However, cross-presentation by liver APCs induces partial T-cell

However, cross-presentation by liver APCs induces partial T-cell activation, which AZD5363 clinical trial is dependent on intercellular adhesion molecule-1 (ICAM-1) expression. These results support a model of liver immunity that achieves primary T-cell activation but fails to induce an effective immune response. APC, antigen-presenting cell;

bm8, B6.C-H-2bm8; CTL, cytotoxic T lymphocyte; DC, dendritic cell; HSCs, hepatic stellate cells; KCs, Kupffer cells; LSECs, liver sinusoidal endothelial cells; mDCs, spleen myeloid dendritic cells; MHC, major histocompatibility complex; OVA, ovalbumin. Eight to 10-week-old C57BL/6 wildtype, OVA transgenic, ICAM-1 deficient, OVA-specific H-2Kb-restricted T-cell receptor (TCR) transgenic (OT-I), and B6.C-H-2bm8 (bm8) mice were used in accordance with

Institutional ABT-888 concentration Animal Care and Use Committee guidelines. Candidate liver APCs were isolated to high purity using a novel multistep isolation technique (Supporting Fig. S1). Spleen mDCs were isolated using magnetic antibody cell sorting against CD11c (MACS, Miltenyi Biotec). CD8+ T cells were isolated from spleen and peripheral lymph nodes of OT-1 mice as described.14 Following isolation, OT-1 CD8+ T cells were labeled with 0.7 mM carboxy-fluorescein-succinimidyl-ester (CFSE) and used in antigen presentation experiments. Antibodies used are described in the Supporting Information. For studying antigen cross-presentation, 2.5 × 104 isolated

liver APCs or spleen DCs were seeded in 96-well round-bottom plates, and soluble OVA protein (final concentration of 100 μg/mL, grade VII) was added. On the following day, OT-I CD8+ T-cells were isolated, CFSE-labeled, Carnitine dehydrogenase and added to the cultures at 105 cells per well. In antibody blocking experiments, antibodies (10 μg/mL) were added 1 hour before the OT-I CD8+ T cells. For cross-presentation from bm8-OVA hepatocytes, 1 day before isolation of APCs, hepatocytes from bm8-OVA mice were isolated and seeded in the plates at 102, 103, or 104 cells per well. All cells were cultured in RPMI supplemented with 10% fetal bovine serum (FBS), 50 μM beta-mercaptoethanol, glutamine, sodium pyruvate, and antibiotics. We characterized the uptake, intracellular processing, and presentation of OVA using fluorescein-OVA, DQ OVA, and H-2Kb-SIINFEKL staining, respectively. DQ is a self-quenched conjugate of OVA that exhibits bright green fluorescence upon proteolytic degradation. We also quantified the relative basal expression of H-2Kb in each cell type. Isolated hepatocytes from bm8-OVA transgenic mice were used as the source of cell-derived antigen. Cells derived from these mice were unable to present the OVA peptide to OT-I CD8+ T cells due to a mutation in the H-2Kb molecule.15 OT-I CD8+ T-cells express transgenic TCRs against the peptide derived from OVA257-264, peptide sequence SIINFEKL, in the context of H-2Kb.

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