Characterization of a soluble B7-H3 (sB7-H3) spliced from the intron and analysis of sB7-H3 in the sera of patients with hepatocellular carcinoma

Abstract

B7-H3 is a recently discovered member of the B7 superfamily molecules and has been found to play a negative role in T cell responses. In this study, we identified a new B7-H3 isoform that is produced by alternative splicing from the forth intron of B7-H3 and encodes the sB7-H3 protein. Protein sequence analysis showed that sB7-H3 contains an additional four amino acids, encoded by the intron sequence, at the C-terminus compared to the ectodomain of 2Ig-B7-H3. We further found that this spliced sB7-H3 plays a negative regulatory role in T cell responses and serum sB7-H3 is higher in patients with hepatocellular carcinoma (HCC) than in healthy donors. Furthermore, we found that the expression of the spliced sb7-h3 gene is higher in carcinoma and peritumor tissues than in PBMCs of both healthy controls and patients, indicating that the high level of serum sB7-H3 in patients with HCC is caused by the increased expression of this newly discovered spliced sB7-H3 isoform in carcinoma and peritumor tissues.

Figure 1. Nucleotide sequence analysis of spliced sb7-h3 gene.

The complete nucleotide sequence is shown. The boxed sequences are primer pair used to amplify b7-h3 gene. The highlighted sequence is the ORF of spliced sB7-H3. The contained exons and intron are labeled on the left.

Figure 2. Spliced sB7-H3 is expressed as a soluble form and generally exists in healthy donors.

A, The schematic diagram of chimeric sB7-H3-Fc: the complete spliced sb7-h3 gene was fused with rabbit fc in one opened reading frame. B, pc-sB7-H3-Fc (lane 1) and vacant vector (lane 3) were transfected into 293T cells, respectively. The supernatant of transfected cells was harvested 24 h post transfection and submitted to western blot assay. Lane 2: prestained protein ladder. C, Investigation of spliced sb7-h3 gene in the PBMCs of healthy donors: Lane 1: negative control; Lane 2–6: randomly selected healthy donors; Lane 7: positive control; Lane 8: DNA ladder.

Figure 3. Functional analysis of spliced sB7-H3 on T cell response.

CFSE-labeled purified T cells were seeded to a 96-well flat-bottomed plate precoated with 1 µg/ml anti-human CD3 (OKT3), plus either 10 µg/ml spliced sB7-H3-Fc or Fc. After culturing for 72 h, T cell proliferation and cytokines in the culture supernatant was analyzed. A, A representative plot of T cell proliferation analyzed with FACS assay. B, Statistical analysis of the mean fluorescence intensity (MFI) on T cells (mean ± s.e.m). C and D, the concentration of IL-2 and IFN-γ in the culture supernatant was analyzed with multiplex kit and analyzed by FlowCytomixPro software. “*” indicated P<0.05.

Figure 4. Comparison in the expression of spliced sB7-H3 between patients and healthy donors.

A, Sera were obtained from healthy donors or patients with HCC. The expression level of spliced sB7-H3 was monitored with sandwich ELISA assay. B, The cDNAs produced from PBMCs of healthy donors or patients with HCC were analyzed for expression of spliced sB7-H3 by real-time PCR assay. A greater than 2-fold increase over health donors was considered clinical significant. C, The expression of spliced sB7-H3 in PBMCs of healthy donors and patients, as well as patients' tumor tissues and peritumor tissues were evaluated with comparative qPCR assay. A greater than 2-fold increase in value over healthy donors was considered clinical significant. D, Spliced sB7-H3 of individual serum from patients enrolled with HCC before and after resection was evaluated with sandwich ELISA assay. The horizontal line indicates the mean value of each group. “*” indicated P<0.05, “**” indicated P<0.01.