Expression Of sPD-1 Research Articles

Surface PD-1 expression in T cells is suppressed by HNRNPK through an exonic splicing silencer on exon 3.

Immunotherapy targeting programmed cell death 1 (PDCD1 or PD-1) and its ligands has shown remarkable promise and the regulation mechanism of PD-1 expression has received arising attention in recent years. PDCD1 exon 3 encodes the transmembrane domain and the deletion of exon 3 produces a soluble protein isoform of PD-1 (sPD-1), which can enhance immune response by competing with full-length PD-1 protein (flPD-1 or surface PD-1) on T cell surface. However, the mechanism of PDCD1 exon 3 skipping is unclear. The online SpliceAid program and minigene expression system were used to analyze potential splicing factors involved in the splicing event of PDCD1 exon 3. The potential binding motifs of heterogeneous nuclear ribonucleoprotein K (HNRNPK) on exon 3 predicted by SpliceAid were mutated by site-directed mutagenesis technology, which were further verified by pulldown assay. Antisense oligonucleotides (ASOs) targeting the exonic splicing silencer (ESS) on PDCD1 exon 3 were synthesized and screened to suppress the skipping of exon 3. The alternative splicing of PDCD1 exon 3 was analyzed by semiquantitative reverse transcription PCR. Western blot and flow cytometry were performed to detect the surface PD-1 expression in T cells. HNRNPK was screened as a key splicing factor that promoted PDCD1 exon 3 skipping, causing a decrease in flPD-1 expression on T cell membrane and an increase in sPD-1 expression. Mechanically, a key ESS has been identified on exon 3 and can be bound by HNRNPK protein to promote exon 3 skipping. Blocking the interaction between ESS and HNRNPK with an ASO significantly reduced exon 3 skipping. Importantly, HNRNPK can promote exon 3 skipping of mouse Pdcd1 gene as well. Our study revealed a novel evolutionarily conserved regulatory mechanism of PD-1 expression. The splicing factor HNRNPK markedly promoted PDCD1 exon 3 skipping by binding to the ESS on PDCD1 exon 3, resulting in decreased expression of flPD-1 and increased expression of sPD-1 in T cells.

An anti-PD-1 antisense oligonucleotide promotes the expression of soluble PD-1 by blocking the interaction between SRSF3 and an exonic splicing enhancer of PD-1 exon 3

PD-1 is a key immune checkpoint molecule. Anti-PD-1 immunotherapy is encouraging in cancer treatment. However, it still needs to be improved. PD-1 has at least five isoforms generated by alternative splicing. An isoform without exon 3 encoding soluble PD-1 (sPD-1) can activate anti-tumor immunity by inhibiting the interaction between cellular surface full-length PD-1 (flPD-1) and PD-L1. However, the regulatory mechanism of exon 3 splicing remains largely unknown. Here, we screened the exon 3 sequence by mutation and searched corresponding splicing factors by SpliceAid database and pulldown assay. The alternative splicing of PD-1 exon 3 was analyzed by RT-PCR. The expression levels of flPD-1 and sPD-1 were analyzed by Western blot, flow cytometry, and ELISA. We discovered that an exonic splicing enhancer (ESE) of exon 3 is essential for its inclusion. Moreover, SRSF3 can bind to this ESE and enhance exon 3 inclusion and flPD-1 expression. We designed and screened out an antisense oligonucleotide (ASO) targeting PD-1 to block the interaction between SRSF3 and ESE, and significantly increase exon 3 skipping and sPD-1 expression, which was verified in various tumor cells in addition to oral cancer cells. Altogether, our results uncovered the regulatory mechanism of human PD-1 exon 3 splicing and sPD-1 expression and further designed a novel anti-PD-1 ASO, which are useful for developing a new method of anti-cancer immunotherapy.

Unbalanced expression of membrane-bound and soluble inducible costimulator and programmed cell death 1 in patients with myasthenia gravis

This study aimed to investigate the possible functions and mechanisms of positive and negative costimulatory molecules in the pathological process of myasthenia gravis (MG). The expression levels of membrane-bound inducible costimulator (ICOS) and programmed cell death 1 (PD-1) in peripheral blood T cells, their corresponding ligands ICOSL and PDL-1 on B cells, and their soluble forms (sICOS, sPD-1, sICOSL, and sPDL-1) in plasma were detected in patients with untreated-stage MG (USMG) and remission-stage MG (RSMG). The results showed that the expression levels of membrane-bound ICOS and PD-1 in the peripheral blood T cells of the USMG group and their corresponding ligands ICOSL and PD-L1 on B cells were significantly increased compared to those in the RSMG group and healthy controls (HCs). The levels of sICOSL and sPD-1 were significantly upregulated in USMG patients compared to those in the RSMG and HC groups, while the levels of sICOS and sPD-L1 were not different. The expression of PD-L1 on CD19+ B cells was positively correlated with the concentrations of AchR Ab in the USMG group. The expression of ICOS and PD-1 in CD4+ T cells and the expression of ICOSL and PD-L1 on CD19+ B cells were positively correlated with the quantitative myasthenia gravis (QMG) scores in the USMG group. Also, in the USMG group, the plasma levels of sICOSL and sPD-1 were positively correlated with the QMG scores. In addition, the percentage of peripheral blood follicular helper T (Tfh) cells in the USMG group was positively correlated with ICOS and PD-1 expression on CD4+ T cells and ICOSL and PD-L1 expression on CD19+ B cells. There were positive correlations between sICOSL and sPD-1 levels and the percentage of peripheral blood Tfh cells and plasma interleukin-21 (IL-21) levels in the USMG group. The results suggest that the positive ICOS/ICOSL and negative PD-1/PD-L1 costimulatory molecule pairs participate in the pathological process of MG. Abnormal sICOSL and sPD-1 expression might interfere with the normal signal transduction of ICOS and PD-1 on Tfh cells, causing excessive activation of Tfh cells and promotion of disease progression. sICOSL and sPD-1 have potential value in monitoring MG disease states.

Analysis of Plasma EBV-DNA and Soluble Checkpoint Proteins in Nasopharyngeal Carcinoma Patients after Definitive Intensity-Modulated Radiotherapy.

Background Tumor immunotherapy and immunological checkpoint-related proteins are research hotspots. Intensity-modulated radiotherapy (IMRT) is the main treatment for nasopharyngeal carcinoma (NPC). Hence, the evaluation of its effect is very important. The aim of this study was to assess the relationship between the concentrations of soluble checkpoint proteins, plasma EBV-DNA, and cytokines in NPC patients treated with IMRT. Methods In this study, the plasma samples of 37 NPC patients and 40 healthy controls were collected. Luminex MAGPIX was used to detect the concentrations of 32 plasma targets, including soluble programmed cell death 1 (sPD-1). RT-qPCR was used to measure EBV-DNA. Results The concentrations of 33 plasma targets were detected in NPC patients before and after IMRT to explore the changes after IMRT. The results showed that IMRT could increase the expression of sPD-1 and significantly reduce the level of EBV-DNA in the plasma of NPC patients. The expression level of sPD-1 in TNM I/II patients was significantly higher than that in III/IV patients. Besides, the concentrations of 12 other targets were significantly different after IMRT, including LAG-3, PD-L1, TIM-3, IFN-γ, IL-12p70, IL-1β, IL-5, IL-6, TNF-α, IL-10, IL-17A, and IL-22. High sPD-1 patients had longer survival than those with low sPD-1. Also, patients with lower EBV-DNA and TNM grades I and II/III had longer survival than those with higher EBV-DNA or TNM IV. Conclusions This study demonstrated that the concentration of sPD-1 was significantly increased and EBV-DNA was significantly reduced in the NPC patients after IMRT. Plasma EBV-DNA level was a highly specific and sensitive biomarker for NPC diagnosis. Both sPD-1 expression and EBV-DNA concentration in plasma were related to the survival of patients.

Reconstructed Adeno-Associated Virus with the Extracellular Domain of Murine PD-1 Induces Antitumor Immunity

The negative signaling provided by interactions of the co-inhibitory molecule, programmed death-1 (PD-1), and its ligands, B7-H1 (PD-L1) and B7-DC (PD-L2), is a critical mechanism contributing to tumor evasion; blockade of this pathway has been proven to enhance cytotoxic activity and mediate antitumor therapy. Here we evaluated the anti-tumor efficacy of AAV-mediated delivery of the extracellular domain of murine PD-1 (sPD-1) to a tumor site. An rAAV vector was constructed in which the expression of sPD-1, a known negative regulator of TCR signals, is driven by human cytomegalovirus immediate early promoter (CMV-P), using a triple plasmid transfection system. Tumor-bearing mice were then treated with the AAV/sPD1 construct and expression of sPD-1 in tumor tissues was determined by semi quantitative RT-PCR, and tumor weights and cytotoxic activity of splenocytes were measured. Analysis of tumor homogenates revealed sPD-1 mRNA to be significantly overexpressed in rAAV/sPD-1 treated mice as compared with control levels. Its use for local gene therapy at the inoculation site of H22 hepatoma cells could inhibit tumor growth, also enhancing lysis of tumor cells by lymphocytes stimulated specifically with an antigen. In addition, PD-1 was also found expressed on the surfaces of activated CD8+ T cells. This study confirmed that expression of the soluble extracellular domain of PD-1 molecule could reduce tumor microenvironment inhibitory effects on T cells and enhance cytotoxicity. This suggests that it might be a potential target for development of therapies to augment T-cell responses in patients with malignancies.

Expression of soluble PD-1 in the sera of patients with recurrent genital herpes and its immunologic significance

Objective To determine the concentration of soluble PD-1 (sPD-1) in the sera of patients with recurrent genital herpes (RCH), and to explore the significance of abnormal expression of sPD-1 in RCH. Methods Serum samples were obtained from 88 healthy blood donors, 74 patients with RCH including 34 cases of outbreak-stage RCH and 40 cases of stable-stage RCH. The serum level of sPD-1 was measured by monoclonal antibody labeling and enzyme linked immunosorbent assay (ELISA). Results A significant difference was observed in the serum level of sPD-1 between the patients with RGH and the blood donors (33.06 ± 17.5 μg/L vs. 53.07 ± 26.3μg/L, P < 0.01) and between the patients with outbreak-stage RGH and those with stable-stage RGH (27.47 ± 12.9 μg/L vs. 37.71 ± 19.6 μg/L, P< 0.01). Conclusions There is a low expression of sPD-1 in patients with RGH, which may be one of the mechanisms underlying the escape of HSV- Ⅱ from immunologic surveillance and development of immunological tolerance.