Dynamics of HSPC repopulation in nonhuman primates revealed by a decade-long clonal-tracking study

Abstract

In mice, clonal tracking of hematopoietic stem cells (HSCs) has revealed variations in repopulation characteristics. However, it is unclear whether similar properties apply in primates. Here, we examined this issue through tracking of thousands of hematopoietic stem and progenitor cells (HSPCs) in rhesus macaques for up to 12 years. Approximately half of the clones analyzed contributed to long-term repopulation (over 3-10 years), arising in sequential groups and likely representing self-renewing HSCs. The remainder contributed primarily for the first year. The long-lived clones could be further subdivided into functional groups contributing primarily to myeloid, lymphoid, or both myeloid and lymphoid lineages. Over time, the 4%-10% of clones with robust dual lineage contribution predominated in repopulation. HSPCs expressing a CCR5 shRNA transgene behaved similarly to controls. Our study therefore documents HSPC behavior in a clinically relevant model over a long time frame and provides a substantial system-level data set that is a reference point for future work.

Figure 1. Longitudinal EGFP Marking in Four Rhesus Macaques

The levels of EGFP expression in granulocytes (Grans; green), monocytes (Monos; brown), lymphocytes (Lymphs; red), red blood cells (RBC; purple), and platelets (PLT; blue) were assayed by flow cytometry, based on the forward scatter versus side scatter plot. PLTs were also gated on CD41+ cells. Data represent longitudinal follow-up for 38 to 145 months post-transplant, depending on the test animal. Data gathered during the ganciclovir IV treatment (see arrow at 23 months post-transplant) and within the two week period after leukapheresis have been excluded. See Table S1 for more details.

Figure 2. Polyclonal Repopulation with Highly Variable Detection Frequencies Among Clones

(A) The total number of unique vector integrants recovered in each animal [Uniq.VI (y-axis) at all time points and for all cell types (see Table 1)] showed a linear correlation with the total number of EGFP+ cells transplanted at the beginning of the experiment (x-axis). (B) The stacked area charts show the average detection frequencies of individual QVI clones in serial peripheral blood analysis, with the most prevalent QVI clone located at the bottom. Total peripheral blood cells (PBC) in animal 95E132, and both granulocytes (Grans) and peripheral blood mononuclear cells (PBMCs) in animals 2RC003, RQ5427, and RQ3570 are shown. The left vertical axis shows the cumulative contribution to overall repopulation by individual QVI clones. The total number of QVI clones recovered in each animal appears beside a black arrow at the right of each chart. A large number (43–71%) of QVI clones (denoted by blue arrows) were detected at an extremely low average frequency of <0.0002, contributing to < 7% of total blood repopulation over the entire course of observation, except in animal RQ3570, where the contribution was <23%. By contrast, the 5% most frequently detected clones (denoted by red arrows) contributed to an average of 49 – 72% of total blood repopulation, depending on the animal and cell type. The mean and standard deviation (±STDEV) of QVIs are noted at the bottom of each chart. See Fig. S1 and Fig. S2 for more details.

Figure 3. Repopulation Kinetics of HSPC Clones

(A) Sequential Expansions of HSPC Clones Over Time. Major clonal kinetics patterns in serial PBC (in animal 95E132) and PBMC and Grans (in animals 2RC003, RQ5427, and RQ3570) were derived using average linkage hierarchical clustering based on clonal frequency profiles over time. The relative frequencies of QVI clones at different time points (shown in months at the top of each chart) appear in a white–black–yellow color scheme, with white denoting zero frequency and yellow the highest frequency (see the numbers at the bottom of each chart). QVIs were grouped into 5 – 8 clusters (denoted by different colored arrows), depending on animal and cell type, using the WGCNA package (Langfelder and Horvath, 2008). All clusters were clearly separable, with a significance level (|Zsummary.qual|) of >10 (Langfelder and Horvath, 2008), except for the cluster-1 in the 2RC003 PBMC data set (only 9 QVI clones). Results showed that different groups of QVI clones, beginning with Cluster 1, expanded sequentially over time. See Fig. S3 for more details. (B) Repopulation of Long-term and Short-term QVI Clones. Long-term (LT-QVIs) and short-term QVI clones (ST-QVIs) were segregated based on the presence of these clones in blood lineages isolated at 116–117 months (95E132), 70–71 months (2RC003), 63–64 months (RQ5427), or 36–37 months (RQ3570) months post-transplant. The relative contribution (y-axis) of LT-QVIs (orange) and ST-QVIs (black) in serial PBC (95E132) or Grans/PBMCs (2RC003, RQ5427, and RQ3570) are shown over time. See Fig. S4 for more details.

Figure 4. Functional Subtypes of Long-term Repopulating Clones

(A–D) Myeloid-biased (My-bi), balanced (Bal), and lymphoid-biased (Ly-bi) subtypes among the LT-QVIs are shown for each animal. The My-bi (blue), Bal (magenta), and Ly-bi (green) groups were determined based on myeloid (GM): lymphoid (BT) cell ratios of ≥ 3:1, between 3:1 and 1:3, and ≤ 1:3, respectively. In each Figure i), the frequency profiles of individual QVIs are shown in a white-black-yellow color scheme for lymphoid cells (BT: CD4+, CD8+, and CD20+ cells), myeloid cells (GM: CD18+ and CD14+ cells) and mPB CD34+ cells near the endpoint, and serial PBC(95E132) or Grans/PBMC (2RC003, RQ5427, and RQ3570). The range of clones detected in CD34+ cells is indicated by a red arrow. The numbers of QVI clones appear in parentheses. In Figures ii) and iii), the ternary diagrams show the lineage output potentials of each clone (indicated by the positions of the circles relative to the points of the triangle). The relative frequencies of clones (indicated by the size of the circles) in mature lineages (ii) are compared to those of identical clones in mPB CD34+ cells (iii). (E) The total contribution to blood repopulation by My-bi, Bal, and Ly-bi QVIs (upper chart) and the average contribution by individual QVIs within each group (lower chart) are shown over time. Error bars indicate standard errors. See Fig. S5 and Fig. S6 for more details.

Figure 5. Summary Diagrams Showing the Relative Proportions of HSPC Subpopulations and Their Contributions to Blood Repopulation Over Time

(A) Summary Diagrams. Figure (i) shows the relative proportions of long-term repopulating cells within the transplanted CD34+ cell pool. Figure (ii) shows the contributions of short-term clones (ST-Clones) and the three subtypes of long-term clones over time. (B) These charts show variable repopulation kinetics among the clones within each subpopulation. The numbers of My-bi, Bal, Ly-bi, and ST-QVI clones in the various kinetics clusters are indicated by bar graphs. The clusters and color codes for each cluster are the same as those in Fig. 3A. (C) The bar graphs show the proportions of My-bi, Bal, and Ly-bi clones present in the CD34+ HSPC pool near the endpoint (denoted by red bars) relative to the total detected in the mature lineages (denoted by black bars) (see Fig. 4A–D(i) for individual clones in mature lineages and CD34+ cells). The long-term HSPC population is relatively enriched for the Bal and the My-bi clones. (D) Comparison of clonal compositions across cell types. The relative frequencies of individual clones are compared between cell types. The Pearson correlations coefficient value (r) is indicated by a rainbow color scheme.

Figure 6. Vector Integration Site Bias Conserved Across Animals and Functional Subgroups

(A–B) Genomic VIS hot-spots in four test animals are shown in (A) in comparison with those of published (pub) and freshly infected naïve CD34+ cells (acute). Hot-spots in My-bi, Ly-bi, Bal, and ST-QVIs (four animals combined) are shown in (B). Each row shows the complete VIS pattern for one data set, where data set names are indicated on the y-axis, and the x-axis gives the genomic location of the VIS in Mb units. Vertical black bars indicate chromosomal boundaries. Color definitions were assigned for each data set independently, based on relative VIS density. Hot-spot regions are colored in red. One of the strongest hot-spots was located at chromosome (chr) 4 (29.94–34.01 Mb) hosting about 4.22% of the total VIS with a density of about 69 VIS per Mb. The second major hot-spot was defined as a 4.99 Mb region at rhesus chr14 (5.98–10.97 Mb) containing 5.54% of VIS at a density of 73 VIS/Mb. Hot-spots among subtypes were significantly correlated, with overlap ranging between 29–79% among subtypes and overlap p-values (based on a Fisher’s exact test for hot-bin overlap) ranging from 1.7×10⁻⁷ to 3.3×10⁻²². See Table S3 and Fig. S7 for more details.