Volume 21, Issue 2, Pages 476-484 (February 2013) Quality Controls in Cellular Immunotherapies: Rapid Assessment of Clinical Grade Dendritic Cells by Gene Expression Profiling  Luciano Castiello, Marianna Sabatino, Yingdong Zhao, Barbara Tumaini, Jiaqiang Ren, Jin Ping, Ena Wang, Lauren V Wood, Francesco M Marincola, Raj K Puri, David F Stroncek  Molecular Therapy  Volume 21, Issue 2, Pages 476-484 (February 2013) DOI: 10.1038/mt.2012.89 Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Contributions of manufacturing, intraindividual and interindividual variability and assay repeatability to the consistency of clinical grade monocyte-derived mature dendritic cells (mDCs) and the starting monocytes. (a) The bar chart shows the 1-intraclass correlation coefficient (ICC) values in different groups of samples based on the analysis of the entire transcriptome dataset. Light gray bars represent mDCs and the dark gray bars monocytes. (b) Similarity matrix analysis of all 25 samples tested based on the entire gene expression profile of each sample. Samples are ordered according to unsupervised hierarchical clustering. Yellow lines represent samples assessing within assay variability, blue lines samples assessing between assay variability, red lines samples assessing manufacturing-related variability, green lines samples assessing intraindividual variability and purple lines samples assessing interindividual variability. The similarity matrix is colored according to Pearson correlation coefficient and the scale is indicated. Molecular Therapy 2013 21, 476-484DOI: (10.1038/mt.2012.89) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Genes contributing to mature dendritic cell (mDC) product variability: genes with the greatest assay-adjusted manufacturing, intradonor and interdonor variability. (a,b) Three-dimensional plot of the 877 genes whose expression was most variable in the DC gene expression data set (one percentile) in at least one factor (manufacturing, intradonor and interdonor). Each genes is represented according to its assay-adjusted variances in the DC dataset: manufacturing-related variability (x-axis), intraindividual-related variability (z-axis) and interindividual-related variability (y-axis). Genes whose expression was most variable in more than one factor are represented in green. Genes most variable in mDC manufacturing samples are shown in blue, genes most variable in intraindividual samples are shown in purple and genes most variable in interindividual samples are shown in orange. For each factor, ellipsoids are depicted to include 2 SD from the mean value of each of the three factors. Each panel shows a different perspective. (c,d) Three-dimensional plots of the expression of the same 877 genes in monocytes rather than in DCs. Genes are represented according to the assay-adjusted variances in the monocyte dataset: manufacturing-related variability (x-axis), intraindividual-related variability (z-axis), and interindividual-related variability (y-axis). Genes whose expression was most variable in more than one factor in DC dataset are shown in green, genes most variable in DC manufacturing samples are shown in blue, genes most variable in DC intraindividual samples are shown in purple, and genes most variable in DC interindividual samples are represented in orange. For each factor, ellipsoids are depicted to include 2 SD from the mean value of each factor. Molecular Therapy 2013 21, 476-484DOI: (10.1038/mt.2012.89) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Genes identified as candidate markers and their properties. (a) Flowchart describing the approach used to select candidate biomarkers. (b) Similarity matrix of the 291 genes induced reproducibly in mature dendritic cells (mDCs) compared to both monocytes and immature DCs with P value <0.001, FDR <0.005, and fold-change >5. Pearson correlation values were calculated based upon mDCs gene expression levels. The genes are sorted according to unsupervised clustering in order to reveal gene correlation networks in the mDCs. (c) The 291 genes are plotted three-dimensionally according to the assay-adjusted variances: manufacturing-related variability (x-axis), intraindividual-related variability (z-axis), and interindividual-related variability (y-axis). Genes included in the first decile according to the index of variability are represented in green, genes in the tenth decile in red and the others in grey. (d) Pearson correlations between the level of expression of genes in the tenth decile of the index of variability and the concentrations of selected cytokines measured in the culture media. Both genes and cytokines are ordered according to unsupervised hierarchical clustering. Molecular Therapy 2013 21, 476-484DOI: (10.1038/mt.2012.89) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Analysis of the variability of expression of the 291 candidate mature dendritic cell (mDC) markers in a clinical dataset. The expression of 291 candidate genes were measured in a clinical dataset made up of 80 different mDC products (14 patients, between 2 and 8 products were manufactured for each patient). The Index of Variability was calculated for each gene. The genes included in the first decile according to their index of variability calculated in the initial mDC dataset are shown by the green bar, genes in the tenth decile by the red bar and the others by the grey bar. The boxes indicate the 25 and 75% percentiles, and whiskers indicate the 10 and 90% percentiles. Molecular Therapy 2013 21, 476-484DOI: (10.1038/mt.2012.89) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions