Programmed cell death protein 1 (PD-1), a costimulatory molecule of the CD28 family, has 2 ligands, PD-L1 and PD-L2. Our previous studies showed that the expression of PD-1 and PD-L1 is up-regulated during viral infection in pigs. Extensive studies have shown that blockade of the PD-1/PD-L1 pathways by anti-PD-L1 antibody or soluble PD-1 restores exhausted T-cells in humans and mice. In the present study the extracellular domains of PD-1 and PD-L1 were used to evaluate the binding of PD-1 and PD-L1 with peripheral blood mononuclear cells (PBMCs). We amplified the cDNA encoding the extracellular domains of PD-1 and PD-L1 to construct recombinant expression plasmids and obtain soluble recombinant proteins, which were then labeled with fluorescein isothiocyanate (FITC). The His-ExPD-1 and His-ExPD-L1 recombinant proteins were expressed in the form of inclusion bodies with a relative molecular weight of 33.0 and 45.0 kDa, respectively. We then prepared polyclonal antibodies against the proteins with a multi-antiserum titer of 1:102 400. Binding of the proteins with PBMCs was evaluated by flow cytometry. The fluorescence signals of His-ExPD-1-FITC and His-ExPD-L1-FITC were greater than those for the FITC control. These results suggest that the soluble recombinant proteins may be used to prepare monoclonal antibodies to block the PD-1/PD-L1 pathway.

OBJECTIVE To investigate protein kinase CK2 (CK2) expression in squamous cell carcinoma (SCC) of cats and to examine effects of CK2 downregulation on in vitro apoptosis and viability in SCC. SAMPLE Biopsy specimens of oral mucosa and testis and blood samples from clinically normal cats, biopsy specimens of oral SCC from cats, and feline SCC (SCCF1) and mammary gland carcinoma (K12) cell lines. PROCEDURES Immunohistochemically labeling for CK2α was performed on biopsy specimens. Sequences of the CK2α subunit gene and CK2α’ subunit gene in feline blood and feline cancer cell lines were determined by use of PCR and reverse-transcription PCR assays followed by direct Sanger sequencing. Specific small interfering RNAs (siRNAs) were developed for feline CK2α and CK2α'. The SCCF1 cells were treated with siRNA and assessed 72 hours later for CK2α and CK2α’ expression and markers of apoptosis (via western blot analysis) and for viability (via 3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium assays). RESULTS CK2α was expressed in all feline oral mucosa samples and 7 of 8 oral SCC samples. Expression of CK2α and CK2α’ was successfully downregulated in SCCF1 cells by use of siRNAs, which resulted in decreased viability and induction of apoptosis. CONCLUSIONS AND CLINICAL RELEVANCE In this study, CK2 appeared to be a promising therapeutic target for SCCs of cats. A possible treatment strategy for SCCs of cats would be RNA interference that targets CK2.