Clonal and imaging analyses of in vivo hematopoiesis, immune cell ontogeny and adoptive cell therapies

National Heart, Lung, And Blood Institute

Investigators

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Abstract

We have utilized molecular techniques to gain new insights into the behavior of hematopoietic stem and progenitor cells (HSPCs) and immune cells 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 30 macaques with barcoded autologous CD34+ cells, and have been able to track hematopoietic output from thousands of individual HSPCs over time for up to 8 years and in multiple lineages in a quantitative and highly reproducible manner. We have recently modified our barcoding strategy to allow simultaneous retrieval of the barcode and single cell RNASeq/ATACSeq 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. The first macaque has now been transplanted with this novel vector, and analyses focusing on emergence of expanded NK cell clones are in progress. 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. We have analyzed tissue-resident NK cells, shown to be important for function NK memory in murine and monkey adoptive transfer studies. We have discovered markedly expanded NK clones in tissues including spleen, lung, gut, and liver, with expanded clones shared across these tissues, suggesting specific homing to all tissues from the blood following expansion. We have also identified a minor phenotypic population in the blood of CD56negCD16neg NK cells that contain these expanded clones, suggesting these cells may be precursors for these tissue resident NK, and documented expression of chemokine-responsive homing molecules on these cells. We hypothesize that expanded NK cell clones might be generated in the context of a response to CMV, based on correlative data in human transplantation and blood donor studies, and we tested this hypothesis via barcoded transplantation in CMV negative macaques, showing specific clonal changes occur in the mature NK cell populations following experimental CMV infection. Single cell RNASeq experiments on NK cells before and after CMV infection document informative clustering and new insights into the relationship between NK subsets and the response of NK cells to CMV. Adaptive/memory NK responses to CMV have been linked to interactions involving HLA-E. We are now working to understand NK response to a vaccine based on a rhesus CMV platform previously shown to confer uniquely potent protection from SIV by our collaborator Louis Picker. We are investigating whether NK clonal expansion arise following vaccination in barcoded macaques, dependent on HLA-E and explaining at least in part the unique response to this promising vaccine platform. 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 compared the impact of specific conditioning regimens on clonal patterns following HSPC engraftment, noting differences between TBI and busulfan, and are now actively extending the studies to antibody-mediated conditioning, achieving high level engraftment of barcoded cells following anti-CD345 conditioning, and more surprisingly, tolerance to foreign trasngenes. We are also applying tracking approaches to the adoptive transfer of natural killer cells and CAR-T cells in the macaque model. We have analyzed the clonal composition of ex vivo expanded NK cells and documented expanded putative adaptive clones are maintained and expanded in vitro, in collaboration with the Richard Childs laboratory. We plan to study the clonal patterns and persistence of NK populations following adoptive transfer. We have also cloned the sodium-iodide symporter gene (NIS) into a CAR lentiviral vector, which will allow in vivo imaging and tracking of adoptively-transferred CAR-T cells, and analysis of the impact of various co-stimulatory domains. This work is in progress in murine xenograft models.