Clonal analysis of in vivo hematopoiesis

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We have utilized molecular techniques to gain new insights into the behavior of hematopoietic stem and progenitor cells (HSPCs) in vivo. We have continued active development and utilization of lentiviral "barcoding" with high-diversity 31-35 base pair genetic barcodes introduced into target cells in order to study in vivo hematopoiesis in the non-human primate model. Our collaborator Rong Lu first devised this very powerful approach and applied it to study murine hematopoiesis. We have now transplanted 27 macaques with barcoded autologous CD34+ cells, and have been able to track hematopoietic output from thousands of individual HSPCs over time (up to 8 years) and in multiple lineages in a quantitative and highly reproducible manner. We are currently modifying our barcoding strategy to allow simultaneous retrieval of the barcode and single cell RNASeq in order to begin to be able to connect our cell fate findings (barcode-defined ontogeny) together with "state" characterization in terms of gene expression and we hope epigenetic marks at a single cell level. The new vector has the barcode placed in a position that allows high expression and retrieval via 10X and other standard single cell RNASeq platforms. Preliminary single cell RNASeq experiments on NK cells before and after CMV infection document informative clustering and new insights into the relationship between NK subsets, as indicated by RNA velocity approaches. We have already made a number of important and novel discoveries, including a surprising life history for mature NK cells, showing that the major fraction of circulating mature cytotoxic NK cells(CD16+CD56-) do not share barcodes with B, T or myeloid cells or their putative precursor CD56brightCD16neg NK cells, even 80 months post-transplant. In vitro and murine models have not previously been able to shed light on NK cell lineage relationships. These circulating NK cells consist of massively-expanded and oligoclonal populations, waxing and waning in a pattern suggesting responses to specific environmental cues such as viral infection or viral reactivation. Our data provides the first direct demonstration of clonal NK responses, providing insights into possible mechanisms for NK memory. We used differentially-expressed KIR surface molecules, previously linked to NK viral and tumor responses, to sort NK cells expressing different KIR, and documented clonal segregation within these specific KIR-expressing NK populations. This is the first direct demonstration of the generation and persistence of clonal populations of NK cells with specific receptor characteristics, presumably epigenetically-maintained. With in vivo NK depletion based on CD16 expression, the same expanded clones arise again, without recruitment from highly polyclonal HSPC but with recruitment from a residual highly proliferative CD16dim NK subset. This work was published recently. We hypothesize that these clones might be generated in the context of a response to CMV, based on correlative data in human transplantation and blood donor studies, and are testing this via barcoded transplantation in CMV negative macaques, showing specific clonal changes occur in the mature NK cell populations, published this FY. We are now working to understand NK response to vaccinations and other challenges, and the involvement of the macaque equivalent of HLA-E in specific memory-like NK responses. We have recently completed a project comparing the clonal behavior of young versus aged HSPC, demonstrating marked differences in clonal patterns, specifically very delayed contributions from multilineage clones with persistence of unilineage contributions, in both myeloid, B and T cell lineages, in contrast to murine models suggesting accumulation of only myeloid-biased clones. In the oldest animal, we observed clonal dropout and clonal expansions, potentially providing a model of human "CHIP" associated with aging (clonal hematopoiesis of indeterminate prognosis). This finding has recently been linked to the presence of the same panel of somatic mutations found in humans with aging, ie "age-related clonal hematopoiesis", in contrast to the lack of these mutations in murine models. We have continued to analyze clonal patterns following engraftment of ex vivo expanded HSPC, CAR-T cells and NK cells, topics of translational and clinical importance. We have also begun to compare the impact of specific conditioning regimens on clonal patterns following HSPC engraftment, noting differences between TBI and busulfan, and extending the studies to antibody-mediated conditioning.