Female BALB/c (6-wk-old) mice were used according to National Institutes of Health guidelines and approved by The University of Texas Southwestern Medical Center.
Isolation and culture of human T cells
Peripheral blood was obtained from healthy donors according to an Institutional Review Board-approved protocol and following participant consent. CD4+ T cells were isolated from the blood samples using CD4 T cell isolation kit (Miltenyi Biotec). Some cells were activated by 1 μg/ml anti-CD3 Ab (eBiosciences) or PMA (5 ng/ml) plus ionomycin (250 ng/ml) (Sigma-Aldrich).
Generation of transfectants
Mouse SD-4- or PD-1-coding sequence was attached with the N-terminal V5 epitope sequence and inserted into a lentiviral vector plasmid (pHR-SIN-CSGW-GFP) by replacing the GFP gene [20
]. These lentiviruses were infected into mouse T cell hybridoma DO11.10 line (from J. Kappler and P. Marrack, National Jewish Medical and Research Center, Denver, CO). Post-infection, V5+
cells were enriched by flow cytometric sorting until they were >90% positive.
Immunoblotting and flow cytometry
At varying time points after activation of human CD4+ T cells with anti-CD3 Ab (1 μg/ml), whole cell extracts were prepared and applied to SDS-PAGE (10 μg/lane) and blotted with mouse anti-CD148, goat anti-syntenin, or rabbit anti-β-actin (each 1 μg/ml) and HRP-secondary Ab. Intensity of Ab-reactive bands was measured by ImageQuant (GE Healthcare) and expression level of CD148 or syntenin is expressed as a value relative to β-actin. These activated cells were also stained with anti-CD148 or control IgG and PE-anti-mouse IgG, followed by flow cytometry.
To examine localization of SD-4 and CD148, activated human CD4+
T cells (2 × 105
) were treated with immobilized DC-HIL-Fc (the extracellular domain fused to the IgG-Fc) [5
] or ICAM1-Fc (20 μg/ml, R & D System) for 30 min, and labeled with anti-SD-4 Ab plus Alexa 488 anti-mouse IgG (2 μg/ml) or with anti-CD148 Ab plus Alexa 546 anti-rabbit IgG (2 μg/ml) (Invitrogen).
To examine localization of SD-4 relative to the IS, DC (1 × 106 cells) were harvested from BM cell (from BALB/c mice) culture of 6 d with 10 ng/ml GM-CSF (PeproTech), pulsed with OVA peptide (2 μg/ml), mixed 1:1 with transfected DO11.10 T cells, and incubated for 5- 30 min at 37°C. The cells were then allowed to settle onto poly-L-lysine-coated slides for 5 min. After fixing with 4% paraformaldehyde, slides were treated with anti-V5 Ab plus Alexa 546 anti-mouse IgG (2 μg/ml) and with anti-CD3 Ab (10 μg/ml) plus Alexa 488 anti-hamster IgG (2μg/ml). Stained cells were examined by confocal microscopy. Co-localization of SD-4 and CD3 (or CD148) was evaluated individually on 42–66 cells using Pearson correlation coefficient r.
Activated human CD4+ T cells (1 × 107) were cultured in a 100 mm petri dish precoated with DC-HIL-Fc or control Ig (20 μg/ml). At varying time points after incubation at 37°C, whole cell extracts (1 × 107 cells/ml) were prepared and incubated with anti-SD-4 Ab or control IgG (2μg/ml) for 3 h on ice, and then precipated with protein A-agarose (Pierce). After washing, immune complexes were eluted by boiling and subjected to SDS-PAGE/immunoblotting.
To assay phosphorylation of CD148 after treating activated CD4+ T cells with immobilized DC-HIL-Fc or control Ig, whole cell extracts were incubated with anti-CD148 Ab (2 μg/ml) and rabbit anti-mouse IgG (4 μg/ml) at 4°C for 3 h, and then precipitated with protein A-agarose. After washing, immune complexes were separated electrophoretically and then blotted using biotinylated anti-phospho-tyrosine (0.5 μg/ml) (Upstate) and HRP-streptavidin. Blotted membranes were reprobed with rabbit anti-CD148 Ab (1 μg/ml) and HRP-secondary Ab.
Human CD4+ T cells (1 × 106/ml) were cultured for 3 d with PMA/ionomycin, and then incubated with immobilized DC-HIL-Fc or control Ig (20 μg/ml) at 37°C for 30 min. An aliquot (5 × 106 cells equivalent) of whole cell extract was immunoprecipitated with anti-CD148 Ab or control IgG (2 μg) and protein A-agarose. PTP activity was measured using the Malachite Green Phosphatase Assay (Upstate Biotechnlogy Inc). Specific activity of CD148 was expressed as OD620 reading after subtracting OD620 reading of control IgG-precipitate from that of anti-CD148 Ab.
Knockdown of gene expression and T cell activation
CD148-siRNA (Cat#sc-35189) or control siRNA (Cat#sc-37007, Santa Cruz Biotechnology) was treated with 15 μl of Metafetene™ Pro (Biotex) for 30 min and then added to human CD4+ T cells (1 × 106). After incubating at 37°C for 4 h, cells were cultured in 10% FCS-RPMI for 2 d in the presence of PMA/ionomycin. Transfected cells were examined by immunoblotting for expression of CD148 or β-actin. For DC-HIL-mediated suppression, siRNA-transfected cells (2 × 105/well) were cultured for 2 d in ELISA wells precoated with a constant dose of anti-CD3 Ab (1 μg/ml) and varying doses of DC-HIL-Fc or control Ig. T cell activation was measured by 3H-thymidine incorporation (pulsing with 1 μCi/well in the last 20 h of the culture period). For the mixed lymphocyte reaction (MLR), transfected T cells (2 or 4 × 105/well) were also co-cultured with CD14+ cells (2 × 105/well) isolated from peripheral blood of an allogenic donor for 6 d. IL-2 production was measured by ELISA.