We then sorted the CD11b+ myeloid cells (Physique S8), with a purity of 90%, and examined their expression of CXCL1 and CXCL2

We then sorted the CD11b+ myeloid cells (Physique S8), with a purity of 90%, and examined their expression of CXCL1 and CXCL2. the differentiation of bone marrow cells in tumor\bearing conditions, which suggests that inhibition of CXCL1 and CXCL2 could 2-MPPA decrease mo\MDSC generation and improve host immunosurveillance. for 20?minutes at 4C using a 3000 nominal molecular\weight limit centrifugal filter (Merck Millipore, Burlington, MA, USA). The concentrated cell\conditioned medium (300?L) was injected i.v. daily for 7?days in the absence or presence of CXCL1 (50?g/mouse) or CXCL2 (50?g/mouse). 2.6. Cytokine array for 2-MPPA cell\conditioned medium For the cytokine array, the conditioned medium collected from B16F10 cells, 4T1 cells and MEF cells was processed according to the manufacturer’s instructions (R&D Systems). 2.7. Induction of mouse bone marrow cells in?vitro Induction of mouse bone marrow cells was carried out as previously described.22 Briefly, mouse 2-MPPA bone marrow cells were flushed out from the femurs and tibias using a syringe with a 26\gauge needle and ground into a single\cell suspension. Erythrocytes were eliminated using hypotonic lysis buffer. The remaining cells were cultured in complete medium supplemented with GM\CSF (10?ng/mL) for 5?days. In a separate experiment, CXCL1 or CXCL2 was added to the induction system. 2.8. Construction of the lentiviral expression plasmid and transfection PLL3.7 Cloning Vector (Addgene, Cambridge, MA, USA) was used to knock down the expression of CXCL1 and CXCL2. The CXCL1 ShRNA sequences were #1: 5\ TGCACCCAAACCGAAGTCATTTCAAGAGAATGACTTCGGTTTGGGTGCTTTTTTC\3 and 5\ TCGAGAAAAAAGCACCCAAACCGAAGTCATTCTCTTGAAATGACTTCGGTTTGGGTGCA\3; and #2: 5\ TGGAGACCACTAAGTGTCAATTCAAGAGATTGACACTTAGTGGTCTCCTTTTTTC\3 and 5\ TCGAGAAAAAAGGAGACCACTAAGTGTCAATCTCTTGAATTGACACTTAGTGGTCTCCA\3. The CXCL2 shRNA sequences were #1: 5\ TGGGTTGACTTCAAGAACATTTCAAGAGAATGTTCTTGAAGTCAACCCTTTTTTC\3 and 5\ TCGAGAAAAAAGGGTTGACTTCAAGAACATTCTCTTGAAATGTTCTTGAAGTCAACCCA\3; and #2: 5\ TGCCAAGGGTTGACTTCAAGTTCAAGAGACTTGAAGTCAACCCTTGGCTTTTTTC\3 and 5\ TCGAGAAAAAAGCCAAGGGTTGACTTCAAGTCTCTTGAACTTGAAGTCAACCCTTGGCA\3. The synthesized shRNAs were cloned into the vectors, and the constructed plasmids and shCtrl plasmid were transfected into 293T cells, together with the packaging plasmid psPAX2 and the envelope plasmid pMD2.G (both from Addgene) by using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). To knock down CXCL1 or CXCL2, the collected supernatant and 4?mg/mL polybrene (Sigma, St Louis, MO, USA) were used to infect the B16F10 cells. Stable cell lines infected with CXCL1 ShRNA (shCXCL1), CXCL2 ShRNA (shCXCL2) or control ShRNA (shCtrl) were separated by flow cytometry sorting. To knock down CXCL1 or CXCL2 in tumor\bearing mice, the collected supernatant was concentrated and i.v. injected into mice four occasions every other day. 2.9. Cell isolation Monocytic MDSC and G\MDSC were sorted by using the AutoMACS sorter (Miltenyi Biotech) with a myeloid\derived suppressor cell isolation kit according to the manufacturer’s instructions. To isolate CD11b+ cells, the primary tumor was minced into small fragments and then digested into a single\cell suspension with 2?mg/mL collagenase II at 37C for 1?hour. The cells were separated into two layers using Ficoll, and the middle layer was collected. Then, CD11b+ cells were isolated by positive selection with the 2-MPPA biotin\conjugated CD11b antibody and streptavidin particles according to the manufacturer’s instructions (BD IMag). 2.10. RNA extraction and real\time PCR Total RNA was extracted with TRIzol (Invitrogen), and the cDNA was synthesized with reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, USA). Real\time PCR analysis was carried out using SYBR Green Grasp Mix (Roche, Basel, Switzerland) on a Roche LightCycler 480 (Roche). Sequences of primers used for PCR were as follows: 5\ATGGCTGGGATTCACCTCAA\3 and 5\CAAGGGAGCTTCAGGGTCAA\3 for CXCL1; 5\GCCCAGACAGAAGTCATAGCC\3 and 5\TCAGTTAGCCTTGCCTTTGTTC\3 for CXCL2; 5\GACAGGGCTCCTTTCAGGAC\3 and 5\CTTGGGAGGAGAAGGCGTTT\3 for Arg1; and 5\TCCCTTCCGAAGTTTCTGGC\3 and 5\CTCTCTTGCGGACCATCTCC\3 for iNOS. Primers used for the housekeeping gene actin were 5\AACAGTCCGCCTAGAAGCAC\3 and 5\CGTTGACATCCGTAAAGACC\3. 2.11. Transwell analysis Sorted mo\MDSC or G\MDSC (5??104) were loaded around the upper wells, and the chemokines, such as CXCL1 or CXCL2, were placed in the lower wells. Based on the size of the cells, a 5\m pore transwell chamber was used for mo\MDSC, and a 3\m pore was used for G\MDSC. The migrated cells were collected in the lower chamber and calculated after incubation at 37C with 5% CO2 for 3?hours. 2.12. Statistical analysis The data were analyzed by Student’s test using GraphPad Prism software. 3.?RESULTS 3.1. Monocytic MDSC expand under tumor\bearing conditions Tumor progression is usually often accompanied by immunity and inflammation, and the immune system is TNFSF8 usually altered by the tumor environment.1 To test the effect of tumors on immune cells, we examined multiple immune cell populations in a.