S performed after exposing LPS-stimulated BM-DCs to many adenosine receptor antagonists (listed under and in Supplemental Table S3) within the presence of NECA or to various receptor subtype specific adenosine receptor agonists. For cytokine detection IL-12p40 ELISA kit (Biolegend, Cat: 431601) was made use of by following manufacturer’s directions. For ELISA experiment 100nM of following receptor subtype-specific agonists and antagonists from Tocris Biosciences were used: A1R agonist, 2′-MeCCPA; A2AR agonist, CGS 21680 hydrochloride; A2BR partial agonist, BAY 60583; A3R agonist, 2-Cl-IB-MECA; A1R Antagonist, PSB36; A2AR Antagonist, SCH 58261; A2B Antagonist, PSB 603. T cell purification and adoptive transfer: Splenic CD8+ T cells from CD45.2+ 2C WT CD90.1+ or 2C A2BR-/- CD90.1- mice have been selected using a CD8+ T cell enrichment kit (StemCell Technologies; Cat:19853). For CFSE labeling, cells at 106 per mL have been stained with CellTrace CFSE staining option (ThermoFisher, Cat:C34554) for 20 minutes in a 37 water bath in line with the manufacturer’s instructions.Phalloidin Fluorescent Dye 206 purified CFSE-labeled T cells in 100 L of PBS had been injected i.CHAPS Technical Information v.PMID:36717102 into CD45.1+ hosts via the retro-orbital venous sinus 1 day before B16 or B16-SIY injection. 3 days later, the proliferation (CFSE dilution), intracellular IFN-+ production and activation (measured by expression of CD69 and CD44) of transferred T cells (gated on CD8+CD45.1- CD45.2+) in spleens were determined by flow cytometry. For adoptive T cell therapy, splenic CD8+ T cells had been chosen from 2C mice having a CD8+ T cell enrichment kit (StemCell Technologies; Cat:19853) and stimulated with 0.five g/ml SIY peptides, 1 g/ml anti-CD28 and ten ng/ml IL-2 in CR10, i.e. RPMI 1640 supplemented with ten FCS (Life Technologies, Cat: 26140079), one hundred IU/ml penicillin, 100 g/ml streptomycin, 2 mM l-glutamine, 25 mM HEPES buffer, and nonessential amino acids for 3 d. Pre-stimulated T cells were injected i.v. at 506 in one hundred L of PBS per mouse by means of the retro-orbital venous sinus into B16-SIY tumor-bearing mice on day 1 or day 13. To examine the role of A2BR on Tregs, tumor-associated or splenic CD4+CD25hi Tregs from WT or A2BR-/- have been sorted by BD Aria. For the colitis model as described(22), mouse splenocytes had been stained with anti-CD45RB, anti-CD25, and anti-CD4, and sorted by BD Aria for CD4+CD45RBhi and CD4+CD25hi T cells (deemed as Tregs), respectively. 405 sorted CD4+CD45RBhi cells in one hundred L of PBS have been transferred i.v into Rag1-/- mice with or with no 105 CD4+CD25hi WT or A2BR-/- Tregs through the retro-orbital venous sinus. Mice created clinical signs of colitis 3.five.five weeks post transfer. Mice had been observed everyday and weighed weekly. To assess the clinical status with the recipient mice, aggregate clinical scores were assigned as described previously (23) around the day of injection, weekly thereafter, and at time of sacrifice. For histological scores, colon tissue sections have been stained with H E as well as alcian blue and periodic acid-Schiff answer. Colitis severity was graded semi-quantitatively from 0 to 4 in a blinded style.Author Manuscript Author Manuscript Author Manuscript Author ManuscriptCancer Immunol Res. Author manuscript; out there in PMC 2022 September 07.Chen et al.PageIn vivo killing assay:Author Manuscript Author Manuscript Author Manuscript Author ManuscriptAnalysis of tumor antigen-specific effector CTL activity in vivo was performed as previously described (20). Briefly, SIY (SIYRYYGL) peptide-pulse.
