lentiCRISPRv2 virus-transduced cells were selected with 2 mg/mL puromycin (Gibco) after 48 h of cell incubation. Additionally, retroviruses were transduced into naïve CD4+ T cells, which already incubated for one day on anti-CD3/anti-CD28 antibodies-coated plates. The medium was replaced with Th0 cell-conditioned media after 24 h of transduction, and cells were incubated for an additional 72 h either on anti-CD3/anti-CD28 antibody-coated plates or 20 ng/ml IL-2 (Peprotech).

Yeast two-hybrid screening

The data from the yeast two-hybrid screening are the same as those presented by Beon, Han et al. [10]. The AH109 Saccharomyces cerevisiae strain (Clontech) underwent co-transformation with the full-length pGBKT7-hIPMK DNA plasmid, harboring the GAL4-DNA binding domain (BD), and a human brain cDNA activation domain (AD) library (Clontech). Selection markers included two different reporter genes, HIS3 and ADE2. Transformants were plated on a selection medium lacking leucine, tryptophan, and adenine or histidine (SD-LWA and SD-LWH). Candidate prey genes from the library were either amplified via PCR or transformed into E. coli to validate interactions, then reintroduced into the AH1009 yeast strain alongside the IPMK bait plasmid. All procedures for yeast two-hybrid screening were outsourced to Panbionet (Pohang, South Korea, http://panbionet.com).

SDS-PAGE and immunoblotting

For immunoblot analysis, cells were washed with PBS and lysed in lysis buffer consisting of 40 mM (pH 7.4) Tris–HCl, 1 mM EDTA, 1.5 mM sodium orthovanadate, 1% NP-40, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, 200 mM NaCl, and protease inhibitor cocktail (Roche). Protein concentrations were determined by Bradford protein assay (Bio-rad) and the subsequent protein lysates were boiled at 95 °C for 5 min with SDS sample loading buffer (25% glycerol, 0.1% bromophenol blue, 60 mM Tris–HCl, 2% SDS, and 14.4 mM β-mercaptoethanol). A total of 10–20 µg of protein lysates was electrophoresed on SDS-PAGE gels and the separated proteins were transferred onto a nitrocellulose membrane. After blocking with 5% skim milk in Tris-buffered saline (TBS-T, 0.1% Tween-20), the membranes were blotted with primary antibodies and HRP-conjugated secondary antibodies. Antibodies against the following proteins were obtained from the indicated sources: Flag (F1804), Tubulin (T5109) (Sigma Aldrich); Sam68 (sc-1238), normal rabbit IgG (sc-2027), GAPDH (sc-32233), LAT (sc-53550) (Santa Cruz Biotechnology); HA (MMS-101R, Biolegend); GST (2622), β-actin (4970), phospho-PLCγ1 (Tyr783) (2821), PLCγ1 (5690), phospho-Src family (Tyr416) (6943), Src (2108), phospho-PKCθ (Thr538) (9377), PKCθ (13643) (Cell Signaling Technology). IPMK (NBP1-32250, Novus Biologicals).

Immunoprecipitation and GST pull-down assays

For immunoprecipitation, 160 µg to 2 mg of total protein lysates were incubated with 0.5 µg to 2 µg of primary antibodies or 10 µL net volume of anti-Flag M2 beads (Millipore) overnight with rotation at 4 °C. A total net volume of 10 µL of TrueBlot beads (Rockland Immunochemicals) was added to lysates with antibodies and incubated for additional 2 h, then, the samples were washed three times with lysis buffer and boiled at 95 °C for 5 min with 10 µL of 1 M DTT and 2x Laemmli sample loading buffer (100 mM Tris–HCl, 20% glycerol, 4% SDS, 28.8 mM β-mercaptoethanol, and 0.05% bromophenol blue).

For the GST pull-down assay, 10 µL net volume of glutathione agarose beads (Incopharm) were added to 1.5 mg to 2 mg of total protein lysates and incubated overnight at 4 °C with rotation, then, the samples were washed three times with lysis buffer and boiled at 95 °C for 5 min with SDS sample loading buffer.

In vitro binding assay

For the in vitro binding assay, recombinant human IPMK protein was purified, as described previously [35]. 500 ng of Flag-Sam68 purified protein (TP300263, OriGene) was incubated with 10 µL net volume of anti-Flag M2 beads in 0.1 M NaCl TGEM buffer (20 mM Tris–HCl, 20% glycerol, 1 mM EDTA, 5 mM MgCl2, 1 mM DTT, 0.2 mM PMSF, and 100 mM NaCl) for 2 h at 4 °C with rotation. Then, the samples were washed with 0.5 M NaCl TGEM buffer and 0.1 M NaCl TGEM buffer, twice each. Afterward, 500 ng human IPMK purified protein was added to the samples with 0.1 M NaCl TGEM buffer and incubated at 4 °C with rotation for an hour. Finally, the samples were washed three times with 0.1 M NaCl TGEM buffer and boiled at 95 °C for 5 min with 10 µL of 1 M DTT and 2x Laemmli sample loading buffer.

In vitro kinase assay

In vitro kinase assay was conducted using PLCγ1, Sam68, Src, and IPMK purified proteins, according to a previously described protocol by Jones N. P. et al. and Rodriguez R. et al. [36, 37]. A total of 100 ng Flag-PLCγ1 (TP316448, OriGene), 150 ng Flag-Sam68 (TP300263, OriGene), and 150 ng GST-Src (10755-H20B, Sino Biological) were incubated at 37 °C for 10 min in kinase assay buffer (50 mM Tris pH 8, 0.2 mM MnCl2, 2 mM MgCl2, 2 mM DTT, and 0.02% TritonX-100) to form a complex. Then, 150 ng His-human IPMK protein, 50 µM ATP, and 1 mM Na3VO4 was added in an iced-cold protein mixture, or kinase assay buffer was added alongside 50 µM ATP, and 1 mM Na3VO4, and used as the control. Kinase assay was incubated at 37 °C for 20 min. Samples were boiled at 95 °C for 5 min with SDS sample loading buffer.

RNA isolation and RT-qPCR

RNA preparation and RT-qPCR protocols have been described previously [3]. Briefly, total RNA was isolated from cells using TRIzol reagent (Molecular Research Center), according to the manufacturer’s protocol. A total of 1 µg of total RNA was used for the synthesis of first-strand cDNA by Superscript III reverse transcriptase (enzynomics). Quantitative real-time PCR analysis was performed using the SYBR Green master mix (Toyobo) and the Step One Plus Real-Time PCR system (Applied Biosystems). The primer sequences for RT-qPCR were as follows: Actb (forward: 5’-GTGGCATCCATGAAACTACA-3’; reverse: 5’-CTCATCGTACTCCTGCTTGC-3’), Ipmk (forward: 5’-CCAAAAT-ATTATGGCATCTG-3’; reverse: 5’-TATCTTTACATCCATTATAC-3’), Il2 (forward: 5’-AACCTGAAACTCCCCAGGAT-3’; reverse: 5’-TCATCGAATTGGCACTCAAA-3’).

Flow cytometry

To assess cytokine production in CD4+ T cells, their stimulation was induced using 50 ng/mL PMA (Calbiochem) and 500 ng/mL ionomycin (Sigma Aldrich) with brefeldin A (Invitrogen; 1:1000) for 3 h. Fixable Viability Dye (65-0866-14, eBioscience) was administered to CD4+ T cells for 30 min to exclude any dead cells. Next, fluorescent-conjugated antibodies were added for 20 min to stain the CD4 cell surface markers. Then, CD4+ T cells were fixed and permeabilized using each previously described step-appropriate buffer (Invitrogen). Finally, CD4+ T cells were incubated with fluorescent-conjugated antibodies for 30 min to stain IL-2 cytokines. Flow cytometry was performed by LSRII Fortessa (BD) and data were analyzed using the FlowJo software (FlowJo). The fluorescent-conjugated antibodies used to detect cell surface markers or cytokines were as follows: anti-mouse CD4 eFluor450 (48-0042-82), anti-mouse CD44 APC eFluor780 (47-0441-82), anti-mouse CD25 PE (12-0251-82), anti-mouse TCRβ (11-5961-82), anti-mouse IL-2 APC (17-7021-82), anti-mouse IFN gamma PE (12-7311-82), anti-mouse IL-4 (12-7041-82), anti-mouse IL-17 A PE-Cyanine7 (25-7177-82, eBioscience); and anti-mouse CD69 Alexa Fluor® 700 (561238), anti-mouse TNF PE (561063, BD Biosciences); phospho-PLCγ1 (Tyr783) Alexa Fluor® 647 (88717, Cell Signaling Technology).

Calcium flux measurement

Calcium flux was measured in naïve CD4+ T cells by following the Fluo4 AM (F14201) and Fura Red (F3020, Invitrogen). A total of 2 µM Fluo4 or 5 µM Fura Red was added to naïve CD4+ T cells, and the cells were incubated at 37 °C for 25 min, followed by washing with DPBS. The calcium flux of anti-CD3 and anti-CD28-coated cells was analyzed in calcium-free HBSS medium (Welgene) by flow cytometry with a measurement time of 300 s (basal time: 0–30 s; activation time (treatment with anti-Armenian hamster IgG secondary antibodies): 50–300 s). Calcium flux measurement using Fura Red was calculated as ratiometric calcium flux (Qdot 655-A/PerCP-Cy5-5-A), with reference to Wendt ER. et al. [38].

Statistical analysis

Data analysis was performed using GraphPad Prism Software 9.0. All data were first analyzed for normality using the Shapiro-Wilk test. Statistical analyses were conducted using various methods, including unpaired two-tailed Student’s t-test, one sample t-test, one-sample Wilcoxon signed rank test, Mann-Whitney test, and one-way or two-way ANOVA. Means are presented as mean ± SD, with p values considered significant as follows: ns, non- significant; *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001.