Anti-4-1BB pre-activated PDCA-1⁺ B cells inhibit in vivo humoral responses.

<p><b>A</b> Cartoon showing the steps involved in purification of PDCA-1⁺ B cells, activation with IL-7 or anti-4-1BB, adoptive transfer, and serum analysis. <b>B</b>, <b>C</b>, PDCA-1⁺ B cells were purified from spleens of naïve B6 mice, cultured for 3d with IL-7 (100 ng/ml) or soluble anti-4-1BB (5 µg/ml), and pulsed with 0.5 mg/ml NP⁴⁰-AECM Ficoll overnight. Ag-pulsed cells (2×10⁶) were injected (i.v.) into syngeneic hosts. Sera collected on d7 were analyzed for the presence of anti-NP IgM <b>B</b>, and IgG3 <b>C</b> Abs by ELISA. The extent of anti-NP Ab production (absorbance) is shown by the bar graphs (mean ± SD). The results of four pooled independent experiments are shown (<i>n</i> = 4). (D–I) PDCA-1⁺ B cells were purified from the spleens of naïve B6 mice, cultured for 3d with IL-7 (100 ng/ml) or soluble anti-4-1BB (5 µg/ml), pulsed overnight with 0.5 mg/ml NP²³-CGG and adoptively transferred (2×10⁶; i.v.) into syngeneic hosts. Sera collected on d7 were analyzed for the presence of anti-NP IgM <b>D</b>, IgG1 <b>E</b>, IgG2a <b>F</b>, IgG2b <b>G</b>, IgG3 <b>H</b>, and IgA <b>I</b> Abs by ELISA. The extent of anti-NP Ab production (absorbance) is shown by the bar graphs (mean ± SD). The results of four pooled independent experiments are shown (<i>n</i> = 4). * <i>p</i><0.05.</p

4-1BB signaling promotes PDCA-1⁺ B cell development.

<p><b>A</b> BM-derived cells from naïve B6 mice were stained with the indicated markers before (upper panels) or after 5d (lower panels) activation with soluble anti-4-1BB (5 µg/ml) and flow cytometry was performed. The frequencies of CD19⁺CD11c⁺ cells, among gated PDCA-1⁺ cells, are indicated. One of three independent experiments is shown (<i>n</i> = 3). <b>B</b>–D BM-derived cells from naïve B6 mice were stained for the indicated markers either with no cell depletion <b>B</b>, after PDCA-1⁺ cell depletion <b>C</b>, or after B220⁺ cell depletion <b>D</b>. The cells were stained either immediately (upper row) or after 7d stimulation with soluble anti-4-1BB (5 µg/ml) (lower row). One of two independent experiments is shown (<i>n</i> = 2).</p

Activation upregulates 4-1BB expression in PDCA-1⁺ cells.

<p><b>A</b> Total BM-derived cells were stimulated with the indicated soluble agonists for 3d, stained with fluorochrome- tagged Abs to 4-1BB, PDCA-1, and IgD, then analysed by flow cytometry. The percentages of 4-1BB⁺ cells in gated PDCA-1⁺IgD⁺ and PDCA-1⁺IgD⁻ fractions are presented as bar graphs (mean ± SD). The results of three pooled independent experiments are shown (<i>n</i> = 3). <b>B</b> Purified splenic PDCA-1⁺ B cells from naïve B6 mice were stimulated with anti-4-1BB (5 µg/ml) for 3d, stained as indicated, and flow cytometry was performed. The frequencies of cells expressing the indicated activation markers were evaluated and are presented as bar graphs (mean ± SD). The results of three pooled independent experiments are shown (<i>n</i> = 3). * <i>p</i><0.05.</p

Anti-4-1BB pre-activated PDCA-1⁺ B cells inhibit in vitro Ig class switching.

<p><b>A</b> PDCA-1⁺ B cells were sort-purified from spleens of naïve B6 mice and stained as indicated, and flow cytometry was performed. One of four independent experiments is shown (<i>n</i> = 4). <b>B</b> Purified splenic PDCA-1⁺ B cells were stimulated with the indicated agonists, after which in vitro Ig class switching was analyzed by flow cytometry. One of four independent experiments is shown (<i>n</i> = 4). <b>C</b> Purified splenic PDCA-1⁺ B cells were stimulated with soluble anti-4-1BB (5 µg/ml) and/or IL-7 (100 ng/ml) for 3d then re-stimulated with LPS/IL-4 for 4d, after which in vitro Ig class switching was analyzed by flow cytometry. One of four independent experiments is shown (<i>n</i> = 4).</p

4-1BB signaling induces selective expression of effector molecules.

<p><b>A</b> Purified splenic PDCA-1⁺ B cells from naïve B6 mice were stimulated with soluble anti-4-1BB (5 µg/ml) for 3d, cell-free culture supernatants were collected, and levels of IFN-α were analyzed by ELISA. These values are presented as bar graphs (mean ± SD). The results of three pooled independent experiments are shown (<i>n</i> = 2). <b>C</b> Total bone marrow-derived cells were stimulated with anti-4-1BB (5 µg/ml). After 6d, cells were collected, washed, stained as indicated, and analyzed. One of three independent experiments is shown (<i>n</i> = 3). <b>D</b> Purified splenic PDCA-1⁺ B cells were stained for the indicated markers either immediately or 3d after stimulation with anti-4-1BB (5 µg/ml). One of two independent experiments is shown (<i>n</i> = 3). <b>C</b> FACS-sorted PDCA-1⁺ B cells from spleens of naïve mice were stained as indicated and flow cytometry was performed. One of three independent experiments is shown (<i>n</i> = 3). <b>E</b> Sorted PDCA-1⁺ B cells were stimulated for 3d with rat IgG or soluble anti-4-1BB (both 5 µg/ml), and cell-free supernatants were collected and analyzed for cytokines using commercially available array screens. The results of three pooled independent experiments are shown (<i>n</i> = 3). <b>F</b> Table showing the position of individual cytokines/chemokines used in the assay. Upregulated molecules in the cells stimulated with anti-4-1BB and their position in the array are highlighted. Pos, positive; Neg, negative.</p

Anti-4-1BB-activated PDCA-1⁺ B cells inhibit Ag-specific T cell responses.

<p><b><u>A</u></b> Purified PDCA⁺ B cells were treated for 3d with indicated markers and Annexin V assay was performed as depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050272#s4" target="_blank">Materials and methods</a>. The events in the boxes represent cells undergoing apoptosis. None, no stimulation for 72 hrs. One of 2 independent experiments is shown (<i>n</i> = 2). <b>B</b> Purified PDCA⁺ B cells from the bone marrows of naïve Balb/C mice were stimulated for 3d with IL-7 (100 ng/ml) or soluble anti-4-1BB (5 µg/ml) and washed, and Ag uptake assays were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050272#s4" target="_blank"><i>Materials and Methods</i></a>. The cells were stained as indicated and flow cytometry was performed. One of two independent experiments is shown. <i>n</i> = 2. <b>C</b> Purified PDCA-1⁺ B cells were stimulated for 3d with IL-7 (100 ng/ml) or soluble anti-4-1BB (5 µg/ml), pulsed with 0.5 mg OVA^323–339 peptide, and incubated with purified CFSE-labeled DO11.10 CD4⁺ T cells for 3d. They were stained as indicated and flow cytometry was performed. One of two independent experiments is shown. <i>n</i> = 2. Gates were set around CFSE⁺ cells, and proportions of Vα2⁺ cells were enumerated. <b>D</b> Purified PDCA-1⁺ B cells from naïve Balb/C mice were pulsed with OVA^323–339 as in experiment <b>B</b>, and adoptively transferred (2×10⁶/mouse) into syngeneic DO11.10 mice. The recipient DO11.10 mice were further treated with BrdU as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050272#s4" target="_blank"><i>Materials and Methods</i></a>. Spleen cells from the treated mice were prepared 3d after cell transfer, stained as indicated, and flow cytometry was performed. One of two independent experiments is shown. Gates were set around Vα2⁺CD4⁺ cells, and Vα2⁺BrdU⁺ cells were enumerated.</p

PDCA-1⁺ B cells constitutively express 4-1BB.

<p>Bone marrow (BM)-derived and spleen cells from naïve B6 mice were subjected to flow cytometry with the indicated markers. <b>A</b> The proportions of 4-1BB⁺ cells in PDCA-1⁺-gated populations are indicated. One of six independent experiments is shown (<i>n</i> = 6). <b>B</b> The different populations of cells from BM and spleen are indicated. One of three independent experiments is shown (<i>n</i> = 3). <b>C</b> Gates were set around the indicated cell populations. The proportion of 4-1BB-expressing cells in each population is indicated. One of three independent experiments is shown (<i>n</i> = 3).</p

Differences in Gastric Carcinoma Microenvironment Stratify According to EBV Infection Intensity: Implications for Possible Immune Adjuvant Therapy

<div><p>Epstein-Barr virus (EBV) is associated with roughly 10% of gastric carcinomas worldwide (EBVaGC). Although previous investigations provide a strong link between EBV and gastric carcinomas, these studies were performed using selected EBV gene probes. Using a cohort of gastric carcinoma RNA-seq data sets from The Cancer Genome Atlas (TCGA), we performed a quantitative and global assessment of EBV gene expression in gastric carcinomas and assessed EBV associated cellular pathway alterations. EBV transcripts were detected in 17% of samples but these samples varied significantly in EBV coverage depth. In four samples with the highest EBV coverage (hiEBVaGC – high EBV associated gastric carcinoma), transcripts from the BamHI A region comprised the majority of EBV reads. Expression of LMP2, and to a lesser extent, LMP1 were also observed as was evidence of abortive lytic replication. Analysis of cellular gene expression indicated significant immune cell infiltration and a predominant IFNG response in samples expressing high levels of EBV transcripts relative to samples expressing low or no EBV transcripts. Despite the apparent immune cell infiltration, high levels of the cytotoxic T-cell (CTL) and natural killer (NK) cell inhibitor, IDO1, was observed in the hiEBVaGCs samples suggesting an active tolerance inducing pathway in this subgroup. These results were confirmed in a separate cohort of 21 Vietnamese gastric carcinoma samples using qRT-PCR and on tissue samples using in situ hybridization and immunohistochemistry. Lastly, a panel of tumor suppressors and candidate oncogenes were expressed at lower levels in hiEBVaGC versus EBV-low and EBV-negative gastric cancers suggesting the direct regulation of tumor pathways by EBV.</p> </div