Feature Matrix for Development of Models for Estimating the Likelihood of PCR Amplification

This data set contains pairs of primers and immunoglobulin heavy chain variable sequences with annotated experimental amplification status according to gel electrophoresis. The data set tabulates the features (e.g. annealing temperature, mismatches) that determine whether a primer leads to the successful amplification of a template

PCR Results for Immunogloublin Heavy Chain Variable Sequences

This data set indicates the amplification status for pairs of primers and immunoglobulin heavy chain variable sequences. CSV files were generated on the basis of raw gel electrophoresis data (XLS file). In the files, cells with an entry of "1" indicate that a template was amplified by the corresponding primer, while a "0" indicates that this was not the case. Measurements were performed in triplicates. Two primer sets were used. Files containing "Tiller" represent primers from Set2, while file containing "Opt" represent primers from set 1. For each primer set, there are five files. Each file represent the labeling of the gel electrophoresis bands by a single person

Enhanced Cross-Presentation and Improved CD8⁺ T Cell Responses after Mannosylation of Synthetic Long Peptides in Mice

<div><p>The use of synthetic long peptides (SLP) has been proven to be a promising approach to induce adaptive immune responses in vaccination strategies. Here, we analyzed whether the efficiency to activate cytotoxic T cells by SLP-based vaccinations can be increased by conjugating SLPs to mannose residues. We could demonstrate that mannosylation of SLPs results in increased internalization by the mannose receptor (MR) on murine antigen-presenting cells. MR-mediated internalization targeted the mannosylated SLPs into early endosomes, from where they were cross-presented very efficiently compared to non-mannosylated SLPs. The influence of SLP mannosylation was specific for cross-presentation, as no influence on MHC II-restricted presentation was observed. Additionally, we showed that vaccination of mice with mannosylated SLPs containing epitopes from either ovalbumin or HPV E7 resulted in enhanced proliferation and activation of antigen-specific CD8⁺ T cells. These findings demonstrate that mannosylation of SLPs augments the induction of a cytotoxic T cell response <i>in vitro</i> and <i>in vivo</i> and might be a promising approach to induce cytotoxic T cell responses in e.g. cancer therapy and anti-viral immunity.</p></div

Uptake of mannosylated and non-mannosylated SLPs.

<p>A) Wildtype or MR-deficient BM-DCs were incubated with 250 ng/ml fluorochrome-labeled OVA, 200 nM mannosylated SLPs or non-mannosylated SLPs for 15 min, chased with medium for 20 min and analyzed by immunofluorescence microscopy. Nuclei stained with DAPI are depicted in blue. B) Wildtype or MR-deficient BM-DCs were incubated with 250 ng/ml fluorochrome-labeled OVA, 200 nM mannosylated SLPs or non-mannosylated SLPs for 15 min. Antigen uptake was monitored by flow cytometry (gated on all living cells). C) Quantification of B) using different antigen concentrations. Depicted are representative results of at least 3 independent experiments. MFI: mean fluorescence intensity.</p

In vivo T cell activation by mannosylated SLPs.

<p>A) Mice were injected i.d. with 75 µg of OVA-specific mannosylated or non-mannosylated SLPs and 20 µg CpG. After 7 days, antigen-specific T cells in the blood were monitored by flow cytometry using epitope-specific tetramers. Cells were gated for CD8. B) Quantitative analysis of epitope-specific T cells in the blood or in the spleen after s.c or i.d. injection of SLPs. Graphs show percentage of tetramer-positive CD8⁺ T cells. C) as in A) using HPV-specific SLPs. D) Quantitative analysis of epitope-specific T cells in the blood or in the spleen after s.c or i.d. injection of HPV-specific SLPs. E) Intracellular cytokine staining of splenic CD8⁺ T cells after i.d. injection of HPV-specific mannosylated or non-mannosylated peptides and CpG as above. F) Quantitative analysis of intracellular cytokines in T cells isolated from the spleen after s.c or i.d. injection of HPV-specific SLPs. Graphs show percentage of IFNγ⁺ TNFα⁺ cells amongst all CD8⁺ T cells. Dot plots depict representative results of 2 independent experiments. Bar graphs depict pooled results of 2 independent experiments (n = 9–10).</p

Overview of the used SLPs.

<p>To study the effect of mannosylation on cross-presentation, we generated mannosylated and non-mannosylated SLPs containing the MHC I-restricted epitope of OVA (OVA_257–264; peptides 1 and 2). Mannosylation was introduced by a bis-mannosylated Lysin residue. Additionally, we generated mannosylated or non-mannosylated SLPs containing the MHC II-restricted epitope of OVA (OVA_323–339; peptides 3 and 4). To analyze intracellular trafficking, we synthesized fluorescently-labeled mannosylated or non-mannosylated SLPs (peptides 5 and 6). The Alexa647 fluorochrome was introduced by conjugation to an Alexa647-labeled cysteine. To study T cell responses against the E7 protein of HPV16, we generated mannosylated or non-mannosylated SLPs containing the MHC I restricted epitope of E7 (E7_43–63; peptides 7 and 8). Epitopes within the SLPs are in bold.</p

Intracellular localization of mannosylated SLPs.

<p>A) Wild-type BM-DCs were incubated simultaneously with Alexa647-labeled SLPs and Alexa488-labeled OVA for 15 min. After medium chase of another 20 min, intracellular localization was determined by immunofluorescence microscopy. B) Wild-type BM-DCs were incubated with fluorochrome-labeled OVA, SLPs, Transferrin and/or Lucifer Yellow for 15 min, chased with medium for another 20 min and stained with antibodies against EEA1, LAMP1 or Lucifer Yellow. Intracellular distribution was analyzed by immunofluorescence microscopy. To analyse co-localization of OVA and SLPs with the indicated markers, the Pearson correlation coefficient (varying between −1 and +1 with −1 for perfect negative correlation, 0 for perfect absence of correlation and 1 for perfect correlation) and the Mander's overlap coefficient (varying between 0 and 1 with 0 for no overlap and +1 for perfect overlap), were calculated. Nuclei stained with DAPI are depicted in blue. PCC: Pearson Correlation Coefficient. MOC: Mander's overlap coefficient.</p

Cross-presentation and MHC II-restricted presentation of mannosylated SLPs.

<p>A) Wildtype or MR-deficient BM-DCs were incubated with OVA or SLPs and incubated with CFSE-labeled OT-I T cells. After 3 days, T cell proliferation was determined by flow cytometry after gating on CD8⁺ cells. B) Quantification of A), depicting the division index and the percentage of dividing OT-I T cells. C) BM-DCs were treated with 100 ng/ml LPS or 50 nM mannosylated or non-mannosylated SLPs. After 24 h, expression of CD40, CD80, CD86 and MHC I were analyzed by flow cytometry. Control: Isotype control. D) BM-DCs were pre-treated with 50 nM mannosylated or non-mannosylated SLPs containing the MHC II epitope of OVA for 1 h before they were loaded with the SIINFEKL short peptide for another 3 h and incubated with CFSE-labeled OT-I T cells. After 3 days, T cell proliferation was determined by flow cytometry after gating on CD8⁺ cells. E) Wildtype or MR-deficient BM-DCs were incubated with OVA or SLPs and incubated with CFSE-labeled OT-II T cells. After 3 days, T cell proliferation was determined by flow cytometry after gating on CD4⁺ cells. F) Quantification of E), depicting the division index and the percentage of dividing OT-II cells. Depicted are representative results of at least 3 independent experiments.</p