Increased B-cell activation in mesenteric lymph nodes of NOD^high mice.

<p>(<b>A</b>) Total cell counts in the indicated lymphoid organs of NOD^low (closed circles) vs. NOD^high (open circles) mice: MLN = mesenteric lymph nodes, PLN = pancreatic lymph nodes, ILN = inguinal lymph nodes. Analysis by 2-way ANOVA with p values given for significant colony differences. (<b>B</b>) Representative flow cytometry dot plots of CD69^high B cells isolated from mesenteric lymph nodes of NOD^low (left) and NOD^high (right) females at six weeks of age. (<b>C</b>) Frequencies of CD69^high cells in gated B220⁺ B cells from mesenteric lymph nodes (MLN) or spleens (SP) of NOD^low (closed circles) vs. NOD^high (open circles) female mice. Individual mice, means and significant p values (p < 0.05, by 2-way ANOVA) are shown.</p

Microbiological characterisation of female NOD^low and NOD^high mice.

<p>(<b>A</b>) Venn diagram depicting microorganisms consistently detected by routine health screens in sentinel mice from the NOD^low and NOD^high colonies (also includes the results of screening for SFB). None of the other viruses, bacteria or parasites that are routinely tested under FELASA guidelines [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181964#pone.0181964.ref030" target="_blank">30</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181964#pone.0181964.ref031" target="_blank">31</a>] were detected in either colony. (<b>B</b> and <b>C</b>) Quantification by qPCR (normalized to EUB) of <i>H</i>. <i>hepaticus</i> (<b>B</b>) and SFB (<b>C</b>) in the feces of individual NOD^low (black circles) and NOD^high (white circles) mice; horizontal bars represent means. Species-specific primers for the 16S RNA gene were used. (<b>D</b>) Metagenomic analysis of bacterial 16S rRNA genes from NOD^low and NOD^high females at 5 weeks of age. Different bacterial clades are color-coded, and log₂-fold mean differences between the detection frequencies in NOD^high and NOD^low mice shown on the y-axis; positive values show over-representation in the NOD^high colony. The dotted horizontal lines represent tenfold differences in either direction; significant colony differences (p < 0.05 after correcting for multiple comparisons) of tenfold or greater are shown as large squares with their names given. (<b>E</b>) Weights of individual age-matched NOD^low and NOD^high females (symbols as in <b>B/C</b>); means (horizontal lines) were compared by Student’s t test.</p

Exposure to a diabetogenic environment after the age of weaning does not modify T1D development in NOD^low mice.

<p>(<b>A</b>) Schematic illustration of continuous co-housing of NOD^low (grey outline) with NOD^high (black outline) mice from 3 weeks of age onwards. (<b>B</b>) Kaplan-Meier analysis of diabetes-free survival in females up to 30 weeks of age, comparing the original NOD^low (black circles, n = 85) and NOD^high (white circles, n = 45) colonies (cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181964#pone.0181964.g001" target="_blank">Fig 1A</a>) with NOD^low (full grey triangles) and NOD^high females (empty grey triangles) that were co-housed from three weeks of age. Despite being in a shared environment, the animals retained the disease incidence curves of their colonies of origin. (<b>C</b>) Diabetes-free survival in females from the original colonies (symbols as in <b>B</b>) was compared with that of NOD^low mice orally gavaged with fecal matter collected from 12-week-old female pre-diabetic NOD^high mice (closed grey diamonds) at three weeks of age and maintained in isolators. Fecal gavage did not raise T1D incidence over that in the NOD^low parental colony (p > 0.05, log rank test), despite successful transmission of <i>H</i>. <i>hepaticus</i> (cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181964#pone.0181964.s004" target="_blank">S3 Fig</a>).</p

T1D is induced in NOD^low mice by anti-PD-L1 antibody, but not by cyclophosphamide.

<p>(<b>A</b>) Normoglycaemic (pre-diabetic) NOD^low (black circles) and NOD^high (white circles) female mice were treated at 16–18 weeks of age with cyclophosphamide, and T1D development was monitored daily up to 25 days post intra-peritoneal injection. (<b>B</b>) Representative FACS plots for CD4 (x-axis) vs. FoxP3 (y-axis) on gated CD4⁺ T cells from NOD^low (left) and NOD^high (right) spleens. (<b>C</b>) Percentages (left) and absolute numbers (right) of FoxP3⁺ CD4 T cells from pancreatic lymph nodes of NOD^low (full circles) and NOD^high (white circles) mice. Statistical analysis was performed using Student’s t test. (<b>D</b>) Normoglycaemic (pre-diabetic) NOD^low females were treated with anti- (α-) PD-L1 (clone MIH5, grey circles) or PBS (black circles) at 12–14 weeks of age, and T1D development was monitored daily up to 25 days post intra-peritoneal injection. In (A) and (D), diabetes-free survival was compared between groups by Kaplan-Meier analysis and log rank test.</p

Characterisation of NOD^low and NOD^high colonies.

<p>(<b>A</b>) Kaplan-Meier survival curves showing diabetes-free survival up 30 weeks of age in 85 NOD^low (black circles) and 45 NOD^high (white circles) female mice. Incidence curves were compared by the log-rank test. From [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181964#pone.0181964.ref028" target="_blank">28</a>] with permission; Copyright 2013, The American Association of Immunologists, Inc. (<b>B</b>) and (<b>C</b>) Percent of islets exhibiting no insulitis, peri- or intra-insulitis in six NOD^low vs. eight NOD^high 6-week-old female mice (<b>B</b>), or in seven NOD^low vs. five NOD^high 30-week-old female mice (<b>C</b>). Each bar represents analysis of three pancreatic sections from an individual mouse. (<b>D</b>) Examples of H&E stained pancreatic sections from 30-week-old NOD^low females showing, on the left, two islets without insulitis and, on the right, one islet each with peri-insulitis (*) and intra-insulitis (**).</p

Survival plot with futility boundary for TKM-130803 recipients.

<p>The red line denotes the futility boundary. The points and dashed line denote the number of survivors at day 14 plotted against the number of day 14 reports.</p

Box and whisker plot of vital signs in TKM-130803 recipients, before, during, and after TKM-130803 infusions.

<p>Heart rate, respiratory rate, mean arterial blood pressure, and tympanic temperature in patients administered TKM-130803 at the following time points: immediately prior to TKM-130803 infusion (PRE), during the infusion, immediately at the end of the infusion (END), and at 1, 2, 4, and 8 h after the end of the infusion. The middle line shows the median value, the box shows the interquartile range, and the whiskers spread from the lower to the upper adjacent values. Outside values, that is, observations that are larger/smaller than the upper/lower adjacent values, are shown as circles.</p

Patient screening and enrolment.

<p>*One patient did not give consent. One patient was not competent to give consent, and a suitable proxy to provide consent could not be identified within inclusion time limits. **Two patients who died within 48 h of admission were excluded from the primary outcome analysis, as specified in the protocol.</p

Baseline demographic and clinical characteristics of trial population.

<p>Baseline demographic and clinical characteristics of trial population.</p

Ebola virus RT-PCR cycle threshold values and RNA copies/ml over time.

<p>Top row: TKM-130803 recipients. Bottom row: Observational patients. RT-PCR Ct upper limit of quantitation (LOQ) = 40. RT-qPCR lower limit of quantitation = 1,000 genome copies. The Ebola virus RT-qPCR quantification is expressed as the number of genome copies/millilitre of plasma. Black diamonds denote results for survivors. Grey circles denote results for non-survivors.</p