Cell Isolation and Culture
Single donor endothelial cells (EC) were isolated from human umbilical cords as previously described , and were grown in M199 medium (Bio Whittaker) containing 20% FCS (GIBCO BRL, Grand Island NY) or 10% human serum, EC growth supplement, 1% penicillin/streptomycin, l-glutamine, and heparin. Single donor fibroblasts and renal tubular epithelial cells (RPTEC) were purchased from Clonetics and cultured in CC-4126 FGM 2 or CC-4127 REGM (Clonetics, Walkersville MD) according to the manufacturers protocol. EC, fibroblasts and RPTEC were treated with IFN (1000U/ml, R&D Systems, Minneapolis, MN) for 72h prior to use. EC were used in subculture 34. Apoptosis was induced in EC monolayers following treatment with TNF- (200 U/ml, Biogen, Cambridge MA) and cyclohexamide (2ng/ml, Sigma, St. Louis, MO) for 7h. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque gradient centrifugation from healthy volunteers. CD4+ T cells were isolated from PBMC by positive selection using anti-CD4-coated magnetic beads (Invitrogen, Grand Island NY) according to the manufacturers protocol. CD4+CD45RA+ and CD4+CD45RO+ cells were isolated from CD4+ cells by negative selection using magnetic beads coated with mouse anti-human CD45RO and CD45RA antibodies (Invitrogen, Grand Island NY).
Lymphocyte transmigration assays
Transmigration experiments were conducted using EC, fibroblasts or RPTEC monolayers cultured on fibronectin (50 g/ml) coated 3m pore size transwell inserts (Costar, Cambridge, MA, USA) as previously described [35, 36]. A total of 3103 EC, fibroblasts or RPTEC were seeded onto inserts and were allowed to grow for 45days. Monolayers were treated with IFN (1000U/ml, R&D systems) for the last 72 culture. Confluence of the monolayers was confirmed by Coomasie staining using standard techniques . Monolayers were labeled with DiOC-16 (5 g/ml, Molecular Probes, Eugene OR, USA), which incorporates into the membranes of cells and has an emission at 501nm. A total of 1x106 PBMC were placed into the upper transwell and cells were collected from the bottom well after 1.5h. Following collection, DiOC uptake was analyzed on CD14+ CD4+ and CD8+ cells by flow cytometry.
Confluent monolayers were harvested in Trypsin/EDTA (Sigma, St. Louis, MO) and were analyzed by indirect immunofluorescence staining and flow cytometry as previously described . Briefly, cells were incubated in optimal concentrations of the primary antibodies anti-HLA DR (LB3.1, ATCC, Manassas, VA), negative control mouse IgG (K16/16, a gift from Michael Gimbrone, Brigham and Womens Hospital) or positive control anti-ICAM-1 (RR1/1, a gift from TS Springer) for 30 mins on ice. Cells were washed and subsequently incubated in FITC-conjugated anti-mouse secondary antibody (Jackson, Immunoresearch, West Grove, PA) for an additional 30 mins on ice, and stained cells were washed and fixed in 1% paraformaldehyde prior to analysis. Leukocytes were stained using Phycoerythrin (PE) conjugated anti-human -CD14, -CD4 and -CD8 antibodies (PharMingen, San Diego CA) or isotype negative controls using standard techniques. All stained cells were analyzed using a FACSCalibur cell sorter (Becton Dickinson, Mountain View, CA) and CellQuest and FlowJo software.
PBMC-allogeneic cell coculture
EC, fibroblasts or RPTEC were cultured to confluence in fibronectin-coated 24-well flat-bottom plates (Costar) coated with fibronectin (50 g/ml) and treated with IFN (1000U/ml) for 72h. PBMC (1x106/well) were added and were cultured for 7days in standard RPMI 1640 medium containing 10% autologous serum. Subsequently, PBMC were harvested by washing with HBSS and CD4+ T cells were isolated by positive selection (Invitrogen, Grand Island NY) and were rested in culture medium containing 2.5% of human T-stim without PHA (Becton Dickinson Labware, Bedford, MA) for 5days. The allogeneic HLA mismatch between PBMCs and stimulator EC, fibroblasts or RPTEC was unknown, but was presumed based on consistency of the response in multiple repeated experiments.
Preparation of Sonicates
Cell membranes were prepared as sonicates from IFN- treated EC, fibroblasts and RPTEC. Exactly 8 106 of each cell type was washed and suspended in 1ml of sterile PBS and was sonicated using a tip sonicator (Branson Sonifer 250) fitted with a 2mm probe. Disrupted cells were centrifuged for 10min at 500g to remove debris. A total of 20l of sonicate was used in each assay. As a quality control, the protein content per ml was measured in occasional sonicate supernatants by standard Bradford assay. Sonicates were frozen at 20C and thawed at 37C before use.
Indirect allorecognition assays
CD4+ T cells (1x105/well) isolated from primary cultures (above) were used with autologous APCs in proliferation assays. Autologous APCs (1x105/well) were isolated from T cell-depleted PBMC, were irradiated (1750rad) and used as stimulators. Assays were performed in round-bottom 96-well plates (Costar). Sonicates from the allogeneic cells, were added (as above) to each culture and, after 6days proliferation was assessed by standard [3H]thymidine incorporation assays (1Ci/well) for the last 16h of the coculture.
ELISpot was performed in 96-well plates (Cellular Technology Ltd., Cleveland, OH) coated overnight with capture cytokine antibodies diluted in sterile PBS. Antibodies were mouse anti-human IFN (clone 2G1, Endogen, Wolburn, MA, 4g/ml), mouse anti-human IL-2 (clone 5355, R&D Systems Inc., Minneapolis, MN, 5g/ml), mouse anti-human IL-4 (clone 8D4-8, BD PharMingen, San Diego, CA, 5g/ml) or rat anti-human IL-5 (clone TRFK5, PharMingen, San Diego, CA, 5 g/ml). After blocking for 1h with PBS/1%BSA, the plates were washed and CD4+ T cells (2x105/well) were cultured with autologous APCs (2x105/well) in the absence or presence of sonicate. The plates were incubated at 37C in 5% CO2 for 24-36h. Detection antibodies were biotinylated mouse anti-human IFN- (clone B133.5, Endogen, 2g/ml), mouse anti-human IL-2 (clone 5334, R&D Systems Inc., 50ng/ml), rat anti-human IL-4 (clone MP4-25D2, BD PharMingen, 2g/ml) and rat anti-human IL-5 (clone JES1-5A10, PharMingen, 2g/ml). After overnight incubation at 4C, the plates were washed and cytokines were detected using streptavidin HRP (Daco, Carpenteria, CA) and 3-amino-9-ethyl carbazole (Pierce Chemical Co., Rockford, IL). Spots were counted using a computer-assisted ELISpot image analyzer (Cellular Technologies Limited).
Statistical analysis was performed using the student t test for two groups of data and by one-way ANOVA for three or more groups. P values <0.05 were considered statistically significant.