Ship-1 conditional knockout mice on a C57/BL6 background were obtained by crossing Foxp3YFP-cre or CD4Cre mice with SHIP-1fl/fl mice from Jackson laboratory. All mice were housed in individual ventilated cages within the animal center of the Children’s Hospital of Chongqing Medical University. Mice at 8-10 weeks old were used unless otherwise noted. For treatment with rapamycin in vivo, mice were consecutively injected with rapamycin (4 mg per kg body weight) by IP for 14 days. All animal experiments were performed according to protocols approved by the Institutional Animal Care and Usage Committee of Children’s Hospital of Chongqing Medical University.
BM Chimera Mice
For generation of bone marrow chimera mice, CD45.1 mice were irradiated with sublethal radiation (6Gy) and then intravenously injected with 1×107 total BM cells containing SHIP-1 KO BM (expressing CD45.2) and WT BM (expressing CD45.1) at a ratio of 1:1. The recipient mice were analyzed after 8 weeks, as described previously(24).
Cell purification and flow cytometry
Lymphocytes from gender-matched Foxp3YFP-cre Ship-1fl/fl mice and Foxp3YFP-cre Ship-1+/+ mice were isolated from the thymus, spleen and lymph nodes according to published protocols(25). For analysis of lymphocyte surface markers, the following antibodies were used: PE anti-ICOS (clone: 7E.17G9, eBioscience), PerCP-anti-7AAD (BBI Life Sciences), PerCP/Cy5.5 anti-CD44 (clone: IM7), APC anti-CD25 (clone: PC61), APC-CY7 anti-TCR-β (clone: H57-597 ), PE-CY7 anti-CD62L (clone: MEL-14 ), APC anti-CD304 (Neuropilin-1) (clone: 3E12), Pacific Blue anti-CD4 (clone: RM4-5 ), Brilliant Violet 510 anti-CD8a (clone: 53-6.7), PE-CY7 anti-PD1 (CD279) (clone: RMP1-30), PE anti-CD4 (clone: GK1.5). All antibodies were from BioLegend unless otherwise stated. For staining cells, antibody incubations were on ice for 30mins in PBS containing 2% FBS. For detection of intracellular proteins, cells were fixed and permeabilized using Foxp3/transcription factor staining buffer set by eBioscience, then stained with the following antibodies: PE anti-CD152 (CTLA - 4) (clone: UC10-4B9), PE anti-Foxp3 (clone: MF-14), APC anti-IL-4 (clone:11B11), PE/Cy7 anti-IFN-γ (clone: XMG1.2), Brilliant Violet 421 anti-IL-17A (clone: TC11-18H10.1). All antibodies were from Biolegend. PE-Cyanine7 Ki-67 monoclonal antibody (SolA15) from eBioscience was used for detecting cell population growth.
Treg cells were sorted using a CD4+CD25+ regulatory T cell isolation kit (Miltenyi Biotec) and activated in plates coated with anti-CD3 ( clone: 145-2C11, Bio X Cell) at 5 mg/ml and anti-CD28 (clone: 37.51, Bio X cell) at 5 mg/ml for 4 hours, then immediately fixed with Phosflow perm buffer (BD Biosciences), permeabilized with Phosflow lyse/fix buffer (BD Biosciences), and stained with: anti-phospho- Akt (pSer 473) (clone: D9E), anti-phospho-S6 (pSer235/236), anti-phospho-PI3 kinase p85 (Tyr458)/p55 (Tyr199), anti-phospho-Foxo1 (Ser256), anti-phospho-mTOR (Ser2448) (clone: D9C2). All antibodies were from Cell Signaling Technology. Flow cytometry data were acquired on a FACS Canto (BD Biosciences) and analyzed using FlowJo software. Cell numbers of various populations were calculated by multiplying the total cell number with the percentages of each individual population from the same mouse, and then averaged.
In vitro Treg suppression assays
For the in vitro Treg cell suppression assay, CD4+CD25+ Treg cells and CD4+CD62LhighCD44low Tnaive cells were sorted by CD4+CD25+ regulatory T cell and naive CD4+ T cell isolation kits (Miltenyi Biotec), respectively. Tnaive (1×105 cells ) and Treg cells (at different ratios with Tnaive cells) were cultured in 96-well flat-bottom plates along with anti-CD3 (clone: 145-2C11, Bio X Cell) at 5 mg/ml and anti-CD28 (clone: 37.51, Bio X Cell) at 5 mg/ml for 72 h. T cell proliferation was determined by using the CellTrace violet cell proliferation kit (C34557, Invitrogen). For drug treatment in vitro, Treg cells were preincubated with vehicle or 100 nM rapamycin (AY 2298, MedChemExpress) for 1 h before stimulation.
Colitis model were performed as previously published literature(26).
MC38 colon adenocarcinoma cells were maintained and cultured according to the literature(27), briefly, the MC38 colon adenocarcinoma cells were cultured in DMEM with 10% FBS and 1% penicillin-streptomycin. Gender-matched Foxp3YFP−cre Ship-1fl/flmice and Foxp3YFP−cre Ship-1+/+ mice were injected subcutaneously with 2 × 105 MC38 colon adenocarcinoma cells in the right flank. Tumors were measured with digital calipers every 3 days, and tumor volumes were calculated by the formula: length × width × (length × width)0.5 × /6. To prepare tumor-infiltrating lymphocytes (TILs), tumors were excised, minced and digested with 0.5 mg/ml collagenase IV (Roche) and 200 U/ml DNase I (Sigma) for 1 h at 37°C. TILs were isolated by density-gradient centrifugation over Percoll (Roche).
In vivo migration ass
CD4+YFP-Foxp3+ Tregs from Foxp3YFP−cre Ship-1+/+ and Foxp3YFP−cre Ship-1fl/fl mice were respectively transferred into WT CD45.1+ mice, together with B cells from CD45.2+ WT mice for normalization. After 5 hours, the ratio of CD45.2+CD4+ YFP-Foxp3+ Treg cells vs. CD45.2+B220+ B cells was analyzed in the spleen, PLN, MLN and blood, and normalized based on the starting ratio 1:2, as previously described(28).
For immunofluorescence staining of mouse kidney sections, the kidneys were embedded in OCT compound and quickly frozen in liquid nitrogen for 1min, then sliced 7 to 10 microns thick. The samples were fixed with cold acetone for 5 min and dried at room temperature. After washing with PBS, the samples were blocked in blocking buffer (5% BSA, 1:100 Fc blocker, in PBS) for 2 hours and stained overnight with AF488 anti-mouse IgG (1:500 in blocking buffer). Images were acquired using confocal microscopy (Nikon A1R).
Serum autoantibody analysis
Autoantibodies to dsDNA were measured with kits from Alpha Diagnostic International.
Splenic CD4+YFP-Foxp3+ Treg cell, CD4SP and CD8SP T cells from WT and Foxp3YFP−cre Ship-1fl/fl, CD4CreShip-1fl/fl mice were sorted on a MoFlow (Beckman-Coulter), then RNA was isolated by RNAPURE kit (RP1202; BioTeke), then the expression of ship1 were analyzed by using following primers: ship1 F- GCAGCAGATGAAGAACAAG, ship1R- CCAAGTGCCAATGAAGAT.
Statistical significance was assessed by two-tailed unpaired Student’s t-test with Prism 7 software, (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001). Data are presented as standard error of the mean (SEM).