Investigation has been conducted in accordance with the ethical standards and according to the Declaration of Helsinki and according to national and international guidelines and has been approved by the author's institutional review board.
Tissues, cell lines and treatments
Stage III/IV human ovarian cancer tissues (n=30) were obtained through Research Pathology at Dartmouth Medical School, Lebanon, NH, after institutional approval. Single-cell suspensions were generated by gently forcing minced fresh specimens through a 70-μm pore mesh and subjected to Ficoll gradient centrifugation. Human ovarian cancer cell lines SKOV3, Ovcar60 and A2008 were grown to 80% confluency in DMEM medium containing 10% FBS prior to RNA isolation.
Peripheral blood lymphocytes were obtained by leukapheresis/elutriation and Miltenyi bead–purified, as described[5
Monocyte-derived dendritic cells (MoDCs) were generated by incubating magnetically purified CD14+ cells for 7 days with granulocyte-macrophage colony stimulating factor (20 ng/mL; PeproTech,) and IL-4 (50 ng/mL; R&D Systems). MoDCs were then stimulated for 3 days with IL-10 (200 ng/ml), IL-6 (10 ng/ml), CCL3 (50 ng/ml), VEGF (50 ng/ml), PLGF-1 (50 ng/ml) or filter-sterilized human ovarian cancer ascites at a 1:1 ratio.
Immunohistochemistry was performed using a purified anti-CD277 antibody (clone eBioBT3.1, eBioscience) and the ABC kit (Chemicon International, Temecula, CA), as reported[3
]. Horse serum (1/10 dilution) was used as a negative control.
Artificial antigen presenting cells (K32) were generated by stably expressing human CD32 on the surface of K562 cells, as previously described[29
]. CD277 was PCR amplified from human spleen cDNA using primers BTN-Fw (5' GTT GGG ACT CAA AGG TGA AGA C 3') and BTN-Rev (5' TGT CTC TAG GGA ATG ATC AGC A 3'). Expression of CD277 on K32 cells was achieved using the MSCV Retroviral Expression System (Clontech), following the recommendations of the manufacturer.
For T cell proliferation experiments, K32 cells expressing or not CD277 were γ-irradiated (100 Gy), washed twice with 1X PBS, and loaded with anti-CD3 alone (100 ng/ml, clone OKT3; eBioscience) or anti-CD3 (25 ng/ml) plus anti-CD28 (100 ng/ml, clone 15E8; Chemicon International) antibodies at room temperature for 10 min. T cells were CFSE-labeled and cocultured with loaded K32 cells at a 10:1 ratio. Proliferation of stimulated T cells was determined 5 days later by FACS on the basis of CFSE dilution.
RNA isolation, cDNA generation and Real Time PCR
In all cases, total RNA was extracted using Trizol (Invitrogen) and total cDNA was generated using SuperScript III Reverse Transcriptase (Invitrogen) and random primers, following the recommendations of the manufacturer. CD277 mRNA levels were quantified by Real Time PCR using TaqMan assays (Applied Biosystems) and primers qBTN-F (5' GGA GGG TGT ATC CTG TAC CAT CA 3'), qBTN-R (5' AAG AAG CAG CAG CAA GAC AGG 3') and the internal probe pBTN (5' CCT GGA AAA GAC AGC CAG CAT 3'). In all experiments CD277 expression was normalized among samples by quantifying the messenger levels of GAPDH using primers GAPDH-F (5' CCT GCA CCA CCA ACT GCT TA 3'), GAPDH-R (5' AGT GAT GGC ATG GAC TGT GGT 3') and the internal probe pGAPDH (5' CCT GGC CAA GGT CAT CCA TGA CAA C 3')
Flow cytometry, Bio-Plex and Immunoblotting
Flow cytometry was performed on a FACSCanto (BD Biosciences, San Jose, CA). Cell populations were sorted from human tumor single-cell suspensions using a FACSAria sorter (BD Biosciences). Anti-human antibodies were specific for CD45 (HI30), DEC205 (MG38), CD3 (UCHT1), CD4 (OKT4), CD11b (ICRF44), CD14 (M5E2) all from BD Biosciences; CD277 (20.1, BT3.1) and CD25 (BC96) antibodies were from eBioscience.
Cytokines in supernatants from stimulated T cells were quantified in a Bio-Plex assay (Bio-Rad, Hercules, CA) using the Human-27-Plex panel. Plates were read in a Bio-Plex Array Reader (Bio-Rad).
Intracellular expression of Annexin V was determined using the Annexin V-FITC Apoptosis Detection Kit (BD Biosciences), following manufacturer's instructions.
To determine c-Flip expression, pellets from CD277-exposed T cells were resuspended in 30 μl Laemmli buffer, boiled for 5 min, loaded onto a 15% sodium dodecyl sulfate–polyacrylamide electrophoresis gel, transferred to a nitrocellulose membrane, blocked, and incubated with an anti-human c-Flip antibody (C-terminus; eBioscence). Immunoreactive bands were developed using horseradish peroxidase–conjugated secondary antibodies (Bio-Rad) and chemiluminescent substrates (Pierce Chemical, Rockford, IL). In all cases, membranes were re-probed with an anti-GAPDH antibody (BioLegend) as loading control.
Soluble proteins and binding assays
The extracellular portion of BTN3A1 (aa 30-254) was PCR amplified from human spleen cDNA using primers eBTN-F (5' aag ctt CAG TTT TC TGT GCT TGG AC 3') and eBTN-R (5' gaa ttc TCT GGG CGC TCC TGA AGA AG 3'), wherein the lower case nucleotides represent HindIII and EcoRI sites, respectively. The amplified product was subcloned in frame into the expression vector pSecTag2B (Invitrogen) following the recommendations of the manufacturer, to generate a secreted protein containing the BTN3A1 extracelullar portion fused to a C-terminal Myc epitope and a poly-histidine tag. HEK293 cells were transfected with the resulting vector and secreted CD277 was purified from culture supernatants 3 days later using the His GraviTrap system (GE Healthcare). To generate a control extracelullar protein (iProt), HEK293 cells were transfected with empty pSecTag2B, which generates an irrelevant His-tagged polypeptide containing a C-terminal Myc epitope. For binding assays, resting or CD3/CD28 bead-activated T cells were incubated with the tagged proteins (0.5 ug/ml) at room temperature for 30 min. Cells were washed stringently and protein binding was assessed by secondary incubation with a FITC-labeled anti-Myc antibody (9E10; Sigma), followed by FACS analysis.
Prediction of structure and statistical analysis
The structure of the extracellular domains of B7-H4 and CD277 were modeled using PD-L1 with Protein Homology/analog Y Recognition Engine (Phyre)[37
]. Predicted structures were overlapped using Rasmol (http://rasmol.org
). Phylogenetic analysis of members of the B7 family was performed with ClustalW[38
Differences between the means of experimental groups were analyzed with the Mann-Whitney test using the GraphPad Prism 4.0 software.