Reagents and antibodies

Reagents and antibodies used in this study were purchased from the indicated companies: recombinant human PD-L1-Fc fusion protein (BPS Bioscience, Catalog #71104), recombinant mouse PD-L1-Fc fusion protein (Sino, Catalog #50010-M02H), anti-human CD3ε (clone OKT3, Biolegend, Catalog #317326), anti-human CD28 (clone CD28.2, Biolegend, Catalog #302934), anti-mouse CD3ε (clone 145-2C11, Biolegend, Catalog #100340), anti-mouse CD28 (clone 37.51, Biolegend, Catalog #102116), PHA (Sigma, Catalog #L8954), polybrene (Millipore, Catalog #3924803), SYBR (Bio-Rad, Catalog #1725125), cycloheximide (Sigma, Catalog #239763), MG132 (Sigma, Catalog #M8699), NH4Cl (Sigma, Catalog #254134), 3-MA (Sigma, Catalog #189490), Pitavastatin calcium (Aladdin, Catalog #P129617), Rosuvastatin calcium (Aladdin, Catalog #R129220), Simvastatin (Aladdin, Catalog #S129538), Lovastatin (Aladdin, Catalog #L107709), Fluvastatin Sodium (Aladdin, Catalog #F129852), human IL-2 (SL Pharm, Catalog #S19991010), human IL-7 (Peprotech, Catalog #200-07), mouse IL-7 (Peprotech, Catalog #217-17) and human IL-9 (Peprotech, Catalog #200-09). Information on the commercially available antibodies used in this study is provided in Supplementary information, Table S3. The antibody that specifically recognizes phosphorylated Y357 of γc was raised by immunizing rabbits with a synthetic peptide of human γc (354HSP(Y-p)WAPPC362) by ABclonal Technology (Wuhan).

Cells

Jurkat cells were obtained from American Type Culture Collection. HEK293 cells were originally provided by Dr. Gary Johnson (National Jewish Health, Denver, CO). HPB-ALL cells were provided by Dr. Hudan Liu (Wuhan university). CTLL2 cells were obtained from Cell Resource Center (IBMS, CAMS/PUMC). Human CD8+ T cells were obtained from Milestone® Biotechnologies. Mouse CD8+ T cells were isolated from the spleen of 6–8-week-old C57BL/6 mice by negative selection magnetic beads (STEM CELL Technologies, Catalog #19853 A). B16F10 cells, CT26 cells and MC38 cells were provided by Dr. Jinfang Zhang (Wuhan University).

Jurkat cells were cultured in RPMI 1640 (GIBCO, Catalog #C11875500BT) supplemented with 10% FBS (Cell Max, Catalog #SA211.02) and 1% penicillin-streptomycin (GIBCO, Catalog #15140-122). HPB-ALL cells were cultured in RPMI 1640 supplemented with 10% FBS, 1% penicillin-streptomycin, 1% non-essential amino acids (GIBCO, Catalog #11140-050), 2 mM L-glutamine (GIBCO, Catalog #35050-061), 1 mM sodium pyruvate (GIBCO, Catalog #11360-070) and 55 μM β-mercaptoethanol (Sigma-Aldrich, Catalog #63689). CTLL2 cells were cultured in RPMI 1640 supplemented with 10% FBS, 1% penicillin-streptomycin and IL-2 (100 IU/mL). Human CD8+ T cells and mouse CD8+ T cells were cultured in RPMI 1640 supplemented with 10% FBS, 1% penicillin-streptomycin, 1% non-essential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, 55 μM β-mercaptoethanol and IL-2 (400 IU/mL). HEK293, B16F10, CT26 and MC38 cells were cultured in DMEM (GIBCO) supplemented with 10% FBS and 1% penicillin-streptomycin. All cells were detected negative for mycoplasma.

Constructs

Mammalian expression plasmids for Flag-, HA-, or Myc-tagged γc, MARCH5, β-actin, IL2RB, IL4R, IL7R, OTUD6B, OTULIN, UCHL5, USP3, USP5, USP11, USP14, SHP2, JAK3 and their mutants, as well as pSuper.Retro-shRNA plasmids for MARCH5 were constructed by standard molecular biology techniques. Guide-RNA plasmids targeting γc, MARCH5, USP5, BATF and SHP2 were constructed into a lentiCRISPR V2 vector, which was provided by Dr. Shu-Wen Wu (Wuhan University).

Transfection

HEK293 cells were transfected by standard calcium phosphate precipitation. The empty control plasmid was added to ensure that each transfection receives the same amount of total DNA.

CRISPR-Cas9 knockout

Double-stranded oligonucleotides corresponding to the target sequences were cloned into the Lenti-CRISPR-V2 vector, which were co-transfected with the packaging plasmids into HEK293 cells. Two days after transfection, the viruses were harvested, ultra-filtrated (0.45-μm filter, Millipore) and used to infect cells in the presence of polybrene (8 μg/mL). The infected cells were selected with puromycin (Jurkat: 1 μg/mL, HPB-ALL: 2 μg/mL, CTLL2: 4 μg/mL) for at least 6 days. The information of gRNA sequences is shown in Supplementary information, Table S4.

Co-immunoprecipitation, ubiquitination and immunoblotting analysis

Cells were lysed in 1 mL of NP-40 lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 1% Triton X-100, 10 μg/mL aprotinin, 10 μg/mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride, PMSF). For each immunoprecipitation reaction, a 0.4 mL aliquot of lysate was incubated with 0.5–2 μg of the indicated antibody or control IgG and 35 μL of a 1:1 slurry of Protein-G Sepharose at 4 °C for 3 h. The Sepharose beads were washed three times with 1 mL of lysis buffer containing 500 mM NaCl. The precipitates were fractionated by SDS-PAGE, and immunoblotting analysis was performed with the indicated antibodies. For ubiquitination assays, the immunoprecipitants were re-extracted in NP-40 lysis buffer containing 1% SDS and denatured by heating for 10 min. The supernatants were diluted with regular lysis buffer until the concentration of SDS was decreased to 0.1%, following by re-immunoprecipitation with the indicated antibodies. The immunoprecipitants were analyzed by immunoblotting with the ubiquitin antibody.

qPCR

Total RNA was isolated for qPCR analysis to measure mRNA abundance of the indicated genes. Data shown are the relative abundance of the indicated mRNAs normalized to that of GAPDH. The qPCR data were collected with Bio-Rad CFX96 (Version 3.1) and analyzed with Bio-Rad CFX Manager (Version 3.1). Gene-specific primer sequences are listed in Supplementary information, Table S5.

ChIP

ChIP was performed according to the manufacture’s instruction. Ten million cells were fixed with 1% formaldehyde for 10 min, quenched with 0.125 M glycine for 5 min at 37 °C and lysed in SDS Lysis Buffer. Cell lysate was sonicated by Bioruptor Pico Sonifier to shear chromatin DNA to a size range of 200–1000 bp. The supernatant was diluted 10-fold in ChIP Dilution Buffer and precleared with 60 mL agarose beads for 30 min. The supernatant fraction was immunoprecipitated with the indicated antibodies (2 μg) against BATF overnight at 4 °C. The antibody-chromatin complexes were pulled down with protein A agarose/salmon sperm DNA beads (Sigma, Catalog #16-157) for 1 h at 4 °C. The de-crosslinked DNA was subjected to qPCR analysis using specific primers listed in Supplementary information, Table S6.

Flow cytometry analysis

Cells were subjected to stain with the indicated antibodies for 30 min in 4 °C. The cells were analyzed and data were acquired with BD Fortessa X-20 and FACSDiva 7 software following the exemplified gating strategy for flow cytometry analysis. The data were processed using FlowJo software. The antibodies used for flow cytometric analyses in this study are provided in Supplementary information, Table S7.

Mass spectrometry

Jurkat cells (1 × 108) were used for mass spectrometry analysis. Endogenous γc was immunoprecipitated with anti-γc and desalted, then analyzed by mass spectrometry. Mass spectrometry analysis was performed as previously described by SpecAlly (Wuhan) Life Science and Technology Company.65,66

Proliferation assay

Cells (5 × 104) were seeded in 6-cm dish for 24 h. Triplicate wells were seeded for each experimental group. The cells were trypsinized, resuspended in DMEM containing 10% FBS, and counted with a Cellometer (Bio Red) every 2 days over a 5 days period.

Human NSCLC samples

The human NSCLC samples were provided by Dr. Bo Zhong (Wuhan University).67 All cases were re-reviewed by pathologists from the Department of Pathology of Tongji Hospital for the confirmation of tumor histology and tumor content. All cases used in this study were performed with written patient informed consents and approved by the Institutional Review Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, and the Medical Ethic Committee of the School of Medicine, Wuhan University.

IHC staining

IHC staining was performed as previously described.68 In brief, the slides were deparaffinized in xylene, and rehydrated sequentially in 100%, 95%, and 75% ethanol for 5 min. The antigen retrieval was performed by heating slides in a microwave for 30 min in sodium citrate buffer (pH 6.0) or 0.5 mM EDTA buffer (pH 8.0). The sections were cooled down naturally to room temperature and quenched in 3% hydrogen peroxide to block endogenous peroxidase activity. The sections were incubated with antibodies overnight at 4 °C. Next, a secondary biotinylated immunoglobulin G antibody solution and an avidin-biotin peroxidase reagent were added onto slides. After washing with phosphate buffer saline, 3,3′-diaminobenzidine tetrachloride was added to the sections, followed by counterstaining with hematoxylin (Beyotime Biotech). The information and dilutions of antibodies are listed in Supplementary information, Table S3. Signals were imaged with an Aperio VERSA 8 (Leica) multifunctional scanner and quantified with the software Image-Pro Plus 6.0.

MARCH5 conditional knockout mice and genotyping

March5flox/flox mice were generated by the Animal Center of Wuhan University Medical Research Institute. Genotyping by PCR was performed using the following primers: F, 5′-AAGGACCTCTTGAACTTGGAAAG-3′; R, 5′-GCCCATACAGTCATGTAGGCAAA-3′. Amplification of the wild-type (WT) allele with primers F and R generates a 1020-bp fragment, whereas amplification of the disrupted allele with primers F and R generates a 1088-bp fragment. To generate MARCH5 hematopoietic-specific knockout mice, March5flox/flox mice were bred to Vav1-Cre mice to generate March5f/f:Vav1-Cre mice. Genotyping of the Vav1-Cre mice by PCR was performed using the following primers which produces a 205-bp fragment: Forward-CGTATAGCCGAAATTGCCAG; Reverse-CAAAACAGGTAGTTATTCGG. To generate MARCH5 T cell-specific knockout mice, March5flox/flox mice were bred to CD4-Cre mice to generate March5f/f:CD4-Cre mice. Genotyping of the CD4-Cre mice by PCR was performed using the following primers which produces a 408-bp fragment: Forward-GCATTACCGGTCGATGCAACGAGTGATGAG; Reverse-GAGTGAACGAACCTGGTCGAAATCAGTGCG. All animal utility was carried out in compliance with the Institutional Animal Care and Use Committee (IACUC) guidelines and approved by the Animal Care and Ethics Committee of Wuhan University Medical Research Institute.

In vivo experimental therapy in syngeneic mouse tumor models

Age- and sex-matched March5+/f and March5+/f:Vav1-Cre mice, C57BL/6J or Balb/c mice (all age of 6–8 weeks) were anaesthetized and subcutaneously injected with the indicated mouse tumor cells (5 × 105 in 200 μL PBS). The mice were euthanized when the tumor size was bigger than 15 mm of the mean tumor diameter or tumor volume reaches 2000 mm3 or deemed as died.

Anti-PD-1 therapy in C57BL/6J and Balb/c mice: mice were intraperitoneally injected with control or anti-PD-1 (BE0273, 100 μg per mouse, dissolved in PBS, BioXCell) every three days (four times in total) five days after B16F10 (C57BL/6J mice) or CT26 (Balb/c mice) cells inoculation. Tumor-bearing mice were euthanized on day 17. Tumor tissues were analyzed by IHC staining and TILs were analyzed by flow cytometry.

The MC38 and B16F10 tumor models in March5+/f and March5+/f:Vav1-Cre mice: on the day of three after tumor cells implantation, tumor sizes were measured every two days by caliper. Tumor-bearing mice were euthanized on day 13. TILs were analyzed by flow cytometry.

PC therapy in C57BL/6J mice: mice were intraperitoneally injected with control or PC (P129617, 5 mg/kg/day, dissolved in PBS, Aladdin) three (MC38 tumor) or five (B16F10 tumor) days after inoculation of tumor cells, and tumor sizes were measured every two days by caliper. Tumor-bearing mice were euthanized on day 13 (MC38 tumor) or day 15 (B16F10 tumor). TILs were analyzed by flow cytometry.

IL-2 and anti-PD-1 therapy in March5+/f and March5+/f:Vav1-Cre mice: mice were intraperitoneally injected with control, IL-2 (50,000 IU per mouse, dissolved in PBS) or anti-PD-1 (100 μg per mouse, dissolved in PBS) five days after inoculation of MC38 cells. Tumor size and mouse survival were measured every two days from day 5.

PC, IL-2 and anti-PD-1 therapy in C57BL/6J mice: mice were intraperitoneally injected with control, PC (5 mg/kg, dissolved in PBS), IL-2 (50,000 IU per mouse, dissolved in PBS) and anti-PD-1 (100 μg per mouse, dissolved in PBS) five days after inoculation of MC38 cells. Tumor size and mouse survival were measured every two days from day 5.

PC, RC and IL-2 therapy in C57BL/6J mice: mice were intraperitoneally injected with PBS, PC (5 mg/kg, dissolved in PBS), RC (20 mg/kg, dissolved in PBS) and IL-2 (50,000 IU per mouse, dissolved in PBS) five days after inoculation of MC38 cells. Tumor size and mouse survival were measured every two days from day 5.

For survival studies, mice were sacrificed when the tumor size was bigger than 15 mm of the mean tumor diameter, tumor volume exceeded 2000 mm3, or tumor had ulcers with a diameter reached 10 mm. Statistical analysis was performed using the GraphPad Prism 8 software. Kaplan–Meier survival curves and corresponding log-rank (Mantel-Cox) tests were used to evaluate the statistical differences between groups in survival studies. There is a significant difference when the P < 0.05.

Isolation of tumor-infiltrated immune cells

Tumor tissues were separated from mice and cut into pieces. The tumor tissues were suspended with 2 mL of tumor digestion buffer (1× HBSS buffer with 5 mg/mL collagenase II and 0.1% DNase I) and rotated for at 37 °C for 1 h. The cell suspension was filtered using a 70-μm filter to obtain single-cell suspension. The lymphocytes were isolated by density-gradient centrifugation using 40% and 70% Percoll (GE). The TILs were stained using fluorescently labelled antibodies for different markers. Cells were analyzed and data were acquired with BD Fortessa X-20 and FACSDiva 7 software following the exemplified gating strategy for flow cytometry analysis. The data were processed using FlowJo software.

Confocal microscopy

H1299 or A549 cells were transfected with the indicated plasmids for 20 h. The cells were fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.3% Triton X-100 in PBS for 15 min. The cells were blocked with 5% BSA in PBS and stained with the indicated primary and secondary antibodies. The nuclei were strained with DAPI for 2 min and then washed with PBS for 3 times. The stained cells were observed with a Zeiss LSM880 confocal microscope under a 63× oil objective.

Statistical analysis

Data were analyzed using Student’s unpaired t-test, multiple t-test or two-way ANOVA with GraphPad Prism 8. The correlation study was analyzed using a Spearman rank correlation test. The number of asterisks represents the degree of significance with respect to P values, with the latter presented within each figure or figure legend. All the biochemical experiments, particularly immunoblotting analysis, were repeated for at least two times with similar results. The reproducibility of other experiments is described in the respective figure legends.