Controlling T‑Cell Activation with Synthetic Dendritic Cells Using the Multivalency Effect

Artificial antigen-presenting cells (aAPCs) have recently gained a lot of attention. They efficiently activate T cells and serve as powerful replacements for dendritic cells in cancer immunotherapy. Focusing on a specific class of polymer-based aAPCs, so-called synthetic dendritic cells (sDCs), we have investigated the importance of multivalent binding on T-cell activation. Using antibody-functionalized sDCs, we have tested the influence of polymer length and antibody density. Increasing the multivalent character of the antibody-functionalized polymer lowered the effective concentration required for T-cell activation. This was evidenced for both early and late stages of activation. The most important effect observed was the significantly prolonged activation of the stimulated T cells, indicating that multivalent sDCs sustain T-cell signaling. Our results highlight the importance of multivalency for the design of aAPCs and will ultimately allow for better mimics of natural dendritic cells that can be used as vaccines in cancer treatment

Comparison of Mitochondrial Mutation Spectra in Ageing Human Colonic Epithelium and Disease: Absence of Evidence for Purifying Selection in Somatic Mitochondrial DNA Point Mutations

<div><p>Human ageing has been predicted to be caused by the accumulation of molecular damage in cells and tissues. Somatic mitochondrial DNA (mtDNA) mutations have been documented in a number of ageing tissues and have been shown to be associated with cellular mitochondrial dysfunction. It is unknown whether there are selective constraints, which have been shown to occur in the germline, on the occurrence and expansion of these mtDNA mutations within individual somatic cells. Here we compared the pattern and spectrum of mutations observed in ageing human colon to those observed in the general population (germline variants) and those associated with primary mtDNA disease. The pathogenicity of the protein encoding mutations was predicted using a computational programme, MutPred, and the scores obtained for the three groups compared. We show that the mutations associated with ageing are randomly distributed throughout the genome, are more frequently non-synonymous or frameshift mutations than the general population, and are significantly more pathogenic than population variants. Mutations associated with primary mtDNA disease were significantly more pathogenic than ageing or population mutations. These data provide little evidence for any selective constraints on the occurrence and expansion of mtDNA mutations in somatic cells of the human colon during human ageing in contrast to germline mutations seen in the general population.</p> </div

Genetic consequences of mutations observed in ageing, population, and disease.

<p>A: The percentage of changes which predict synonymous, non-synonymous and premature termination codons or frameshifts in protein encoding genes. Contingency analysis with Bonferroni correction for multiple testing was carried out on the frequencies of the changes in each gene type (ageing n = 81, population n = 155, disease n = 76). Thresholds for statistical significance are; ***<0.0003, ** 0.003, * = 0.017. B: Frequency of termination codon and frameshift mutations in inherited and sporadic disease-causing mtDNA mutations. Chi-squared analysis showed a significantly higher frequency such mutations in sporadic than inherited cases (p = 0.003).</p

Gene location and types of mutations observed in ageing, population, and disease.

<p>A: Gene location of mutations. Data are represented as the percentage of the total coding region mutations. Contingency analysis with Bonferroni correction for multiple testing was carried out on the frequencies of the changes in each gene type (ageing n = 117, population n = 182, disease n = 176). Thresholds for statistical significance are; ***<0.0003, ** 0.003, * = 0.017. B: Types of changes observed in ageing, population and disease. Data are represented as the percentage of the total coding region mutations. Contingency analysis with Bonferroni correction for multiple testing was carried out on the frequencies of the changes in each mutational category. Thresholds for statistical significance are; ***<0.0003, ** 0.003, * = 0.017.</p

Exclusion of mitochondrial DNA (mtDNA) mutations occurring in the germline or in early embryogenesis.

<p>(<b>A</b>) Somatic mtDNA frequency (mtDNA mutations present in colon only) measured by NGS. There was a significantly higher mutation frequency in the subjects >70 years (P = 0.0351, unpaired t-test). (<b>B</b>) Percentage of synonymous/polymorphic variants and non-synonymous mtDNA mutations which were of germline or early embryological origin compared to those which were somatic in adults <26 years of age and over 70 years of age. There was a significantly lower frequency of non-synonymous mtDNA mutations in the somatic mtDNA mutation groups compared to the germline or early embryological mtDNA mutation group (p = 0.041 Fisher's, exact test).</p

Analysis of mitochondrial DNA point mutation frequency with age by Random Mutation Capture (RMC).

<p>(<b>A</b>) Schematic diagram describing the RMC methodology. (<b>i</b>) Schematic diagram of the structure of the human colorectal crypt. (<b>ii</b>) Schematic diagram showing mtDNA isolation. Colonoscopic biopsies are homogenized and the mitochondrial fraction isolated by differential centrifugation. MtDNA is then prepared by phenol/chloroform extraction and quantified using real-time PCR (standard curve method). (<b>iii</b>) MtDNA is digested for 10 hours with <i>Taq1α</i>. PCR is then carried out over the restriction site. Only molecules with mutations in the restriction site are able to successfully amplify. (<b>iv</b>) Agarose gel showing PCR products from a typical RMC run. Each reaction contained ∼10000 target base pairs. 488 base pair bands show amplified mutated molecules (wells 4,6,13,16 and 20). The wild-type control well (Wt) shows complete digestion of wild-type DNA following PCR. (<b>v</b>) Example electropherograms showing mutations (asterisks) within the restriction site (highlighted in blue). (<b>B</b>) Frequency of all RMC detected mtDNA mutations in human colorectal mucosa (n = 207). There was no correlation between mtDNA mutation frequency and age (Pearson correlation = 0.127 (P = 0.07)). (<b>C</b>) Data from (<b>B</b>) presented on a log 10 scale to show the spread of the data. Note that the zero values cannot be displayed in this way therefore n = 175. (<b>D</b>) Frequency of all mtDNA mutations detected in human colonic mucosa, grouped by decade. Subjects were grouped as follows, 17–26 (n = 12), 27–36 (n = 19), 37–46 (n = 58), 47–56 (n = 51), 57–66 (n = 43), 67–77 (n = 23). There were no significant differences between any of the groups (P = 0.343, One Way ANOVA).</p