Figure 2.

Clonal contributions to hematopoietic lineages in peripheral blood over time

(A) Heatmaps depicting the top contributing clones for various PB lineages: T, B, Monocyte (Mo), Granulocyte (Gr), CD16⁺ NK, and CD56⁺NK over time (m: months post-transplantation) for each busulfan-conditioned animal (11021142, 10U004, H84D) and a representative TBI-conditioned animal (ZG66). Each row of the heatmap represents a unique barcoded clone. Each column is purified lineage sample at various time points. The heatmap for each animal was generated by selecting the top 10 most abundant clones contributing in each sample (designated by the asterisks in that sample) and plotting the abundance of these clones across all samples, with a color scale indicating the fractional contribution of each barcode to all barcodes retrieved from that sample. (B) Autocorrelation plots showing the Spearman correlation between all clonal contributions in each PB Gr sample and the previous time point. The y axis represents the autocorrelation R value. Samples with close to identical clonal contributions will have an autocorrelation close to 1 and very dissimilar clonal contributions will have an autocorrelation near 0. Samples from the three busulfan-conditioned animals (colored lines) and five TBI-conditioned animals (ZG66, ZH19, ZJ31, ZH33, ZK22, shown in black lines) were analyzed. (C) The top three graphs show the number of unique barcodes (y axis) in PB lineages over time (x axis) retrieved from each busulfan-conditioned animal (11021142, 10U004, and H84D). The bottom graph compares the number of unique barcodes in PB Gr between busulfan-treated animals (colored lines) and five TBI-conditioned animals (black lines). (D) The top three graphs display the Shannon diversity indices for PB lineages over time in busulfan-conditioned animals. The bottom plot directly compares the Shannon diversity indices for PB Gr between busulfan (colored lines) and five TBI (black lines)-conditioned animals. (E) The top three graphs show the cumulative number of unique barcodes retrieved from PB lineages in busulfan-conditioned animals. The bottom graph directly compares the number of cumulative barcodes retrieved from PB Gr samples between busulfan (colored lines), and five TBI (black lines)-conditioned animals. (F) Normalized unique barcodes retrieved from PB Gr of three busulfan (colored lines) and five TBI (black lines)-conditioned animals over time. Normalization: unique barcode counts divided by the vector marking level (NE vector VCN) (busulfan) or % GFP marking level (TBI) from the same sample. (G) Normalized cumulative barcode number retrieved from the longest time point PB Gr (11021142 at 18 months, 10U004 at 22.5 months, and H84D at 14 months) from busulfan-conditioned animals and equivalent time points from TBI-conditioned animals (ZG66 at 17 months, ZH19 at 15.5 months, ZH33 at 23 months, ZJ31 at 20 months, and ZK22 at 15.5 months). Normalization: cumulative barcode number from the last time point divided by the NE vector VCN or % GFP marking from the last time point sample. Unpaired t test with p < 0.05 is considered as significant.