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Clonal tracking methods provide quantitative insights into the cellular output of genetically labeled 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. The R package barcodetrackR facilitates the analysis and visualization of clonal tracking data, and it includes a graphical user interface to allow researchers without programming experience to utilize various quantitative tools.

Ex vivo hematopoietic stem and progenitor cell (HSPC) expansion platforms are under active development, designed to increase HSPC numbers and thus engraftment ability of allogeneic cord blood grafts or autologous HSPCs for gene therapies. Murine and in vitro models have not correlated well with clinical outcomes of HSPC expansion, emphasizing the need for relevant pre-clinical models. Our rhesus macaque HSPC competitive autologous transplantation model utilizing genetically barcoded HSPC allows direct analysis of the relative short and long-term engraftment ability of lentivirally transduced HSPCs, along with additional critical characteristics such as HSPC clonal diversity and lineage bias. We investigated the impact of ex vivo expansion of macaque HSPCs on the engineered endothelial cell line (E-HUVECs) platform regarding safety, engraftment of transduced and E-HUVEC-expanded HSPC over time compared to non-expanded HSPC for up to 51 months post-transplantation, and both clonal diversity and lineage distribution of output from each engrafted cell source. Short and long-term engraftment were comparable for E-HUVEC expanded and the non-expanded HSPCs in both animals, despite extensive proliferation of CD34+ cells during 8 days of ex vivo culture for the E-HUVEC HSPCs, and optimization of harvesting and infusion of HSPCs co-cultured on E-HUVEC in the second animal. Long-term hematopoietic output from both E-HUVEC expanded and unexpanded HSPCs was highly polyclonal and multilineage. Overall, the comparable HSPC kinetics of macaques to humans, the ability to study post-transplant clonal patterns, and simultaneous multi-arm comparisons of grafts without the complication of interpreting allogeneic effects makes our model ideal to test ex vivo HSPC expansion platforms, particularly for gene therapy applications., Graphical Abstract, The rhesus macaque barcoded autologous competitive repopulation preclinical transplantation model was utilized to study the engraftment and safety of hematopoietic stem and progenitor cells expanded in co-culture with E4ORF-1 gene transduced human umbilical vein endothelial cell as compared to non-expanded cells, documenting polyclonal long-term multilineage hematopoiesis.

The anisotropic ductility of a direct laser deposited 17-4 PH cubic part was investigated. Anisotropic elongations in the specimens from varied surfaces of the part were obtained: ~ 6.2%, ~ 1.5%, and ~ 4.5% in XY, YZ, and XZ samples, respectively. Furthermore, various orientations of ferrite were found in different specimens, taking the loading direction as reference. A finite element analysis depending on actual microstructures and dislocation density revealed that the orientation of ferrite caused the ductility anisotropy. The orientation of ferrite affected its plastic deformability and the deformation compatibility between phases during the uniaxial loading. The ferrite parallel to the tensile direction in the YZ sample had the worst deformability and induced severe strain localization and stress triaxiality, which resulted in inferior ductility. The ferrite perpendicular to the tensile direction showed the best deformability, whereas strain localization remained intense in the XZ sample owing to the unmatched deformability of martensite. The inclined ferrite in the XY sample exhibited moderate deformability and was found to enhance the plastic flow of martensite, leading to the best deformation compatibility and ductility.

Macrophages are critical orchestrators of tissue immunity, from the initiation and resolution of antimicrobial immune responses to the subsequent repair of damaged tissue. Murine studies have demonstrated that tissue-resident macrophages are comprised of a mixture of yolk sac-derived cells that populate the tissue before birth and hematopoietic-derived replacements that are recruited in adult tissues both at steady-state and in increased numbers in response to tissue damage or infection. While resident macrophages in some murine tissues are readily turned over and replaced, other tissues primarily retain their original, yolk sac-derived complement of macrophages. How this translates to species that are constantly under immunologic challenge, such as humans, is unknown. To understand the ontogeny and longevity of tissue-resident macrophages in nonhuman primates (NHPs), we employ a model of autologous HSPC transplantation with HSPCs genetically modified to express individual bar-coded markers allowing for subsequent analysis of clonal differentiation of leukocyte subsets. We study the contribution of HSPC to tissue-macrophages and their clonotypic profiles relative to leukocyte subsets across tissues and peripheral blood. We also use in vivo bromodeoxyuracil infusions to monitor tissue macrophage turnover in NHPs at steady state. We find that, in all anatomic sites we studied, HSPC contribute to tissue-resident macrophage populations. Their clonotypic profile is dynamic and overlaps significantly with the clonal hierarchy of contemporaneous monocytes in peripheral blood. Moreover, we find evidence of tissue-macrophage turnover at steady state in otherwise unmanipulated NHPs. These data demonstrate the life span of tissue-resident macrophages can be limited and they can be replenished from HSPCs. Thus, in primates not all yolk-sac derived tissue-macrophages survive for the duration of the host’s life.

Recent advances in high-throughput genomics have enabled the direct tracking of outputs from many cell types, greatly accelerating the study of developmental processes and tissue regeneration. The capacity for long-term self-renewal with multilineage differentiation potential characterises the cellular dynamics of a special set of developmental states that are critical for maintaining homeostasis. In haematopoiesis, the archetypal model for development, lineage-tracing experiments have elucidated the roles of haematopoietic stem cells to ongoing blood production and the importance of long-lived immune cells to immunological memory. An understanding of the biology and clonal dynamics of these cellular fates and states can provide clues to the response of haematopoiesis to ageing, the process of malignant transformation, and are key to designing more efficacious and durable clinical gene and cellular therapies.

The classical model of hematopoietic hierarchies is being reconsidered on the basis of data from in vitro assays and single cell expression profiling. Recent experiments suggested that the erythroid lineage might differentiate directly from multipotent hematopoietic stem cells / progenitors or from a highly biased subpopulation of stem cells, rather than transiting through common myeloid progenitors or megakaryocyte-erythrocyte progenitors. We genetically barcoded autologous rhesus macaque stem and progenitor cells, allowing quantitative tracking of the in vivo clonal output of thousands of individual cells over time following transplantation. CD34+ cells were lentiviral-transduced with a high diversity barcode library, with the barcode in an expressed region of the provirus, allowing barcode retrieval from DNA or RNA, with each barcode representing an individual stem or progenitor cell clone. Barcode profiles from bone marrow CD45-CD71+ maturing nucleated red blood cells were compared with other lineages purified from the same bone marrow sample. There was very high correlation of barcode contributions between marrow nucleated red blood cells and other lineages, with the highest correlation between nucleated red blood cells and myeloid lineages, whether at earlier or later time points post transplantation, without obvious clonal contributions from highly erythroid-biased or restricted clones. A similar profile occurred even under stressors such as aging or erythropoietin stimulation. RNA barcode analysis on circulating mature red blood cells followed over long time periods demonstrated stable erythroid clonal contributions. Overall, in this nonhuman primate model with great relevance to human hematopoiesis, we documented continuous production of erythroid cells from multipotent, non-biased hematopoietic stem cell clones at steady-state or under stress.

Lentiviral vectors (LVs) are used for delivery of genes into hematopoietic stem and progenitor cells (HSPCs) in clinical trials worldwide. LVs, in contrast to retroviral vectors, are not associated with insertion site-associated malignant clonal expansions and, thus, are considered safer. Here, however, we present a case of markedly abnormal dysplastic clonal hematopoiesis affecting the erythroid, myeloid, and megakaryocytic lineages in a rhesus macaque transplanted with HSPCs that were transduced with a LV containing a strong retroviral murine stem cell virus (MSCV) constitutive promoter-enhancer in the LTR. Nine insertions were mapped in the abnormal clone, resulting in overexpression and aberrant splicing of several genes of interest, including the cytokine stem cell factor and the transcription factor PLAG1. This case represents the first clear link between lentiviral insertion-induced clonal expansion and a clinically abnormal transformed phenotype following transduction of normal primate or human HSPCs, which is concerning, and suggests that strong constitutive promoters should not be included in LVs.

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 cellular therapies and in corresponding animal models, providing valuable insight into lineage reconstitution, clonal dynamics, and vector genotoxicity. However, the absence of a toolbox by which to interrogate these data has precluded the development of standardized analytical frameworks within the field. Thus, we developed barcodetrackR, an R package that provides users with tools for the analysis and visualization of clonal dynamics across time and cellular compartments in clonal tracking experiments. Here, we demonstrate the utility of barcodetrackR in exploring longitudinal clonal patterns and lineage relationships in the context of 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.

Age-associated changes in hematopoietic stem and progenitor cells (HSPCs) have been carefully documented in mouse models but poorly characterized in primates and humans. To investigate clinically relevant aspects of hematopoietic aging, we compared the clonal output of thousands of genetically barcoded HSPCs in aged vs young macaques after autologous transplantation. Aged macaques showed delayed emergence of output from multipotent (MP) clones, with persistence of lineage-biased clones for many months after engraftment. In contrast to murine aging models reporting persistence of myeloid-biased HSPCs, aged macaques demonstrated persistent output from both B-cell and myeloid-biased clones. Clonal expansions of MP, myeloid-biased, and B-biased clones occurred in aged macaques, providing a potential model for human clonal hematopoiesis of indeterminate prognosis. These results suggest that long-term MP HSPC output is impaired in aged macaques, resulting in differences in the kinetics and lineage reconstitution patterns between young and aged primates in an autologous transplantation setting.

By virtue of their direct cytotoxicity to transformed and virus infected cells, Natural Killer (NK) cells play crucial roles in immunity. NK cells modulate and coordinate innate and adaptive responses through the release of chemokines and cytokines. Although NK cells are endowed only with germ-line encoded receptors, evidence has been accumulating, that subsets of NK cells can bestow adoptively transferable, long-lasting and antigen-specific immune responses to certain haptens and viruses. Growing evidence suggests that adaptive immune responses lie on a spectrum. Rechallenge of cells, canonically belonging to the innate immune system, can result in enhanced responsiveness - a process termed 'trained immunity' and thought to be maintained by epigenetic and metabolic reprogramming. In previous work, our lab studied the role of NK cell responses to rhesus cytomegalovirus (rhCMV) in a genetic barcoding model. We found that new clones arose in the CD16 + NK compartment after primary rhCMV infection. There was rapid clearance without the emergence of new clones in subsequent rechallenge with rhCMV. In this study we used 3'-end single cell RNA-seq (3'-scRNA-seq) with CITE-seq to profile NK and T cells from an initially CMV-naïve rhesus macaque (RM) at four time points before and after primary and secondary infections with rhCMV. We immunophenotypically sorted NK and T cells from peripheral Blood (PB) samples at 'baseline', 30 days after initial rhCMV infection ('primary infection'), ca. 500 days after initial rhCMV infection ('steady state') and 10 days after rmCMV reinfection ('secondary infection'). Alongside the PB samples at 'steady state' and after 'secondary infection', we also sorted NK and T cells from lymph nodes (LN). We applied CD16 and CD56 CITE-seq antibodies to NK cells from all samples; NK cells from the 'steady state' and 'secondary infection' samples were also labeled with CX3CL1 CITE-seq antibodies. We multiplexed NK and T cells from each time point in 4:1 ratios before preparing 3'-scRNA-seq libraries. We used scanpy and scvi-tools as well as custom python code to demultiplex NK from T cells, harmonize 3'-scRNA-seq with CITE-seq data and integrate the 6 different samples. We used scvelo and cellrank to compute RNA velocities and infer trajectories, respectively. We obtained a total of 35,523 high-quality cells. We identified 20 clusters of NK and T cells, on the basis of community detection via the Leiden algorithm. All clusters contained cells from both tissue sources. The 4 clusters characterized by expression of CD56 exhibit higher expression of KLRC1 (protein: NKG2A), IL7R and the transcription factors LEF1 and MYC. The 8 clusters of CD16 + cells are distinguished by high expression of the transcription factors ZEB2 and TBX21/T-BET, cytotoxicity markers, GZMB and PRF1, and activating receptors, KLRC2 (protein: NKG2C), KLRC3 (protein: NKG2E) and NCR3 (protein: NKp30). An adaptive population of NK cells is identified on the basis of high KLRC2 and low FCER1G expression. We analyzed changes in the proportions of cells in each cluster of the time course of CMV infection using a binomial generalized linear model. Clusters associated with proliferation and acute inflammation were increased in proportion after primary rhCMV infection; the proportion of the adaptive population did not significantly change during the acute phase of primary infection but increased markedly by the later 'steady state' samples. RNA velocity and inferred developmental trajectories suggest transitions between the adaptive, proliferating CD16 + and mature effector subsets; the predominant path into the adaptive population occurring from the proliferating CD16 + subset after primary infection. There is a notable paucity of inferred transitions between the CD56 + and CD16 +subpopulations under all the experimental conditions we observed. We have characterized the single cell transcriptional states and dynamics of RM NK cells in response to rhCMV infection. We focus on a subset transcriptionally resembling a previously identified subset with adaptive function and find it arises from a proliferating population of effector cells after primary infection. This may be analogous to the dedifferentiation of effector CD8 T cells into memory T cells proposed by Youngblood et al. Confirmatory experiments to analyze the reconstitution of the CD16 + compartment after treatment with a depleting antibody are on-going. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Recent phenotypic, functional and transcriptomic analyses of natural killer (NK) cells in human and animals have established the presence of tissue resident NK(trNK) cells with specific characteristics and a central role in NK memory. The lack of endogenous clonal markers on NK cells impedes understanding the clonal genesis of trNKs. Transplantation of lentivirally-barcoded autologous hematopoietic stem and progenitor cells (HSPCs) has allowed tracking of NK cells at a clonal level in rhesus macaques (RM). We reported large KIR-restricted clonal expansions of mature CD56 -CD16 +NK in the peripheral blood (PB) of RM, clonally distinct from myeloid, T, B and CD56 +16 -NK, persisting for months to years, suggesting self-renewal independent of ongoing production from HSPCs (Wu et al, Cell Stem Cell, 2014 and Science Imm, 2018). We have now used this model to investigate the clonal distribution of NK cell populations in bone marrow (BM), liver, spleen, lymph nodes (LN), jejunum, colon and bronchoalveolar lavage (BAL). Serial of tissue biopsies were obtained from 3 barcoded monkeys by endoscopy and laparotomy overtime at steady state post transplantation , as well as tissues from necropsy. We compared clonal patterns between various trNKs and PB NKs collected from barcoded RM. Tissue or PB NK were defined as CD3-CD14-CD20-NKG2+ (NKG2A+ and NKG2C+), and NK subsets were further sorted for CD16, CD56, CD49a (putative liver tissue memory-like NK marker), and CXCR3 (critical for NK cell migration into tumor or normal tissues). The same expanded CD56 -CD16 +NK clones found in the PB were also detected at high abundance within BM, LN, liver and/or spleen CD56 -CD16 +NK, but not found in tissue CD56 +16 -NK and CD56 -16 -NK subsets. The liver and spleen bulk NK clonal patterns were highly correlated, and distinct from other tissues. We also observed tissue specific barcoded NK clones in BAL, jejunum and colon samples with no or very low abundance in other tissues and PB. Strikingly, a group of markedly expanded trNK clones, distinct from the expanded CD56 -CD16 +NK clones present concurrently or previously in PB, were present and shared across all tissues examined. These clones were enriched in CD56 -16 + trNK and absent in CD56 +16 - trNK. Notably, in both tissue and PB these clones were expanded in NKG2+ CD56-CD16- NK. These common expanded trNK cells were specifically enriched in both tissue and PB CD56 -16 -CXCR3 +NK, suggesting a role for this chemokine receptor and the ability of these clones to move between tissues. In contrast, CD49a expression did not enrich for these expanded clones. Clonally-expanded and persistent mature trNK cells, shared across multiple tissues but not present within PB mature CD56 -CD16 +NK subsets, combined with prior functional data suggesting NK memory is restricted to liver or other trNK cells, suggests these clonally-expanded trNK cells may be of interest. The pattern of shared clones across tissues, together with identification of a rare PB CD56-CD16-NK subpopulation harboring the clones, suggests preferential hematogenous homing of these clones to multiple tissues. Further analyses of gene expression and clonal dynamics are ongoing and should shed light on the ontology of trNK cells, with implications for the development of NK based immunotherapies and NK memory. Disclosures No relevant conflicts of interest to declare.

The present work thoroughly investigated the heterogeneous mechanical properties along the building direction in a direct laser deposited 17-4 PH part by tensile tests. From top to bottom (1# - 5#) in the as-built cuboid, the yield strength enhanced from ~729 MPa to ~1050 MPa, while the elongation decreased from ~4.69% to ~1.50%. The microstructure of samples was composed of main martensite, reversed austenite, and a large amount of NbC nano-particles. The discrepancy in chemical composition and dislocation density between the phases existed due to the heat accumulation as the increasing building height. The various reduced fraction of austenite in different samples after tensile tests, implying the diverse extent of strain-induced phase transformation. Besides, the average Schmid factor of the 1# sample was higher; thus its grain orientation was more conducive to plastic deformation, leading to lower yield stress. The kernel average misorientation (KAM) was higher and distributed uniformly near the fracture of the 1# sample, and showed better deformation compatibility in the microstructure. Based on the combination of microstructural models and the dislocation density micromechanical model, multiscale finite element analysis further found that larger particles advanced the movements of dislocations, inducing more intense plastic flow within the austenite grains, which enabled greater fractions of transformation in austenite to improve the overall elongation of the sample.

In this issue of JEM, Wu et al. use genetic barcoding of macaque hematopoietic stem cells to demonstrate that, after transplantation, HSCs are very asymmetrically distributed and uncover a thymus-independent pathway for mature T cell production in the bone marrow, In this issue of JEM, Wu et al. (https://doi.org/10.1084/jem.20171341) use genetic barcoding of macaque hematopoietic stem cells to demonstrate that, after transplantation, HSCs are very asymmetrically distributed and uncover a thymus-independent pathway for mature T cell production in the bone marrow.

Natural killer (NK) cells have long been considered short-lived effectors of innate immunity. However, recent animal models and human studies suggest that subsets of NK cells have adaptive features. We investigate clonal relationships of various NK-cell subsets, including the adaptive population, by taking advantage of naturally occurring X-linked somatic PIGA mutations in hematopoietic stem and progenitor cells (HSPCs) from patients with paroxysmal nocturnal hemoglobinuria (PNH). The affected HSPCs and their progeny lack expression of glycosylphosphatidylinositol (GPI) anchors on their cell surface, allowing quantification of PIGA-mutant (GPI-negative) HSPC-derived peripheral blood cell populations. The fraction of GPI-negative cells within the CD56dim NK cells was markedly lower than that of neutrophils and the CD56bright NK-cell compartments. This discrepancy was most prominent within the adaptive CD56dim NK-cell population lacking PLZF expression. The functional properties of these adaptive NK cells were similar in PNH patients and healthy individuals. Our findings support the existence of a long-lived, adaptive NK-cell population maintained independently from GPIposCD56dim.

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.

Single-cell lineage tracking strategies enabled by recent experimental technologies have produced significant insights into cell fate decisions, but lack the quantitative framework necessary for rigorous statistical analysis of mechanistic models describing cell division and differentiation. In this paper, we develop such a framework with corresponding moment-based parameter estimation techniques for continuous-time, multi-type branching processes. Such processes provide a probabilistic model of how cells divide and differentiate, and we apply our method to study hematopoiesis, the mechanism of blood cell production. We derive closed-form expressions for higher moments in a general class of such models. These analytical results allow us to efficiently estimate parameters of much richer statistical models of hematopoiesis than those used in previous statistical studies. To our knowledge, the method provides the first rate inference procedure for fitting such models to time series data generated from cellular barcoding experiments. After validating the methodology in simulation studies, we apply our estimator to hematopoietic lineage tracking data from rhesus macaques. Our analysis provides a more complete understanding of cell fate decisions during hematopoiesis in nonhuman primates, which may be more relevant to human biology and clinical strategies than previous findings from murine studies. For example, in addition to previously estimated hematopoietic stem cell self-renewal rate, we are able to estimate fate decision probabilities and to compare structurally distinct models of hematopoiesis using cross validation. These estimates of fate decision probabilities and our model selection results should help biologists compare competing hypotheses about how progenitor cells differentiate. The methodology is transferrable to a large class of stochastic compartmental and multi-type branching models, commonly used in studies of cancer progression, epidemiology and many other fields.

Recent functional, gene expression, and epigenetic studies have suggested the presence of a subset of mature natural killer (NK) cells responsible for maintaining NK cell memory. The lack of endogenous clonal markers in NK cells impedes understanding the genesis of these cell populations. In humans, primates, and mice, this phenotype and memory or adaptive functions have been strongly linked to cytomegalovirus or related herpes virus infections. We have used transplantation of lentivirally-barcoded autologous hematopoietic stem and progenitor cells (HSPC) to track clonal hematopoiesis in rhesus macaques and previously reported striking oligoclonal expansions of NK-biased barcoded clones within the CD56−CD16+ NK cell subpopulation, clonally distinct from ongoing output of myeloid, B cell, T cell, and CD56+16− NK cells from HSPC. These CD56−CD16+ NK cell clones segregate by expression of specific KIR surface receptors, suggesting clonal expansion in reaction to specific environmental stimuli. We have now used this model to investigate the impact of rhesus CMV(RhCMV) infection on NK clonal dynamics. Following transplantation, RhCMVneg rhesus macaques display less dominant and oligoclonal CD16+ NK cells biased clones compared to RhCMVpos animals, however these populations of cells are still clearly present. Upon RhCMV infection, CD16+ NK cells proliferate, followed by appearance of new groups of expanded NK clones and disappearance of clones present prior to RhCMV infection. A second superinfection with RhCMV resulted in rapid viral clearance without major change in the mature NK cell clonal landscape. Our findings suggest that RhCMV is not the sole driver of clonal expansion and peripheral maintenance of mature NK cells; however, infection of macaques with this herpesvirus does result in selective expansion and persistence of specific NK cell clones, providing further information relevant to adaptive NK cells and the development of NK cell therapies.

Lentiviral vectors (LV) have been used for the delivery of genes into hematopoietic stem and progenitor cells (HSPC) in clinical trials worldwide. LV, in contrast to retroviral vectors, have not been associated with insertion site-associated malignant clonal expansions, and thus have been considered safer. Here, however, we present a case of markedly abnormal dysplastic clonal hematopoiesis impacting the erythroid, myeloid and megakaryocytic lineages in a rhesus macaque transplanted with HSPCs that were transduced with a LV containing a strong retroviral murine stem cell virus (MSCV) constitutive promoter-enhancer in the LTR. 9 insertions were mapped in the abnormal clone, resulting in overexpression and aberrant splicing of several genes of interest, including the cytokine stem cell factor and the transcription factor PLAG1. This case represents the first clear link between a lentiviral insertion-induced clonal expansion and a clinically abnormal transformed phenotype following transduction of normal primate or human HSPC, and are thus concerning, and suggest that strong constitutive promoters should not be included within LV vectors.

Laser direct metal deposition (DMD) is an efficient and flexible additive manufacturing technique which has broad application prospects, but it is also limited due to defects and mechanical anisotropy. Laser remelting (LR) is a process that after each layer is deposited, re-scan the deposition layer with the same slice data but without powder deliver, and it is often used during selective laser melting. Herein, LR process has been applied during the DMD process of a 17-4 PH steel to enhance the densification level and relieve the mechanical anisotropy. It is found that the thermal history, porosity and microstructural evolution are dependent on the LR energy density. Moreover, the roughness of top surface of the deposited layer and intralayer porosity decrease with increase of the laser remelting energy density. While for interlayer defects, there is an optimal LR energy density corresponding to the lowest interlayer porosity. Furthermore, LR process can enhance the holding time at high temperature, even sometimes heat the sample above Ac1, resulting in change in contents of austenite and carbide. In addition, LR process greatly dilutes the pre-solidified texture. It was also proved that the flat defects in-plane to the interlayer and the anisotropy of the crystallographic orientations are extremely harmful for the mechanical isotropy of the DMDed samples. Finally, sample manufactured with laser remelting at 15.0 J/mm2 has the lowest porosity and most random crystallographic orientations resulting in near isotropous tensile strength, but the contribution of laser remelting to eliminate anisotropy in elongation is limited because of the interlayer defects.

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 following 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 (KIRs). These clonally distinct NK cell subpopulation patterns persisted for over 4 years, including following transient in vivo anti-CD16 mediated depletion and subsequent regeneration. Profound and sustained IL-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.

Critically short telomeres activate p53-mediated apoptosis, resulting in organ failure and leading to malignant transformation. Mutations in genes responsible for telomere maintenance are linked to a number of human diseases. We derived induced pluripotent stem cells (iPSCs) from 4 patients with aplastic anemia or hypocellular bone marrow carrying heterozygous mutations in the telomerase reverse transcriptase (TERT) or the telomerase RNA component (TERC) telomerase genes. Both mutant and control iPSCs upregulated TERT and TERC expression compared with parental fibroblasts, but mutant iPSCs elongated telomeres at a lower rate compared with healthy iPSCs, and the deficit correlated with the mutations' impact on telomerase activity. There was no evidence for alternative lengthening of telomere (ALT) pathway activation. Elongation varied among iPSC clones derived from the same patient and among clones from siblings harboring identical mutations. Clonal heterogeneity was linked to genetic and environmental factors, but was not influenced by residual expression of reprogramming transgenes. Hypoxia increased telomere extension in both mutant and normal iPSCs. Additionally, telomerase-mutant iPSCs showed defective hematopoietic differentiation in vitro, mirroring the clinical phenotype observed in patients and demonstrating that human telomere diseases can be modeled utilizing iPSCs. Our data support the necessity of studying multiple clones when using iPSCs to model disease.

Retroviral vectors are an efficient and widely employed means of introducing an exogenous expression cassette into target cells. These vectors have been shown to integrate semi-randomly into the cellular genome, and can be associated with genotoxicity due to impact on expression of proximate genes. Therefore, efficient and accurate integration site analysis, while quantifying contributions of individual vector-containing clones, is desirable. Linear amplification-mediated polymerase chain reaction (LAM-PCR) is a widely used technique for identifying integrated proviral and host genomic DNA junctions. However, LAM-PCR is subject to selection bias inherent in the reliance of the assay on the presence of a restriction enzyme-cutting site adjacent to a retrievable integration site, and it is further limited by an inability to discriminate prior to sequencing between the flanking genomic DNA of interest and uninformative internal vector DNA. We report a modified restriction enzyme-free LAM-PCR (Re-free LAM-PCR) approach that is less time and labor intensive compared to conventional LAM-PCR, but in contrast to some other nonrestrictive methods, compares in efficiency and sensitivity, excludes retrieval of uninformative internal vector sequences, and allows retrieval of integration sites unbiased by the presence of nearby restriction sites. However, we report that Re-free LAM-PCR remains inaccurate for quantitation of the relative contributions of individual integration site-containing clones in a polyclonal setting, suggesting that bias in LAM-PCR retrieval of integration sites is not wholly explained by restriction enzyme-related factors.

Virus-based vectors are widely used in hematopoietic stem cell (HSC) gene therapy, and have the ability to integrate permanently into genomic DNA, thus driving long-term expression of corrective genes in all hematopoietic lineages. To date, HSC gene therapy has been successfully employed in the clinic for improving clinical outcomes in small numbers of patients with X-linked severe combined immunodeficiency (SCID-X1), adenosine deaminase deficiency (ADA-SCID), adrenoleukodystrophy (ALD), thalassemia, chronic granulomatous disease (CGD), and Wiskott-Aldrich syndrome (WAS). However, adverse events were observed during some of these HSC gene therapy clinical trials, linked to insertional activation of proto-oncogenes by integrated proviral vectors leading to clonal expansion and eventual development of leukemia. Numerous studies have been performed to understand the molecular basis of vector-mediated genotoxicity, with the aim of developing safer vectors and lower-risk gene therapy protocols. This review will summarize current information on the mechanisms of insertional mutagenesis in hematopoietic stem and progenitor cells due to integrating gene transfer vectors, discuss the available assays for predicting genotoxicity and mapping vector integration sites, and introduce newly-developed approaches for minimizing genotoxicity as a way to further move HSC gene therapy forward into broader clinical application.

The full-length AML1-ETO ( AE ) fusion gene resulting from t(8;21)(q22;q22) in human acute myeloid leukemia (AML) is not sufficient to induce leukemia in animals, suggesting that additional mutations are required for leukemogenesis. We and others have identified activating mutations of C-KIT in nearly half of patients with t(8;21) AML. To test the hypothesis that activating C-KIT mutations cooperate with AE to cause overt AML, we generated a murine transduction and transplantation model with both mutated C-KIT and AE . To overcome the intracellular transport block of human C-KIT in murine cells, we engineered hybrid C-KIT (HyC-KIT) by fusing the extracellular and transmembrane domains of the murine c-Kit in-frame to the intracellular signaling domain of human C-KIT. We showed that tyrosine kinase domain mutants HyC-KIT N822K and D816V, as well as juxtamembrane mutants HyC-KIT 571+14 and 557-558Del, could transform murine 32D cells to cytokine-independent growth. The protein tyrosine kinase inhibitor dasatinib inhibited the proliferation of 32D cells expressing these C-KIT mutants, with potency in the low nanomolar range. In mice, HyC-KIT N822K induced a myeloproliferative disease, whereas HyC-KIT 571+14 induces both myeloproliferative disease and lymphocytic leukemia. Interestingly, coexpression of AE and HyC-KIT N822K led to fatal AML. Our data have further enriched the two-hit model that abnormalities of both transcription factor and membrane/cytosolic signaling molecule are required in AML pathogenesis. Furthermore, dasatinib prolonged lifespan of mice bearing AE and HyC-KIT N822K-coexpressing leukemic cells and exerted synergic effects while combined with cytarabine, thus providing a potential therapeutic for t(8;21) leukemia.

All- trans retinoic acid (ATRA)/arsenic trioxide (ATO) combination-based therapy has benefitted newly diagnosed acute promyelocytic leukemia (APL) in short-term studies, but the long-term efficacy and safety remained unclear. From April 2001, we have followed 85 patients administrated ATRA/ATO with a median follow-up of 70 months. Eighty patients (94.1%) entered complete remission (CR). Kaplan–Meier estimates of the 5-year event-free survival (EFS) and overall survival (OS) for all patients were 89.2% ± 3.4% and 91.7% ± 3.0%, respectively, and the 5-year relapse-free survival (RFS) and OS for patients who achieved CR ( n = 80) were 94.8% ± 2.5% and 97.4% ± 1.8%, respectively. Upon ATRA/ATO, prognosis was not influenced by initial white blood cell count, distinct PML-RAR α types, or FLT3 mutations. The toxicity profile was mild and reversible. No secondary carcinoma was observed, and 24 months after the last dose of ATRA/ATO, patients had urine arsenic concentrations well below the safety limit. These results demonstrate the high efficacy and minimal toxicity of ATRA/ATO treatment for newly diagnosed APL in long-term follow-up, suggesting a potential frontline therapy for de novo APL.

The classical model of hematopoietic hierarchies is being reconsidered, based on data from in vitro assays and single cell expression profiling. Recent experiments suggest that erythroid and megakaryocytic lineages might differentiate directly from multipotent hematopoietic stem/progenitor cells (HSPC) or from a highly biased subpopulations of HSPC, rather than transiting through a common MEP or CMP. We examined the clonal ontogeny of the erythroid lineage using genetic barcoding of rhesus macaque HSPC (Wu Cell Stem Cell, 2014; Koelle Blood, 2017), allowing quantitative and sensitive tracking of the in vivo clonal output of thousands of individual HSPC over time following autologous transplantation. CD34+ HSPC were lentivirally-transduced with a high diversity barcode library, with the barcode in an expressed region of the provirus, allowing barcode retrieval from DNA or RNA, with each barcode representing an individual HSPC clone. CD34+ HSPC were purified from bone marrow(BM) of 3 macaques at 3-45 months post-transplant, and plated in CFU assays. 240 colonies each of CFU-E, CFU-G and CFU-GM were plucked individually, and each colony type was pooled before DNA extraction for barcode retrieval, along with purification and barcode retrieval from concurrent BM CD34+ cells and both blood and BM T cells (T), B cells (B), granulocytes (Gr), and monocytes (Mono). The majority of barcodes retrieved from pooled CFU-E were also detected in pooled CFU-G and CFU-GM, along with purified T cells, B cells, Mono and Gr, suggesting a shared unbiased precursor pool. A small fraction of clones unique to CFU-E were identified, however, unique clones were also detected in CFU-G and in CFU-GM pools, likely reflecting low frequency clones that were to be represented randomly in the pooled CFU of each lineage. To overcome the sampling bias inherent in colony assays on any reasonable colony number, we FACS purified CD71+/CD45- nucleated maturing erythroid lineage cells (nRBC) from the BM, and compared nRBC to other lineages purified concurrently from the same BM sample. There was very high correlation of barcode contributions between BM nRBC and other BM-produced lineages, with the highest correlation between nRBC and both Gr and Mono (r> 0.9), whether at earlier or later time points. We investigated whether RNA barcode retrieval could be utilized for clonal tracking, allowing analysis of anucleate circulating RBC and thus a more global analysis of hematopoiesis compared to local BM production at a limited number of sites. We have reported that clonal output from individual HSPC remains highly geographically restricted within the BM for months-years post-transplant. We compared fractional contributions of DNA and RNA barcodes retrieved from the same sample of each lineage. There was very high correlation between DNA and RNA barcode contributions to T, B, NK, Gr and Mono lineages (r= 0.85±0.04), suggesting the differentiation pathway for these lineages does not impact significantly on expression level of barcodes from the proviral promoter, and RNA fractional contributions in these lineages reflect the clonal representation of cells in a sample. However, nRBC DNA and RNA barcode contributions were less closely related (r= 0.62), suggesting that erythroid differentiation was more likely to alter expression from loci in a manner disconnecting RNA barcode expression from clonal representation of cells in a sample, and suggesting that RNA barcode retrieval may not be ideal for comparing erythroid cells to other lineages. However, tracking RNA barcodes can be used to assess clonal stability in circulating RBC over time, and revealed very stable clonal contributions to erythropoiesis for as long as 4 years post-transplant. Finally, we used RNA barcode retrieval to compare clonal contributions between circulating platelets and other lineages. Whether DNA or RNA was used for T, B, Gr, and Mono clonal mapping, at steady state platelet RNA barcodes were clonally closely related to other lineages. But preliminary data suggests that a unique set of clones is newly recruited to contribute only to platelets following inflammatory stimuli. The presence of a separate pool of platelet-biased HSPC contributing following inflammation has been suggested by prior in vitro assays, but our model may provide the first clonal in vivo confirmation of a unique inflammation-related platelet-biased HSPC pool. Disclosures Dunbar: Novartis/GSK to institute: Research Funding.

SummaryAnalysis 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.

Induced pluripotent stem cell (iPSC) therapies offer a promising path for patient-specific regenerative medicine. However, tumor formation from residual undifferentiated iPSC or transformation of iPSC or their derivatives is a risk. Inclusion of a suicide gene is one approach to risk mitigation. We introduced a dimerizable-“inducible caspase-9” (iCasp9) suicide gene into mouse iPSC (miPSC) and rhesus iPSC (RhiPSC) via a lentivirus, driving expression from either a cytomegalovirus (CMV), elongation factor-1 α (EF1α) or pluripotency-specific EOS-C(3+) promoter. Exposure of the iPSC to the synthetic chemical dimerizer, AP1903, in vitro induced effective apoptosis in EF1α-iCasp9-expressing (EF1α)-iPSC, with less effective killing of EOS-C(3+)-iPSC and CMV-iPSC, proportional to transgene expression in these cells. AP1903 treatment of EF1α-iCasp9 miPSC in vitro delayed or prevented teratomas. AP1903 administration following subcutaneous or intravenous delivery of EF1α-iPSC resulted in delayed teratoma progression but did not ablate tumors. EF1α-iCasp9 expression was downregulated during in vitro and in vivo differentiation due to DNA methylation at CpG islands within the promoter, and methylation, and thus decreased expression, could be reversed by 5-azacytidine treatment. The level and stability of suicide gene expression will be important for the development of suicide gene strategies in iPSC regenerative medicine.

Stem cell gene therapy using integrating retroviral vectors to modify hematopoietic stem and progenitor cells (HSPC) has shown great promise to treat patients with a variety of acquired and inherited immune system and hematologic diseases. However activation of proto-oncogenes via insertional mutagenesis related to the presence of enhancers within integrating vectors has resulted in the development of leukemia in some patients. The risk appears lower with enhancer-deleted retroviral or HIV-based lentiviral vectors. However, to date, there is no predictive or quantitative preclinical in vitro or animal model to assess genotoxicity of integrating vectors, given the difficulty in studying HSPCs in vitro and differences between rodent and human hematopoiesis. An approach to predict the risk of genotoxic adverse events is desirable before moving a new vector into clinical trials. We are utilizing cellular “barcoding” technology to investigate the impact of different vector designs on the clonal behavior of HSPC during in vitro and in vivo expansion as a rapid quantitative surrogate assay for genotoxicity. We have begun by studying a standard HIV-derived lentiviral vector backbone now being developed for clinical applications containing one of three promoter/enhancers with a predicted wide range of genotoxicities, from the least genotoxic EF1a promoter, to a predicted medium genotoxic MSCV enhancer, to the most genotoxic very strong viral enhancer termed SFFV, already associated with a high rate of leukemia incidence in human clinical trials targeting HSPC. Each vector has a high diversity “barcode” (a random sequence tag) inserted into the vector LTR, which will uniquely tag each individual transduced HSPC, and can be retrieved via low cycle PCR and high-throughput Illumina sequencing to quantitate the clonal pattern of contributions from each transduced barcoded HSPC. In addition, actual insertion sites can be retrieved and compared with barcode patterns to identify expanded clones. We have used these barcoded vectors to transduce rhesus macaque CD34+ HSPC in vitro, then expanded transduced cells in culture for 2-3 weeks, and currently in process of bioinformatics analysis of the clonal patterns and diversity with each of the three vectors. The same in vitro approach can be applied to human HSPC. By the time of the ASGCT meeting, we will have in vivo data on clonal pattern generated by each vector type from a rhesus macaque transplanted with autologous CD34+ HSPC split into three fractions, transduced with each of the three vectors, and reinfused simultaneously. In summary, this project will investigate whether HSPC genotoxicity due to integrating vectors can be measured and predicted using a barcoding approach, and provide insights into the relative genotoxicity of a number of different integrating vector designs now under development for clinical applications.

Induced pluripotent stem cell (iPSC)-based cell therapies have a great potential for regenerative medicine, but are also potentially associated with tumorigenic risks. Current rodent models are not the optimal predictors of efficiency and safety for clinical application. Therefore, we developed a clinically relevant non-human primate model to assess the tumorigenic potential and in vivo efficacy of both undifferentiated and differentiated iPSCs in the autologous settings without immunosuppression. Undifferentiated autologous iPSCs indeed formed mature teratomas in a dose-dependent manner. However, tumor formation was accompanied by an inflammatory reaction. On the other hand, iPSC-derived mesodermal stromal-like cells formed new bone in vivo without any evidence of teratoma formation. We therefore show for the first time in a large animal model that closely resembles human physiology that undifferentiated autologous iPSCs form teratomas, and that iPSC-derived progenitor cells can give rise to a functional tissue in vivo.

The classical model of hematopoiesis states that differentiation proceeds from hematopoietic stem cells (HSC) to mature blood cells via specified multipotent and bipotent progenitors, such as the common myeloid progenitor (CMP), common lymphoid progenitor (CLP), erythrocyte-megakaryoctye progenitor and granulocyte-monocyte progenitor. However, recent studies question this assumption and suggest that these intermediates are neither required nor prevalent. As an example, analyses of binarized data from murine barcoding experiments (Perie et al, Cell Reports, 2014) raise the possibility that hematopoiesis progresses via a random loss of potentials rather than discrete steps. Additionally, Notta et al (Science, 2015) showed that oligopotent progenitor cells form only a negligible component in the hierarchy by studying the distribution of progenitors in human marrow, leading them to infer that HSC and earliest multipotent progenitors differentiate directly into unipotent cells. Although these data challenge fundamental beliefs, the quantitative contributions of HSC and progenitors to cell lineages could not be tracked in individual mice or persons over time. We developed a statistical method to infer the rates and probabilities of cell fate decisions in a class of stochastic branching models and used this to analyze sequence data from a rhesus macaque transplanted with lentivirally barcoded CD34+ HSC and progenitor cells. The macaque's blood granulocytes (Gr), monocytes (Mo), B cells, T cells, and NK cells were tracked over 30 months. Our quantitative framework is based on computing correlations between pairs of observable mature blood cell types across all independently barcoded lineages. The method also accounts for experimental uncertainties intrinsic to blood sampling, cell purification and PCR amplification. Specifically, our approach relies on a loss function estimator that minimizes residuals between empirical pairwise correlations across barcode lineages and analytical model-based correlations derived generally for continuous-time multi-type branching processes. We integrate over sampling distributions accounting for noise in experimental protocol and CBC counts. Candidate models represent possible hematopoietic structures and allow an arbitrary number of progenitor and mature cell types descended from each HSC. We identify best-fitting fate decision rates and initial marking levels with corresponding confidence intervals via nonlinear least squares and can assess whether a given model is statistically consistent with the data. This is the first statistical method to our knowledge for fitting stochastic models of hematopoiesis to lineage barcoding time-series, and together with the rhesus macaque data, enables quantitative analysis of in vivo dynamics in a large animal model. Using this new approach, we confirmed the major finding in Wu et al. (Cell Stem Cell, 2014) of a distinct NK cell ontogeny, i.e., that CD16+ blood NK cells do not overlap in origin with T and B cell lineages. We estimate that 13.9% of HSC and 86.1% of progenitors were initially barcoded, which is consistent with the finding by Wu that the percentage of blood cells expressing GFP stabilized at 13% after 6 months. Additionally, we estimate that HSC self-renew approximately once every 12 weeks, which is consistent with the range estimated in previous primate studies based on telomere studies (Shepherd, Blood, 2007). These initial analyses help validate our method. We then showed that Gr and Mo cells derive from a common precursor in vivo (correlation ρ ≈.9 across time). We also estimated progenitor differentiation rates and showed that Gr and Mo cells are produced up to 10- to 100-fold more rapidly than T, B and NK cells, and that each progenitor committed to the Gr/Mo lineage (i.e., CFUGM) produces thousands of mature cells per day. Importantly, we tested models requiring an ordered differentiation through defined intermediaries and found that they did not suitably fit the data compared to models allowing for non-restricted pathways. Together these analyses challenge the classic model of blood cell differentiation and provide new insights into the structure of hematopoiesis. Disclosures Dunbar: GSK/Novartis: Research Funding.

Natural killer (NK) cells are defined as lymphocytes capable of target cell killing and cytokine production independent of somatic antigen receptor recombination. Challenging the paradigm of continuous production of non-specific cytotoxic NK cells from undifferentiated precursors, a subset of murine NK cells sharing many properties with cytotoxic effector T cells was shown to specifically respond to a viral infection, resulting in expansion and protective immunity upon adoptive transfer, uncovering unexpected "adaptive" capabilities for NK cells. In humans, CD56dimCD16+ NK cells expressing NKG2C and variegated patterns of KIR are associated with Cytomegalovirus (CMV) infection, and may represent human analogs of murine adaptive NK cells. In contrast to CD56bright and canonical CD56dim NK cells, we recently showed that human adaptive NK cells lack expression of specific signaling proteins and transcription factors, and epigenetically resemble CD8+ effector T cells, suggesting shared pathogen-driven developmental pathways (Schulms et al, 2015). NK cell heterogeneity, ontogeny, and mechanisms sustaining NK cell memory are poorly understood, particularly in humans, because in contrast to adaptive T and B cells, clonal tracking of NK cells based on receptor gene structure is not possible. We applied autologous transplantation of genetically barcoded CD34+ rhesus macaque hematopoietic stem and progenitor cells (HSPC) to interrogate the landscape of NK cell production and expansion at a clonal level. We followed 3 monkeys for up to 4 years post-transplantation, and observed a distinct clonal ontogeny for CD56-CD16+ (corresponding to human CD56dimCD16+ NK) versus CD56+CD16- rhesus NK cells. CD56-CD16+ NK were oligoclonal, and dominated by small numbers of highly biased clones, a pattern showing little overlap with highly polyclonal multipotent contributions to T, B, myeloid and CD56+CD16- NK cell lineages. There were clear expansions and contractions of groups of these highly biased CD56-CD16+ NK cell clones over time, with individual clones contributing up to 20% to this lineage, similar to patterns of waxing and waning T cell clones, suggesting responses of both cell types to extrinsic cues such as viruses. In contrast, all other lineages, including CD56+CD16- NK cells, were characterized by very stable contributions from long-term engrafting multipotent HSPC clones over time. The highly biased, greatly expanded, and waxing and waning CD56-CD16+ NK cell clones were specifically enriched in a subpopulation of CD56+CD16- NK with high KIR and low SYK expression,and decreased methylation at IFNG the CNS1 locus, analogous to human CMV-associated adaptive NK cells. This population of cells was increased in CMV+ compared to CMV- macaques; our transplanted macaques were CMV+. Canonical CD56+CD16- NK cells, lacking an adaptive phenotype, had clonal patterns similar to CD56+CD16- NK cells. We profoundly depleted CD56-CD16+ NK cells with an anti-CD16 antibody, and observed rapid regeneration of the same CD56-CD16+ NK clonal pattern, without recruitment of contributions from multipotent HSPC via CD56+CD16- putative precursor NK cells. There was a marked increase in cycling of CD56-CD16dim NK cells just prior to reappearance of CD56-CD16bright NK cells, and no change in cycling of CD56+CD16- cells. Thus, adaptive NK cell clones, both at steady state and under regenerative stress do not appear to require ongoing production from HSPC via CD56+CD16- precursors. These results suggest that adaptive NK cells or their immediate precursors have acquired self-renewal properties and high proliferative potential, with clonal restriction potentially based selection and expansion of cells with specific, epigenetically-controlled receptor expression able to interact with external stimuli such as viruses. In conclusion, via tracking of individual NK cell clonal histories in rhesus macaques, we provide the first direct evidence for persistence of adaptive NK cell clones in any species, offering potential insights into the acquisition of NK cell memory. Disclosures Dunbar: GSK/Novartis: Research Funding.

The development of ex-vivo HSPC expansion techniques is particulary relevant for improving cord blood transplantation and gene therapy. Despite successful long-term, multilineage reconstitution of expanded human cord blood HSPC in immunodeficient mice, early phase clinical trials have failed to demonstrate improved outcomes. Thus, it is critical to develop a robust pre-clinical model to study ex-vivo expansion strategies, particularly function of long term HSPC and engrafment of all lineages, difficult in xenograft models. We have previously used retroviral insertion site retrieval and autologous competitive transplantation in rhesus macaques to track hematopoietic engraftment and ontogeny at a single HSPC level, in addition to comparing ex-vivo expanded and unexpanded HSPC (Gomes et al, Mol Ther). More recently we have developed retrieval of 31bp diverse barcodes as a more robust and quantitative in vivo HSPC tracking approach (Wu et al, Cell Stem Cell). Modified human endothelial cells (HUVEC) with the ability to be maintained in serum free media for prolonged periods have shown to support and expand human HSPC in murine Butler et al, Blood). We have developed a barcoded rhesus autologous transplant model to evaluate expansion of HPSC on HUVEC versus unexpanded or cytokine expanded rhesus HSPC. We first tested the feasibility of rhesus CD34+ cell expansion on HUVEC versus cytokines/fibronectin for 8 days. We also evaluated for the diversity of expanded HSPC by retrieving transduced viral barcode DNA tags from individual cells by low cycle PCR followed by Illumina sequencing. On average, rhesus CD34+ cells expanded 76 fold (± 53) on HUVEC versus 13 fold (±7) in cytokines (n=4; p=0.03). By morphologic assessment, the HUVEC expanded cell fraction contained higher numbers of progenitors and differentiating cells, with up to 97% CD34+CD45+ cells in the HUVEC expanded fraction and 65% in the cytokine expanded fraction. The HUVEC expanded rhesus CD34+ cells were capable of multi-lineage colony formation after 8 days of expansion. Within the HUVEC expanded cell fractions, fold expansion was similar whether CD34+ cells were frozen after (C1) or before (C2) transduction-expansion. However, viability and percentages of CD34+CD45+ cells were higher in C2 compared to C1 (55% versus 26%). GFP transduction efficiency was also higher in C2 compared to C1 (43% vs. 28%). We demonstrated a slightly lower percentage but higher absolute number of CD34+CD38-CD45RA-CD90+CD49f+Rho-low putative long-term HSC after expansion on HUVEC compared to in cytokines over 26 days in culture (0.018% vs. 0.016% pre and post expansion, respectively). Retrieved barcode analysis and quantitation revealed that in the HUVEC expanded fraction, the top 20 clones constituted up 15% of the total valid reads by day 8, in contrast to only 3.9% of total clones in the cytokine expanded fraction. Cells have been transplanted into autologous macaques, in a competitive model comparing non-expanded, cytokine-expanded and HUVEC-expanded conditions, and in vivo expansion and barcode analysis will be presented. Our data thus far show that peripheral blood CD34+ cells from rhesus macaques expand more robustly in the presence of HUVEC and cytokines as compared to expansion in cytokines alone, and barcode analysis suggests that the HUVEC fraction selectively expanded a subset of highly proliferative cells, which may represent true HSCs within the CD34+ cell fraction.