Interrogation of clonal tracking data using barcodetrackR.

Clonal tracking methods provide quantitative insights into the cellular output of genetically labelled progenitor cells across time and cellular compartments. In the context of gene and cell therapies, clonal tracking methods have enabled the tracking of progenitor cell output both in humans receiving therapies and in corresponding animal models, providing valuable insight into lineage reconstitution, clonal dynamics, and vector genotoxicity. However, the absence of a toolbox for analysis of clonal tracking data has precluded the development of standardized analytical frameworks within the field. Thus, we developed barcodetrackR, an R package and accompanying Shiny app containing diverse tools for the analysis and visualization of clonal tracking data. We demonstrate the utility of barcodetrackR in exploring longitudinal clonal patterns and lineage relationships in a number of clonal tracking studies of hematopoietic stem and progenitor cells (HSPCs) in humans receiving HSPC gene therapy and in animals receiving lentivirally transduced HSPC transplants or tumor cells.

Clonal expansion and compartmentalized maintenance of rhesus macaque NK cell subsets.

Natural killer (NK) cells recognize and eliminate infected and malignant cells. Their life histories are poorly understood, particularly in humans, due to lack of informative models and endogenous clonal markers. Here, we apply transplantation of barcoded rhesus macaque hematopoietic cells to interrogate the landscape of NK cell production, expansion, and life histories at a clonal level long term and after proliferative challenge. We identify oligoclonal populations of rhesus CD56-CD16+ NK cells that are characterized by marked expansions and contractions over time yet remained long-term clonally uncoupled from other hematopoietic lineages, including CD56+CD16- NK cells. Individual or groups of CD56-CD16+ expanded clones segregated with surface expression of specific killer immunoglobulin-like receptors. These clonally distinct NK cell subpopulation patterns persisted for more than 4 years, including after transient in vivo anti-CD16-mediated depletion and subsequent regeneration. Profound and sustained interleukin-15-mediated depletion was required to generate new oligoclonal CD56-CD16+ NK cells. Together, our results indicate that linear NK cell production from multipotent hematopoietic progenitors or less mature CD56+CD16- cells is negligible during homeostasis and moderate proliferative stress. In such settings, peripheral compartmentalized self-renewal can maintain the composition of distinct, differentiated NK cell subpopulations.

Geographic clonal tracking in macaques provides insights into HSPC migration and differentiation.

The geographic distribution of hematopoiesis at a clonal level is of interest in understanding how hematopoietic stem and progenitor cells (HSPCs) and their progeny interact with bone marrow (BM) niches during regeneration. We tagged rhesus macaque autologous HSPCs with genetic barcodes, allowing clonal tracking over time and space after transplantation. We found marked geographic segregation of CD34+ HSPCs for at least 6 mo posttransplantation, followed by very gradual clonal mixing at different BM sites over subsequent months to years. Clonal mapping was used to document local production of granulocytes, monocytes, B cells, and CD56+ natural killer (NK) cells. In contrast, CD16+CD56- NK cells were not produced in the BM, and in fact were clonally distinct from multipotent progenitors producing all other lineages. Most surprisingly, we documented local BM production of CD3+ T cells early after transplantation, using both clonal mapping and intravascular versus tissue-resident T cell staining, suggesting a thymus-independent T cell developmental pathway operating during BM regeneration, perhaps before thymic recovery.

Quantitative stability of hematopoietic stem and progenitor cell clonal output in rhesus macaques receiving transplants.

Autologous transplantation of hematopoietic stem and progenitor cells lentivirally labeled with unique oligonucleotide barcodes flanked by sequencing primer targets enables quantitative assessment of the self-renewal and differentiation patterns of these cells in a myeloablative rhesus macaque model. Compared with other approaches to clonal tracking, this approach is highly quantitative and reproducible. We documented stable multipotent long-term hematopoietic clonal output of monocytes, granulocytes, B cells, and T cells from a polyclonal pool of hematopoietic stem and progenitor cells in 4 macaques observed for up to 49 months posttransplantation. A broad range of clonal behaviors characterized by contribution level and biases toward certain cell types were extremely stable over time. Correlations between granulocyte and monocyte clonalities were greatest, followed by correlations between these cell types and B cells. We also detected quantitative expansion of T cell-biased clones consistent with an adaptive immune response. In contrast to recent data from a nonquantitative murine model, there was little evidence for clonal succession after initial hematopoietic reconstitution. These findings have important implications for human hematopoiesis, given the similarities between macaque and human physiologies.

Clonal tracking of rhesus macaque hematopoiesis highlights a distinct lineage origin for natural killer cells.

Analysis of hematopoietic stem cell function in nonhuman primates provides insights that are relevant for human biology and therapeutic strategies. In this study, we applied quantitative genetic barcoding to track the clonal output of transplanted autologous rhesus macaque hematopoietic stem and progenitor cells over a time period of up to 9.5 months. We found that unilineage short-term progenitors reconstituted myeloid and lymphoid lineages at 1 month but were supplanted over time by multilineage clones, initially myeloid restricted, then myeloid-B clones, and then stable myeloid-B-T multilineage, long-term repopulating clones. Surprisingly, reconstitution of the natural killer (NK) cell lineage, and particularly the major CD16(+)/CD56(-) peripheral blood NK compartment, showed limited clonal overlap with T, B, or myeloid lineages, and therefore appears to be ontologically distinct. Thus, in addition to providing insights into clonal behavior over time, our analysis suggests an unexpected paradigm for the relationship between NK cells and other hematopoietic lineages in primates.