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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.

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.

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.

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.

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.

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.

AML1-ETO fusion gene is generated from chromosomal translocation t(8;21) mainly in acute myeloid leukemia M2 subtype (AML-M2). Its spliced variant transcript, AML1-ETO9a, rapidly induces leukemia in murine model. To evaluate its clinical significance, AML1-ETO9a expression was assessed in 118 patients with t(8;21) AML-M2, using qualitative and nested quantitative reverse transcriptase (RT)-PCR methods. These cases were accordingly divided into the AML1-ETO9a-H group (n=86, positive for qualitative RT-PCR, with higher level of AML1-ETO9a by quantitative RT-PCR) and the AML1-ETO9a-L group (n=32, negative for qualitative RT-PCR, with lower but still detectable level of AML1-ETO9a by quantitative RT-PCR). C-KIT expression was significantly increased in the AML1-ETO9a-H group, as compared with the AML1-ETO9a-L group. Of the 36 patients harboring C-KIT mutations, 32 patients overexpressed AML1-ETO9a (P=0.0209). Clinically, AML1-ETO9a-H patients exhibited significantly elevated white blood cells count, less bone marrow aberrant myelocytes, increased CD56 but decreased CD19 expression (P=0.0451, P=0.0479, P=0.0149 and P=0.0298, respectively). Moreover, AML1-ETO9a overexpression was related to short event-free and overall survival time (P=0.0072 and P=0.0076, respectively). Taken together, these data suggest that AML1-ETO9a is correlated with C-KIT overexpression/mutations and indicates poor disease outcome in t(8;21) AML-M2.

Hematopoietic stem and progenitor cells (HSPC) primarily reside in bone marrow (BM) niches, but are also found in peripheral blood (PB) in small numbers. Large numbers of HSPC can be pharmacologically mobilized from the BM into the PB and home back to BM niches following transplantation. We used the rhesus macaque to study the process of hematopoiesis in both space and time following autologous transplantation, as a model with great relevance to humans. We labeled individual CD34+ HSPC and their progeny with genetic "barcodes" via lentiviral transduction with a very high diversity barcode library, allowing tracking of the output from individual HSPC clones in vivo following autologous transplantation, in a quantitative and sensitive manner (Wu et al., 2014). In this study we investigated the pattern of HSPC clonal output in various anatomic niches, sampling left (L) vs right (R) iliac crest BM, PB and lymph node (LN) over time post ablative autologous transplantation, for all major hematopoietic lineages and CD34+ HSPC. We tracked thousands of HSPC clones in 6 rhesus macaques from 3.5 months (m) to up to 18.5m post transplantation. L and R iliac crest BM and PB were serially collected and CD34+ HSPC along with CD3+CD4+ T, CD3+CD8+ T, CD20+ B, CD14+ monocytes (Mo), CD33+ granulocytes (Gr), and two NK cell subsets (CD3-CD20-CD14-CD16+/ or CD56+) were purified. In all animals, there was marked geographic segregation of CD34+ HSPC for at least 6m post-transplant, with individual clones localized only to the L or R side but not both (Pearson correlation at 3.5-6m: r=0.019±0.08 for CD34+ L vs R, n=6), despite rapid expansion of CD34+ HSPC during early engraftment. This result suggests that during this phase of recovery, HSPC spread contiguously in the BM, and there is little geographic "mixing" via egress into and re-entry from the PB. With time between 7m and 18.5m post-transplant, the distribution of CD34+ HSPC became more homogeneous, with clones detected on both L and R (r=0.52±0.22 at 12-18.5m, n=3). The geographic restriction of clones at earlier time points suggests that release and homing may be dependent on local contiguous niche occupancy status, with migration only occurring following hematopoietic recovery. We next examined the clonal distribution of the more mature lineage-committed cells in the L vs R BM and in PB. Gr, Mo and B cells were produced locally in BM, with the clonal pattern of each lineage matching the CD34+ cells collected from the same side BM time point through 6m (r>0.80 for each lineage vs same side CD34+ cells), and distinct from same lineages (r Our model for the first time documents the dynamics of HSPC geographic distribution and migration in primates following transplantation, findings with direct clinical relevance, and provides new insights into hematopoietic lineage development, including a potential novel T cell development pathway in the BM. Our findings may also help explain the extremely patchy distribution of hematopoiesis in humans following transplantation or in the setting of marrow failure or aging, and suggest that analysis of individual BM samples may not fully reflect ongoing global hematopoiesis. Disclosures No relevant conflicts of interest to declare.

NK cells play a central role in innate immunity, specifically in tumor surveillance and microbial pathogen control. Recent murine models and human studies have identified subsets of NK-cells with apparent memory cell function, strongly linked to CMV infection in humans and termed "adaptive" NK. Our recent clonal tracking studies following autologous hematopoietic stem cells transplantation (HSCT) of genetically-barcoded CD34+ cells in macaques revealed distinct clonal ontogeny of a subset of NK cells within the primate equivalent of the human CD56 dim population (Wu et al., Cell Stem Cell, 2014), with little clonal overlap with T-, B-lymphoid or myeloid cells, suggesting a separate precursor pool for this NK subtype. Peripheral blood CD 56bright -NK cells have been previously hypothesized to be precursors for the main population of circulating cytotoxic CD56dim cells. To further investigate NK-cell ontogeny and clonal relationships in humans we took advantage of naturally-occurring somatic mutations in the X-linked phosphatidylinositol glycan class A (PIGA) gene in patients with the hemolytic disorder paroxysmal nocturnal hemoglobinuria. This gene codes for an enzyme required for cell surface localization of glycosylphophatidylinositol (GPI)-anchored proteins, and thus loss of function mutations result in hematopoietic cells lacking GPI-anchored proteins, and red cell hemolysis. PNH patients have not been reported to have immune dysfunction and can have stable disease for many years. Membrane bound GPI anchors can be detected on any cell via flow cytometry using a labeled inactive aerolysin (FLAER), and serves as a marker for the fraction of cells comprising the PIGA mutant (GPI negative) clonal compartment. The PNH clone sizes contributing to peripheral blood cells are variable in but can reach almost 100% in some patients, and can be stable over decades. We selected 9 PNH patients with GPI negative granulocytes ranging from 5% to 98% and a median time from diagnosis of 43.7 months (15-100) for this study. NK cells were defined as CD56+/CD16+/CD3-/CD20- lymphocytes. We observed disproportionally fewer GPI negative NK cells compared to granulocytes (Fig 1), with the discrepancy most marked the major peripheral blood CD56 dim population (mean 65% vs 25% GPI negative granulocytes, p = 0.0028, paired t-test), in contrast to 46% GPI negative cells in the CD56bright population (p=0.057). Due to the prolonged life span of memory T and B cells, fewer GPI negative B and particularly T-lymphocytes have been reported in PNH patients. In our cohort 3.4% of CD3+ T-cells and 13.2% of CD20+ B-cells were GPI negative (p=0.0005 and 0.0014, respectively versus granulocytes). Compared to the NK subsets, the CD56 bright population showed the most significant differences (p = 0.0063 versus CD3 and p=0.0151 versus CD20). To further characterize the phenotype of the GPI positive versus negative CD56dim cells, we analyzed cells co-expressing either the terminal differential marker CD57, the inhibitory receptor NKG2A, or activating receptor NKG2C for FLAER positivity. Prior studies have suggested that the human CMV-linked adaptive NK subset is CD57+, NKG2A- and NKG2C+. The NKG2C+ CD 56dim population was highly enriched for GPI positive cells (p=0.0024 vs granulocytes, Fig 1). This profile was most prominent in CMV-IgG positive patients who had also significantly more GPI positive CD 56dim/CD57+ cells compared to granulocytes (p=0.008). Interestingly, one CMV positive patient (#5) had a complete lack of NKG2C expression, most likely due to homozygous loss of function mutation, and this patient had almost 100% GPI negative NK cells, matching his neutrophil pattern. Compared to granulocytes, NKGA2A+ or CD57+ positive CD56dim cells were also mostly GPI negative (p=0.0081 and 0.028). Circulating NK cell turnover has been estimated to be about 14 days. The PNH patients studied had documented clonal PIG-A mutations for many years. Our observation that the majority of CD56dim NK cells, specifically the NKG2C subset, are not progeny of the same progenitors producing CD56bright NK cells or myeloid cells based on clonal disparity regarding the PNH clone is suggestive of an independent, very long-lived or self-renewing NK cell progenitor for a CMV-linked CD56dim/CD57+/NKG2C+ memory NK cell compartment. These observations provide novel further insights into the human adaptive NK cell subset. Figure 1. Figure 1. Disclosures Winkler: Novartis: Research Funding; GSK: Research Funding. Townsley:Novartis: Research Funding; GSK: Research Funding.

Aging of the hematopoietic system is associated with a number of observations, including diminished regenerative potential, skewed lineage differentiation, increased incidence of anemia, and higher rates of neoplastic transformation. Despite advanced age being a strong poor prognostic factor, an increasing number of older patients are receiving hematopoietic stem and progenitor cell (HSPC) transplantation. Most previous investigations of the effects of aging on hematopoiesis have been obtained in murine models. The rhesus macaque is a powerful model to study human hematopoiesis and aging, based on a close phylogenetic relationship to humans, and similar telomere lengths, lifespans and aging phenotypes. To quantitatively elucidate the age-related changes that compromise hematopoietic function at a clonal level, we applied a genetic barcoding approach to quantitatively track the clonal behavior of HSPCs in young versus old macaques following autologous transplantation. We delivered high diversity barcodes via lentiviral transduction of CD34+ HSPC (detailed in our prior study: Wu et al Cell Stem Cell, 2014), allowing quantitative tracking of the output of thousands of individual HSPC clones labeled by unique barcodes, over time and in various lineages post-transplantation. We successfully transplanted barcoded HSPCs into 2 macaques aged 19 and 25 years, constituting "old" macaques based on an average lifespan in captivity of 20-30 years, and compared results to clonal patterns observed in 5 "young" macaques aged 3-5 years. Both old macaques engrafted promptly, and CD3+ T cells, CD3-CD20+ B cells, CD33+ Granulocytes (Gr), CD3-CD20-CD14+ Monocytes (Mo), and CD3-CD20-CD14-CD16+/or CD56+ NK cells were purified from the peripheral blood monthly following transplantation. In terms of overall polyclonality and diversity (Shannon index), analyzed through 4 months to date, there were no marked differences between the old and young recipients, with thousands of individual clones contributing to hematopoiesis in both sets of animals during the initial post-transplant time period studied. However, there were marked differences in the patterns of clonal lineage relationships between young and old animals, as assessed via pairwise Pearson correlations of all contributing clones as well as clustering algorithms allowing interrogation of patterns of clonal behavior. In both young and old, there was no correlation (i.e. no shared clones and thus no shared progenitors) between lineages at 1m, and clones contributing at 1m did not contribute to any lineage at 3m or later, indicating the existence of short-term, lineage-restricted progenitors in both age groups. By 3m in young animals, B and Gr/Mo became correlated, and by 3-6m, B/T/Mo/Gr multilineage clones appeared and constituted the majority of hematopoietic output. However, in old animals clones contributing to Gr/Mo versus B or T lineages remained almost completely distinct or markedly biased, up to 4m studied to date, without evidence for multi-lineage clones (Fig 1). In both young and old animals, the NK lineage remained clonally distinct, as previously reported for young animals. In summary, we transplanted 2 aged macaques with barcoded CD34+ HSPCs, and discovered a pattern of clonal reconstitution distinct from that in young animals, with persistent unilineage or highly-biased myeloid and B lymphoid progenitors in the aged animals. Longer follow-up will be required to determine if this biased pattern persists, and will be presented. This approach should improve our understanding of disorders of hematopoiesis in the elderly, and help improve transplantation and other therapies in this vulnerable patient population. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Natural Killer (NK) cells are a component of the innate immune system that can lyse virally infected cells and tumor cells. Although IL-2 activated and ex vivo expanded NK cells are being developed for adoptive cell therapy of cancer, recent data suggests significant heterogeneity exists within these NK cell populations as defined phenotypically, functionally and developmentally. In order to maximize the efficacy of adoptive NK cell therapy, further insights into the ontogeny and behavior of various NK subsets are needed. NK cells have been historically defined as lymphocytes expressing CD56 but lacking CD3. However, we now report the identification of a population of human peripheral blood mononuclear cells that lack expression of the canonical NK markers CD56 and CD16, but otherwise share phenotypic and functional properties with NK cells, which we term "double negative (DN)" killer cells. Besides CD56 and CD16, these cells lack other standard lymphocyte markers including CD3, CD14, CD19, CD20, and CD33, although the majority of these DN cells express surface markers considered to be NK-specific, including CD8α, CD94, CD224, NKp30, CD127, NKp46, NKG2D, and NKG2A. We sorted CD3- CD19- CD14- CD20- human PBMNCs into CD16- CD56Bright, CD16+ CD56dim, CD16+ CD56- and CD16-CD56-(DN) cellular subsets by flow cytometry, then expanded these subsets in vitro by co-culture with irradiated EBV-transformed LCL feeder cells and IL-2 (500IU/mL). DN cells expanded 40-60 fold after 14 days in culture, with 55% of cells retaining the DN phenotype while 40% of cells acquired expression of CD56 but not CD16 (termed CD56SP). Generation of CD56SP cells from DN cells cultured in vitro suggests that circulating DN cells contain NK cell precursors. When DN cells were expanded for 14 days in culture and the residual remaining DN cells were then resorted and re-expanded for an additional 14 days, exponential cellular growth continued. After this addition 14 days of culture, 10-40% of cells became CD56SP NK cells, while the remainder remained DN. Repeated cycles of resorting residual DN cells demonstrated that the DN subset could continually regenerate additional DN cells as well as produce CD56SP NK cells for at least 45 days, suggesting unique proliferative and differentiative properties of this cell population. CD56SP NK cells generated in a 14 day cell culture from DN cells had up-regulation of a variety of cell surface receptors including CD30 (+57%), CD127 (+0.7%), CD161 (+69%), CD57 (+5.7%), CD8α (+40%), CD244 (+99%), NKp30 (+79%), NKp44 (+53%), CD25 (+3.5%), CD69 (+95%), and had higher intracellular IFN-γ (+35%), and perforin (+60%), suggesting both NK differentiation and education occurred in these progeny (figure). In contrast, expression of inhibitory receptors in the DN population, such as CD94 (88%), CD158e (12%) and NKG2A (24%) showed minimal change in expression levels in their progeny DN cells or CD 56SP NK cells at culture-day 14, consistent with prior studies showing inhibitory receptors are less environmentally influenced. CD56SP cells generated from the DN cells also had higher levels of bone marrow and lymph node homing receptors CXCR4 and CCR7 and possessed superior degranulation capacity against K562 cells compared to pre or post expanded DN or pre-expanded CD56SP cells (figure). Further, gene expression analysis comparing DN vs. CD56SP cells at day 0 and DN and CD56SP cells generated from DN cells at day 14, showed DN cells had a gene expression profile with enhanced expression of genes involved in processes such as cell division and replication, while CD56SP cells generated from DN cells had a more cytotoxic or "active phenotype". Conclusion: CD56- 16- DN cells share many characteristics with canonical NK cells, have long-term in vitro differentiation and proliferation capacity, and may represent a cell population that is a self-renewing progenitor for highly activated and cytotoxic CD56SP NK cells. Isolation and expansion of a progenitor population with long-term NK cell repopulating potential in adoptive NK cell immunotherapy trials could offer advantages over trials transferring more differentiated NK cells with limited in vivo proliferative capacity. Disclosures No relevant conflicts of interest to declare.

Patients with GATA2 deficiency specifically lack CD56bright/CD16- natural killer (NK) cells, while the CD56dim/CD16+ (DP) subset is preserved. CD56bright/CD16- cells have been postulated to be the precursor population for more mature and cytotoxic CD16+NK cells. The mechanism by which an immature population disappears while a putatively more mature population is preserved is not understood. Our group has recently shown that CD16+/CD56dim/- NK cells have a distinct ontogeny, showing little overlap with CD56+/CD16- NK cells, which share clonal derivation with myeloid, T and B lineages (Wu et al, Cell Stem Cell 2014). We hypothesize that the missing CD56bright NK population in GATA2 deficiency is due to a maturation defect in a specific hematopoietic stem/progenitor population, separate from those relatively preserved progenitors producing CD16+ NK cells. We performed ex vivo expansion of NK cells from 4 healthy volunteers and 5 patients with GATA2 mutations and compared phenotypes at baseline and upon expansion. Sorted peripheral blood total NK (TNK) cells (CD3, CD20, CD14 depleted and expressing CD16 and/or CD56) and double negative (DN) cells (CD3-/CD20-/CD14- and CD16-/CD56-) were cultured in the presence of IL2 and irradiated Epstein-Barr virus-transformed lymphoblastoid cells. After 14 days of culture, each cell culture was characterized and re-sorted into TNK cells and DN cells, and cultured for an additional 14 days with restimulation. Subject 1 had GATA2 c.229+1 G>A mutation and presented with pancytopenia, recurrent warts, classic B/NK lymphopenia/monocytopenia and MDS with trisomy 8. Subjects 2 and 5, and subjects 3 and 4 were from the same families, respectively, and all shared the same intron 5 GATA2 c.1017+572C>T mutation. The proband, subject 2 had low grade MDS, recurrent warts and classic B/NK lymphopenia and monocytopenia. However, subjects 3, 4 and 5 had normal peripheral blood counts and bone marrow morphology, with only mild B or NK lymphopenia. TNK cells from subject 1 had profound lack of CD56bright cells at baseline, and showed decreased ex vivo expansion efficiency; 18.5-fold increase at day 14 compared to a 39.4-fold increase for control TNK cells. Even after expansion, the CD56bright population was still absent. When DN cells from subject 1 were placed into culture, they did not give rise to a CD56brightpopulation after 14 or 28 days, while control DN cells did give rise to a clear CD56bright population, also expressing additional NK-defining markers such as NKG2D and NKp46. TNK cells from patients with GATA2 intron 5 mutations had a less striking phenotype and were more variable. Subject 2 had greatly decreased CD56bright NK cells at baseline, in contrast to her asymptomatic father, subject 5, and to subjects 3 and 4 from another family. Ex vivo expansion kinetics for the TNK cells from all 4 subjects were similar to controls. By day 14 in culture, their TNK cells showed preserved generation of CD56bright/+ cells and DP cells. DN cells from subject 2 showed no CD56 expression upon expansion at day 14 and only a minor CD56dim population at day 28. Subjects 3, 4 and 5 showed comparable phenotypes at baseline and upon expansion until day 14 (Figure 1). GATA2 DN cells sorted and expanded twice from DN cells at days 1 and 14, at day 28 had much lower CD56bright NK cells (1.1±0.6% vs. 10.1±3.9%; p CD107a functional analysis showed impaired NK degranulation capacity upon K562 co-incubation in expanded GATA2 TNK cells and DN cells compared to their controls at day 14 (12.4±0.6 vs. 3.5±0.1 vs. 6.7±0.1 (p Our findings suggest that NK differentiation defects in GATA2 deficiency may be due to a differentiation block from a precursor present in the DN subset which is impaired in giving rise to CD56bright/CD16- cells upon prolonged culture, and also potentially in vivo. The differences between the GATA2c.229+1 G>A and GATA2 c.1017+572C>T (intron 5) samples in both phenotype and expansion characteristics correlate with the clinical severity of patients, and suggest a mutation-specific, dose-dependent role of GATA2 in NK development, as well as other modifying factors. Disclosures Townsley: GSK: Research Funding.

Although pilot clinical trials have shown adoptive NK cell transfer can result in tumor regression in humans with cancer, additional insight from animal models are needed to optimize NK cell proliferation in vivo as well as to improve their homing and retention in the tumor microenvironment. Mice have provided fundamental insights into NK cell biology, although significant divergence from humans as a consequence of evolution limits their usefulness to optimize adoptive transfer of ex vivo expanded NK cells in humans. Rhesus macaques (RM) express orthologues to most human MHC class I and II genes, and unlike mice, express KIRs and CD56, making them phenotypically and functionally similar to human NK cells. Further, unlike murine NK cells, cultured macaque NK cells can be expanded ex vivo for prolonged periods of time augmenting their cytotoxicity. Although recent studies have provided detailed characterization of freshly isolated RM NK cells, little information exists on the phenotype and function of these cells following ex vivo expansion. We investigated the feasibility of expanding RM NK cells ex vivo using culture conditions similar to those used to expand clinical grade human NK cells and characterized the cytotoxic function of subsets of these cells based on CD16 and CD56 expression. NK cells were enriched from RM PBMCs by either bead immunodepletion of CD3+ cells or by flow sorting) CD3-/CD20-/CD14-/CD16+ single positive (CD16SP) or CD3-/CD20-/CD14-/CD16-/CD56+ single positive (CD56SP) or CD3-/CD20-/CD14-/CD16+/CD56+ double positive (CD16/56DP) NK cells. NK cells were expanded in vitro by co-culture with an irradiated human EBV lymphoblastoid cell line (EBV-LCL) in media containing 500IU/uL of human IL-2. In contrast to humans, where resting NK cells from the peripheral blood are mostly CD16/56DP with smaller SP populations, the majority of NK cells from RM peripheral blood were CD16SP (Figure). Despite this difference, RM NK cells appeared phenotypically similar to human NK cells following ex vivo expansion, and had similar expansion kinetics, typically yielding a 100-fold increase in cells that were 99% CD3- and mostly CD16/56DP or CD56SP with a minor fraction of CD16SP cells. Using NKG2A as a pan NK cell marker as well as expression levels of CD16 and CD56, we evaluated for changes in the cytotoxic function associated with ex vivo expansion in 5 distinct NK cell subsets; CDSP16bright, CD16SPdim, CD56SPbright, CD56SPdim, and CD16/56DP. After 7 days expansion, CD16SP populations contracted substantially in contrast to CD16/56DP and CD56SP populations which increased. After 14 days of expansion, the CD16SP population represented Conclusion These data show RM NK cells can be expanded ex vivo using culture conditions similar to those used to expand clinical grade human NK cells. RM NK cells expanded using EBV-LCL feeder cells are phenotypically similar to expanded human NK cells and based on CD16 and CD56 expression, contain subpopulations that have varying degrees of cytotoxicity against K562 targets. The similarities between expanded RM and human NK cells establish this animal model as having excellent potential to optimize adoptive NK cell transfer in humans. We aim to establish this model and test expansions in vivo. Disclosures: No relevant conflicts of interest to declare.

Natural killer (NK) cells are cytotoxic leukocytes defined in humans and rhesus macaques by the expression of CD56 and/or CD16, and the absence of T, B, and myeloid markers. Functionally, they are defined by lysis of tumor targets in a major histocompatibility complex (MHC)–unrestricted fashion, and production of cytokines critical to innate immunity. Ex vivo expanded NK cells are being developed as adoptive transfer therapeutics for a variety of malignancies. However, the ontogeny and hematopoietic lineage relationships of human NK cells have been difficult to study, given differences in phenotype and function between human and murine NK cells, poor NK engraftment and development in human xenografts, and restricted development of NK cells from hematopoietic stem and progenitor cells (HSPC) in vitro. In addition, several phenotypic subsets of NK cells have been described, and their relationships and relative locations within the hematopoietic hierarchy are supported by limited in vivo data. We “barcoded” individual CD34+ HSPC from 3 macaques using lentiviral vectors carrying highly diverse oligonucleotides, allowing precise quantitation of clonal contributions via low cycle PCR amplification followed by high-throughput sequencing and computational analysis (Lu et al, Nat Biotech). We quantitated barcode levels, and thus individual HSPC clonal contributions, over time and between lineages, following TBI and infusion of autologous barcoded CD34+ cells. Reconstitution of the NK compartment was clonally distinct from T, B and myeloid (My) lineages. Following reconstitution of NK, T, B and My from uni-lineage progenitors at 1-2 months(m), by 3-4 m, as expected from murine and in vitro models, multi-lineage clones began to contribute and dominate, first B/My, then B/T/My. However, NK cells remained clonally distinct through 9 m, despite overall clonal diversity and marking levels in NK lineage similar to B/T and My. Even the most abundant clones in the NK lineage were not found contributing in any significant way to B/T or My lineages. Shared B/T/My/NK clones finally began to contribute more extensively at 9-14 m. We fractionated peripheral blood NK cells into 3 NK subsets and compared them to overall PB and lymph node NK cells at 4.5-6.5 m. The majority (75-90%) of PB rhesus NK cells are found in the CD16+/CD56- PB subset (corresponding to human CD16+/CD56dim cytotoxic NK), and this subset accounted for the clonal pattern in overall NK cells (Pearson r=0.98 vs overall NK cells), and the disparity from B/T/My, with r values all Disclosures: No relevant conflicts of interest to declare.

Abstract 2047 Induced pluripotent stem cell (iPSC) based therapies offer a promising path for patient-specific regenerative medicine. However, residual undifferentiated iPSCs within the graft present a potential risk of tumor development. Including a suicide gene system in the transplanted cells would allow the elimination of the transplant in case of an adverse event. For this purpose we propose the use of an inducible caspase 9 (iCasp9) suicide gene system (Di Stasi et al., 2011), consisting of human caspase 9 fused to a modified human FK-binding protein. After conditional iCasp9 dimerization by a synthetic small molecule chemical inducer (AP1903) the marked cells undergo rapid apoptosis (Clackson et al., 1998). To explore the system in the context of iPSC, we studied in vitro and in vivo purging strategies as well as therapeutically modalities using lentiviral constructs expressing iCasp9 either from a constitutional promoters or a pluripotency-specific promoter in a teratoma mouse model. We designed lentiviral vectors consisting of the iCasp9 gene linked via a 2A peptide to human CD19, driven by either CMV, EF1α, or embryonic stem cell-specific EOS (3+) promoters (Hotta et al., 2009). The iCasp9 lentiviral constructs were introduced into either into mouse iPSCs (C57Bl/6) or rhesus monkey fibroblasts that were subsequently used to derive monkey iCasp9-iPSCs for future nonhuman primate model. In mature rhesus monkey fibroblasts, cells expressing iCasp9/CD19 driven by either CMV or EF1α promoters underwent rapid and complete apoptosis with exposure to the AP1903 dimerizer (0.5μM to 10 μM), whereas the EOS (3+) promoter drove minimal iCasp9-CD19 expression in fibroblasts, as expected. Murine iPSCs transduced with the CMV-iCasp9 construct demonstrated silencing of the transgenes and no significant apoptosis induction with exposure to AP1903 (10 μM). In contrast, both the EF1α and the EOS (3+) promoters highly expressed iCasp9, allowing for successful induction of apoptosis in iPSCs following AP1903 treatment, with dosages ranging from 0.5μM to 10 μM in vitro, cell apoptosis were analyzed by Annexin V/7AAD using flow cytometry. We next tested the iCasp9 suicide gene system in an in vivo NSG mouse teratoma assay. For this we chose three different approaches: 1) In vitro treatment with AP1903(10 μM) of the iPSCs 4 hours prior to cells injection to NSG mouse (in vitro purging iCasp9-CD19 expressing cells); 2) Immediate I.V. injection of AP1903 (1mg/kg, single does) after injection of the iPSCs into NSG mouse (in vivo purging) ; and 3) Treatment with AP1903(1mg/kg) one week after iPSC injection (in vivo purging after initial teratoma formation). A non-iCasp9 mouse iPSC clone was used as a control in the assay. The results showed that mice injected with iPSCs expressing EOS (3+) or EF1α iCasp9 that underwent in vitro purging with 10 μM AP1903 did not show teratoma formation until 2 months, whereas the non-iCasp9 mouse iPSCs control mice reached the end point size of the teratoma after two weeks(1.494±0.29 g, mean±SD). Mice treated with a single dose of 1mg/kg AP1903 post iPSC cell injection demonstrated markedly delayed teratoma formation. Mice treated one week post iPSC injection with a daily AP1903 regimen (1mg/kg) for four days demonstrated inhibition of further tumor growth, but did not fully ablate existing tumors. In conclusion, we have shown that the iCasp9/AP1903 system is effective in eliminating iPSCs in vitro and in vivo. Furthermore, we show that efficient transgene expression and subsequent elimination of pluripotent cells depends on the internal promoter of the viral construct. Currently, we are investigating why the induction of the suicide gene failed to ablate existing tumors efficiently (insufficient dosage, silencing of transgenes in vivo). Disclosures: No relevant conflicts of interest to declare.

Abstract 51 Telomeres are ribonucleoprotein structures located at the end of linear chromosomes that serve to maintain genomic integrity and cellular proliferative capacity. In highly proliferative cells, the enzyme complex telomerase is responsible for the maintenance and elongation of telomeres, as the process of DNA replication inherently results in loss of terminal nucleotides. Critically short telomeres and deficiency in telomerase activity are etiologic contributors in the bone marrow failure syndromes, idiopathic pulmonary fibrosis, and liver cirrhosis. Although dysfunctional telomere machinery clearly is pathogenic in humans, important clinical features such as highly variable penetrance of phenotype and organ specificity are not well understood. A major obstacle in the investigation of these diseases is the lack of primary tissue, especially early in the clinical course. Direct reprogramming of somatic cells to a pluripotent phenotype (induced pluripotent stem cells, iPSc) by forced expression of a set of defined transcription factors may allow investigation of these phenotypes and specificity in a patient-specific manner. One hallmark of all pluripotent cells, embryonic stem cells and iPSc, is the maintenance of telomere length, most likely due to upregulation of telomerase. Thus, these cells are good candidates through which to directly investigate the effect of loss-of-function mutations within the telomerase complex. Using forced expression of the reprogramming factors Oct4, Sox2, Klf4, and c-myc via retroviral (single transgenes) or lentiviral (polycistronic) gene transfer, we derived multiple iPSc lines from dermal fibroblasts of patients harboring heterozygous loss-of-function mutations in the telomerase genes TERT([R1084P], [R889X]) and TERC([-58C>G]), as well as from healthy subjects. These mutations were shown in vitro to reduce telomerase activity of the mutant allele. Generated iPSc lines morphologically resembled human ES cells, expressed endogenous pluripotency markers (such as TRA 1–60, TRA 1–81, SSEA4, NANOG, OCT4), and showed a similar mRNA expression profile as compared to embryonic stem cells in microarray analysis. Over multiple passages (currently up to 40), iPSc retained their self-renewal capacity and formed teratomas in immune-compromised NSG mice. We randomly chose three iPSc lines from each patient and healthy controls to study telomere dynamics. Telomerase-mutant iPS cells elongated their telomeres during the first 10 passages compared to parental (telomerase negative) fibroblasts, as determined by quantitative real-time PCR and Southern blot. However, telomere elongation was significantly less than in iPSc derived from healthy individuals (p=0.003). Moreover, the pattern and extent of elongation varied among different iPSc lines harboring the same mutation. Telomerase mRNA expression was lower in telomerase-mutant iPS cells than in healthy controls. Additionally, telomerase activity, measured by standard TRAP assay, in early telomerase-mutant iPS cells was reduced relative to control iPSc, but later passage cells tended to have similar activity, suggesting a passage-effect on telomerase activity levels. In conclusion, iPSc can be derived from human telomerase-deficient cells. These cells elongate telomeres to a lesser extent than iPSc from healthy controls, indicating that functional telomerase is the main mechanism of telomere elongation in iPSc. Therefore these cells could be valuable tools in the study of human telomerase deficiencies. Unlike previous studies (Agarwal et al., Nature 2010) investigating iPSc from patients with X-linked dyskeratosis congenita (in which a loss-of-function mutation within DKC1 results in short telomeres), we did not observe significant upregulation of TERC as a compensating mechanism during reprogramming in our telomerease-mutant iPSc. Furthermore, in contrast to the DKC1-mutant iPSc described by Batista et al. (Nature 2011), TERT/TERC mutant iPSc did not show signs of impaired proliferation or self-renewal capacity in long-term culture, likely reflecting clinical differences among patients with TERT/TERC and DKC1 mutations. However, elongation patterns and telomerase activity levels are heterogeneous among clones and passages, indicating the importance of kinetic studies and sample size when studying telomere dynamics in iPSc. Disclosures: Dunbar: ASH: Honoraria.

Abstract 1051 Poster Board I-73 Oligomerization through the NHR2 domain is essential for AML1-ETO's inhibition of granulocytic differentiation and enhanced clonogenic potential of primary bone marrow cells. We show here that Oridonin interferes with AML1-ETO oligomerization through its cleavage fragment DAML1-ETO, which consists of the amino acids (aa) 188-752 of the parental oncoprotein or aa 40-604s of the wild-type ETO. DAML1-ETO interacts with the parental AML1-ETO through NHR2 and exerts dominant negative effects on AML1-ETO with regard to DNA binding, transregulatory activity on target genes and regulation of leukemic cell survival, differentiation and proliferation both in vitro and in vivo. Moreover, Oridonin can activate retinoic acid and cAMP/PKA pathways, and potentiate differentiation induced by all-trans retinoic acid (ATRA) and G-CSF. Consistently, combined use of Oridonin, ATRA and G-CSF significantly prolongs lifespan of t (8;21) leukemic mice and, interestingly, we find that this treatment targets the Lin-/Sca-1+/C-KIT+ and Lin-/Sca-1-/C-KIT+ leukemia initiating cells. These data suggest that Oridonin, and potentially other small molecules, can inhibit AML1-ETO oligomerization and leukemogenic function, thus providing a targeted therapy that activates key regulatory pathways for myelomonocytic cell differentiation and apoptosis. Disclosures: No relevant conflicts of interest to declare.

We read with interest the article by Ma et al. on how a high-fat diet reduced regulatory T cells (Tregs) in liver and influenced susceptibility to endotoxin-induced liver injury.1 This interesting finding may also explain a previous report on increased sensitivity in mice with fatty liver to concanavalin A–induced hepatitis.2 The authors attributed the mechanism of Treg loss to oxidative stressinduced apoptosis. Recently, several agents were found that regulate Treg survival and function. Among them, leptin, a neuroendocrine and immune mediator,3 acts as a negative signal for the proliferation of Tregs.4 The suppressive effect of leptin on Tregs was mediated by the modulation of cyclin-dependent kinase inhibitor p27 (p27kip1) levels and phosphorylation of extracellular-related kinase 1 (ERK1) and ERK2. Serum leptin levels can be considered as a signal to the body of its energy reserves.5 Previous studies have clearly shown that serum leptin levels correlate directly with the severity of hepatic steatosis.6 Therefore, based on these findings, it will be interesting and important to verify the role elevated leptin levels play in Treg loss in mice with fatty livers. In addition, a significant increase of the percentage of peripheral Tregs was observed in mice with a genetic deficiency of leptin (ob/ob mice).4 One of the most prominent phenotypes of ob/ob mice was fatty liver. So, further research should be carried out to determine the percentage of Tregs in liver of ob/ob mice. These studies will improve our knowledge about the interaction between hepatocytes and immune cells in the liver.

AML1-ETO fusion gene is generated from chromosome translocation t(8;21) in acute myeloid leukemia (AML). It is recently reported that its spliced variant AML1-ETO9a induced leukemia rapidly in murine model. Here we detected AML1-ETO9a expression in 58 of 71 (81.7%) patients with t(8;21) AML by using qualitative RT-PCR. Quantitative RT-PCR revealed significantly higher C-KIT levels in the AML1-ETO9a positive group than in the negative group (P=0.0001). Among 29 cases harbored C-KIT mutations, 28 (96.6%) expressed AML1-ETO9a. Clinically, patients expressing AML1-ETO9a exhibited significantly elevated white blood cell counts and a short overall survival time (P=0.0455 and 0.0039, respectively). Morphologically, although there is no difference of leukemic blasts percentage between AML1-ETO9a positive and negative cases (P=0.1169), the latter possessed more aberrant myelocytes (P=0.0462). Taken together, AML1-ETO9a correlated with C-KIT overexpression/mutation and indicated poor disease outcome in t(8;21) AML.