Human MHC-II-restricted epitopes predicted from the SIM2 long peptide using IEDB tool.

<p>Only the top epitopes having the lowest percentile score and lowest IC50 are selected. One epitope is shown for each HLA allele out of 137 predicted binders.</p>a<p>Percentile Rank – Percentage of all peptides binding with this efficacy or lower.</p>b<p>CombLib IC40 – Predicted peptide concentration required to bind 50% of MHC molecules.</p

SIM2_230–256 induces an IFN-γ and CD4 IL-2 response.

<p>IFN-γ production by splenocytes in mice immunized with various treatments. Mice were immunized with either the 9aa SIM2₂₃₇ epitope combined with HBV or SIM2_240–254, or the SIM2_230–256 peptide alone. IFN-γ production was measured by ELISPOT. IL-2 production by CD4 T-cells. CD4 T-cells were sorted from the spleens of immunized mice and tested for reactivity to HBV₁₂₈ and SIM2_240–254 by IL-2 ELISPOT.</p

IFN-γ production by CD8 T-cells from SIM2-immunized mice.

<p>Mice were immunized with either SIM2₂₃₇, SIM2₂₃₇+HBV₁₂₈, SIM2₂₃₇+SIM2_240–254 or SIM2_230–256. Splenocytes were harvested and incubated overnight with T2 cells loaded with the SIM2₂₃₇ peptide. IFN-γ was measured by flow cytometry. FACS plots show the median IFN-γ production for each group (A) and replicate data obtained from each group (B).</p

Human SIM2 gene expression analysis in various cancers.

<p>SIM2 gene expression data were extracted from the Oncomine Research Edition. Microarray datasets that show a 2-fold change in SIM2 expression between cancer and control groups and a p value<0.01 are highlighted. (<b>A</b>) Comparison of SIM2 gene expression between cancer and control specimens. Red color indicates SIM2 overexpression and the blue color indicates SIM2 down-regulation in cancer. Numbers in the boxes indicate the number of datasets showing statistical significance. Box plots were obtained from the datasets selected in (<b>A</b>) to highlight significant overexpression of SIM2 in Prostate Carcinoma (1. Prostate Gland (n = 23), 2. Prostate Carcinoma (n = 65); <i>P</i> = 2.41×10⁻¹⁴, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093231#pone.0093231-Yu1" target="_blank">[40]</a>) (<b>B</b>); Colon Carcinoma (1. Colon (n = 10), 2. Colon Carcinoma (n = 5); P = 1.65×10⁻¹²<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093231#pone.0093231-Skrzypczak1" target="_blank">[41]</a>). (<b>C</b>); Breast Carcinoma (1. Breast (n = 4), 2. Invasive Breast Carcinoma (n = 154); P = 2.25×10⁻⁴, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093231#pone.0093231-Gluck1" target="_blank">[42]</a>) (<b>D</b>); Oligodendroglioma (1. Brain (n = 23), 2. Oligodendroglioma (n = 50); P = 3.31×10⁻⁹<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093231#pone.0093231-Sun1" target="_blank">[43]</a>) (<b>E</b>); and Pancreatic Carcinoma (1. Pancreas (n = 16), 2. Pancreatic Carcinoma (n = 36); P = 3.01×10⁻⁷<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093231#pone.0093231-Pei1" target="_blank">[44]</a>) (<b>F</b>).</p

Splenocytes from SIM2_230–256-immunized mice response to PC3-A2.1 cells.

<p>Splenocytes from HHD mice immunized with HBV and various SIM2_230–256 peptides or HBV alone were co-cultured with PC3, PC3-A2.1 (<b>A</b>) or LNCaP (<b>B</b>). Production of IFN-γ by splenocytes in response to these tumor cell lines was assessed by ELISPOT. Data is representative of 2 experiments and shows mean ± standard deviation.</p

Validation of highly differentially expressed genes between WT and MARCO^−/− DC in response to TLR challenge.

<p>DC were cultured overnight in the absence and presence of different TLR agonists. RT-PCR was performed to measure gene expression. *P<.05 for MARCO^−/−<b>vs.</b> WT DC. Data show 3 WT and 3 MARCO^−/− samples where each sample represents a pool of 3 splenocyte preparations.</p

Expression of MARCO receptor in splenic and bone marrow-derived DC.

<p>(<b>A</b>) MARCO gene expression was determined in BMDC at various time points following treatment with various TLR agonists. Raw data from gene expression dataset GSE17721 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104148#pone.0104148-Amit1" target="_blank">[20]</a> were analyzed to extract MARCO expression values. Data were processed for normalization using the RMAexpress tool and gene annotation using the MeV software. (<b>B</b>) MARCO expression as determined by RT-PCR is shown in TLR agonist-activated DC2.4 cell line (left panel), splenic DC from WT and MARCO^−/− DC from 3 individual mice (middle panel), and TLR agonist-activated splenic DC (right panel). GAPDH expression was used for normalization. Data shown as Mean ± SD from triplicates. *P<.05; **P<.01.</p

Comparison of Upstream Regulator status between WT and MARCO^−/− DC.

<p>Differentially expressed genes (fold change of 2 or higher) were processed through Ingenuity Pathway Analysis to predict the downstream regulators whose activation status was affected by the absence of MARCO in resting cells (<b>A</b>) or LPS-challenged cells (<b>B</b>). The Venn diagram in (<b>C</b>) shows the transcription factors that respond to LPS in WT (WT_LPS) and MARCO^−/− (MARCO_LPS) DC. Transcription factors that reached the significant activation z-score of −2 or +2 are shown. (<b>D</b>) Shown are representative microRNAs that reached the significant activation z-score of −2 or +2. The IPA tool predicts a microRNA to be activated when enough differentially downregulated genes fall among the targets for this microRNA. The inhibition status is attributed when the opposite occurs.</p

Impact of MARCO on TLR gene expression in DC.

<p>TLR2-9 gene expression was determined in unstimulated splenic WT and MARCO^−/− DC. Data show the mean ± SD of 3 WT and 3 MARCO^−/− samples where each sample represents a pool of 3 splenocyte preparations. *P<.05. GAPDH expression was used for normalization.</p

Differentially expressed genes in WT and MARCO^−/− DC cells.

<p>High purity DC preparations were isolated from splenocytes from 5–6 mice per group by positive selection with CD11c antibody and incubated overnight in media containing PBS or LPS (100 ng/ml). Total RNA was extracted and subjected to gene expression profiling. (<b>A</b>) Venn diagrams showing the numbers of genes that differ in expression by a factor of at least 2 between WT and MARCO^−/− DC without and with LPS (left diagram), and numbers of genes that are differentially upregulated (middle diagram) or downregulated (right diagram) in WT and MARCO^−/− DC following LPS exposure. (<b>B)</b> Top 15 differentially expressed genes that characterize MARCO vs. WT, WT_LPS vs. WT, MARCO_LPS vs. MARCO, and MARCO_LPS vs. WT_LPS. Data shown represent fold change of gene expression.</p