The C-Type Lectin OCILRP2 Costimulates EL4 T Cell Activation via the DAP12-Raf-MAP Kinase Pathway

<div><p>OCILRP2 is a typical Type-II transmembrane protein that is selectively expressed in activated T lymphocytes, dendritic cells, and B cells and functions as a novel co-stimulator of T cell activation. However, the signaling pathways underlying OCILRP2 in T cell activation are still not completely understood. In this study, we found that the knockdown of OCILRP2 expression with shRNA or the blockage of its activity by an anti-OCILRP2 antagonist antibody reduced CD3/CD28-costimulated EL4 T cell viability and IL-2 production, inhibit Raf1, MAPK3, and MAPK8 activation, and impair NFAT and NF-κB transcriptional activities. Furthermore, immunoprecipitation results indicated that OCILRP2 could interact with the DAP12 protein, an adaptor containing an intracellular ITAM motif that can transduce signals to induce MAP kinase activation for T cell activation. Our data reveal that after binding with DAP12, OCILRP2 activates the Raf-MAP kinase pathways, resulting in T cell activation.</p></div

Membrane re-localization of OCILRP2 via treatment with an anti-CD3/CD28 mAb.

<p>EL4 cells were incubated with plate-bound CD3/CD28 antibodies, isotype-matched mIgGs, or PMA/ionomycin for 48 h. The cells (1×10⁶ cells) were then incubated with an anti-OCILRP2 antagonist antibody for 30 min on ice, followed by staining with an FITC-labeled second antibody. The cells were analyzed with Cellquest software using a FACS Calibur flow cytometer.</p

Membrane co-localization of OCILRP2 and DAP12 under anti-CD3/CD28 mAb treatment in EL4 T cells.

<p>EL4 T cells pre-transfected with pEGFP-siOCILRP2 or pre-coated with the OCILRP2 Ab were cultured overnight in the presence or absence of an anti-CD3/CD28 mAb and then stained for OCILRP2 (green) and nuclear stained with DAPI (blue) and DAP12 (red) to study OCILRP2 and DAP12 protein expression and localization. Unstimulated EL4 T cells exhibited OCILRP2 protein expression in the cytoplasm (upper panels). In contrast, CD3/CD28-activated EL4 T cells showed intracellular and membrane OCILRP2. OCILRP2/DAP12 co-localization appears in yellow. Each picture is representative of the vast majority of the observed cells on the slides.</p

OCILRP2 co-stimulation affects EL4 cell viability.

<p>Six-well flat-bottom microtiter plates were pre-coated with anti-CD3/CD28 antibodies or anti-CD3/CD28/OCILRP2 antibodies (a) or anti-CD3/CD28/IL-2 antibodies (b) overnight at 4°C. Cells were transfected with pEGFP-siOCILRP2 plasmid and then stimulated with the anti-CD3/CD28 antibodies (a). Then, 100 µL of the cell suspension (5,000 cells/well) was dispensed into each well of a 96-well plate. After incubation for 24, 48, and 72 h (37°C), 10 µL of CCK-8 solution was added to each well, and the cells were incubated for 1 h (37°C). The absorbance was measured at 450 nm using a spectrophotometer (Beckman Coulter DU 640, USA). *P<0.05.</p

mRNA expression in EL4 cells stimulated with or without anti-CD3/CD28 antibodies or anti-CD3/CD28/OCILRP2 antibodies.

<p>RNA from un-stimulated or CD3/CD28 antibody- or CD3/CD28/OCILRP2 antibody-stimulated EL4 cells was isolated using the RNeasy kit (Qiagen). cDNA of IL-2, NF-κB, NFAT, MAPK3, and MAPK8 or the control household gene <i>β-actin</i> was amplified using SYBR Premix Ex Taq (RRO41A, TaKaRa, Shiga, Japan), and expression was monitored using an Rotor-gene 6000 real-time platform (Corbett Research, Australia). Ct values were normalized for the expression of the <i>β-actin</i> gene. *P<0.05.</p

Interaction of OCILRP2 and DAP12 in GST pull-down and co-immunoprecipitation assays.

<p>The GST pull-down assay was carried out using purified beads that contained GST, GST-DAP12, or GST-OCILRP2. Precipitated OCILRP2 or DAP12 was detected by western blotting using an anti-OCILRP2 or anti-DAP12 antibody, respectively (a, b). 293T cells were grown in 6-cm dishes and transfected with the pCDNA3-HA-OCILRP2 and pDsRed-C1-DAP12 plasmids, respectively. OCILRP2 and DAP12 were detected by western blotting using an anti-HA antibody or an anti-DAP12 antibody (c). Schematic diagram of OCILRP2 predicted by SMART software. The green column represents the transmembrane region (amino acids 57–80) (d). The GST pull-down assay was carried out using purified beads that contained GST, full-length GST-OCILRP2, GST-OCILRP2i (aa 1–57), or GST-OCILRP2e (aa 80–221). Precipitated DAP12 was detected by western blotting using an anti-DAP12 antibody (e).</p

IL-2 secreted from EL4 cells after stimulation with an anti-CD3/CD28 mAb or PMA/ionomycin.

<p>EL4 cells were incubated with or without anti-CD3/CD28 antibodies (at various concentrations) or PMA (10 ng/mL) and ionomycin (100 ng/mL) for 12, 24, and 48 h. Supernatants were obtained by centrifugation for 3 min using a microcentrifuge. Cytokine IL-2 secreted into the supernatant was determined by ELISA.</p

Western blot analysis of Raf, Erk, Jnk, and Akt phosphorylation in EL4 cells.

<p>EL4 T cells were incubated in DMEM medium with an immobilized anti-CD3/CD28 mAb in the presence or absence of si-OCILRP2. After incubation at 37°C for 48 h, the T cells were lysed, and aliquots of 20 µg of the whole-cell lysate were analyzed by western blotting using antibodies against phospho-Erk, Erk, phospho-Jnk, and Jnk (a) and phospho-Raf and phosphor-Akt (b). The data are expressed as a percentage of the level of active Erk or Jnk of the total Erk or Jnk. Each data point represents the mean ± S.E. from three separate experiments.</p

The Two-Component Signal Transduction System ArlRS Regulates <em>Staphylococcus epidermidis</em> Biofilm Formation in an <em>ica</em>-Dependent Manner

<div><p>Due to its ability to form biofilms on medical devices, <em>Staphylococcus epidermidis</em> has emerged as a major pathogen of nosocomial infections. In this study, we investigated the role of the two-component signal transduction system ArlRS in regulating <em>S. epidermidis</em> biofilm formation. An ArlRS-deficient mutant, WW06, was constructed using <em>S. epidermidis</em> strain 1457 as a parental strain. Although the growth curve of WW06 was similar to that of SE1457, the mutant strain was unable to form biofilms <em>in vitro</em>. In a rabbit subcutaneous infection model, sterile disks made of polymeric materials were implanted subcutaneously followed with inoculation of WW06 or SE1457. The viable bacteria cells of WW06 recovered from biofilms on the embedded disks were much lower than that of SE1457. Complementation of arlRS genes expression from plasmid in WW06 restored biofilm-forming phenotype both <em>in vivo</em> and <em>in vitro</em>. WW06 maintained the ability to undergo initial attachment. Transcription levels of several genes involved in biofilm formation, including <em>icaADBC</em>, <em>sigB</em>, and <em>sarA</em>, were decreased in WW06, compared to SE1457; and <em>icaR</em> expression was increased in WW06, detected by real-time reverse-transcription PCR. The biofilm-forming phenotype was restored by overexpressing <em>icaADBC</em> in WW06 but not by overexpressing <em>sigB</em>, indicating that ArlRS regulates biofilm formation through the regulation of <em>icaADBC</em>. Gel shift assay showed that ArlR can bind to the promoter region of the <em>ica</em> operon. In conclusion, ArlRS regulates <em>S. epidermidis</em> biofilm formation in an <em>ica</em>-dependent manner, distinct from its role in <em>S. aureus</em>.</p> </div

Regulation of expression of biofilm-related genes by ArlRS.

<p>(A) Effect of ArlRS deficiency on transcription levels of the biofilm-related genes. Gene expression profiles of the <i>arlRS</i>-deficient strain WW06 and the wild-type parent strain SE1457 in the mid-log growth phase were analyzed using RT-qPCR. The experiment was performed in triplicate and the expression ratios of the biofilm-related genes in WW06 and SE1457 are represented as mean ± standard deviation. (B) Effect of <i>arlRS</i> mutation on PIA synthesis. PIA was extracted from cells grown for 24 h under biofilm conditions in 6-well cell culture plates. PIA was detected by dot blot analysis using wheat germ agglutinin coupled to horseradish peroxidase. The PIA-deficient <i>S. epidermidis</i> mutant Δ<i>icaC</i> was used as a negative control, and RP62A was used as a positive control.</p