Biology subjects

Dunbar, C. E.

Publications and source records attributed to Dunbar, C. E..

7 recordsLinked to original sources

bioRxiv2024

Low Kit expression identifies Hematopoietic Stem Cell subsets with enhanced lymphoid potential in mice and humans

Hematopoietic stem cells (HSCs) with multilineage potential are critical for effective T cell reconstitution and restoration of the adaptive immune system after allogeneic Hematopoietic Cell Transplantation (allo-HCT). The Kitlo subset of HSCs is enriched for multipotential precursors,1, 2 but their T-cell lineage potential has not been well-characterized. We therefore studied the thymic reconstituting and T-cell potential of Kitlo HSCs. Using a preclinical allo-HCT model, we demonstrate that Kitlo HSCs support better thymic recovery, and T-cell reconstitution resulting in improved T cell responses to infection post-HCT. Furthermore, Kitlo HSCs with augmented BM lymphopoiesis mitigate age-associated thymic alterations, thus enhancing T-cell recovery in middle-aged hosts. We find the frequency of the Kitlo subset declines with age, providing one explanation for the reduced frequency of T-competent HSCs and reduced T-lymphopoietic potential in BM precursors of aged mice.3, 4, 5 Chromatin profiling revealed that Kitlo HSCs exhibit higher activity of lymphoid-specifying transcription factors (TFs), including Zbtb1. Deletion of Zbtb1 in Kitlo HSCs diminished their T-cell potential, while reinstating Zbtb1 in megakaryocytic-biased Kithi HSCs rescued T-cell potential, in vitro and in vivo. Finally, we discover an analogous Kitlo HSC subset with enhanced lymphoid potential in human bone marrow. Our results demonstrate that Kitlo HSCs with enhanced lymphoid potential have a distinct underlying epigenetic program.

immunology↗

bioRxiv2024

In vivo tracking of ex vivo generated 89Zr-oxine labeled plasma cells by PET in a non-human primate model

B cells are an attractive platform for engineering to produce protein-based biologics absent in genetic disorders, and potentially for the treatment of metabolic diseases and cancer. As part of pre-clinical development of B cell medicines, we demonstrate a method to collect, ex vivo expand, differentiate, radioactively label, and track adoptively transferred non-human primate (NHP) B cells. These cells underwent 10- to 15-fold expansion, initiated IgG class switching, and differentiated into antibody secreting cells. Zirconium-89-oxine labeled cells were infused into autologous donors without any preconditioning and tracked by PET/CT imaging. Within 24 hours of infusion, 20% of the initial dose homed to the bone marrow and spleen and distributed stably and equally between the two. Interestingly, approximately half of the dose homed to the liver. Image analysis of the bone marrow demonstrated inhomogeneous distribution of the cells. The subjects experienced no clinically significant side effects or laboratory abnormalities. A second infusion of B cells into one of the subjects resulted in an almost identical distribution of cells, suggesting a non-limiting engraftment niche and feasibility of repeated infusions. This work supports the NHP as a valuable model to assess the potential of B cell medicines as potential treatment for human diseases.

bioengineering↗

bioRxiv2023

Investigation of the Impact of Clonal Hematopoiesis on Severity and Pathophysiology of COVID-19 in Rhesus Macaques

Clinical manifestations of COVID-19 vary widely, ranging from asymptomatic to severe respiratory failure with profound inflammation. Although risk factors for severe illness have been identified, definitive determinants remain elusive. Clonal hematopoiesis (CH), the expansion of hematopoietic stem and progenitor cells bearing acquired somatic mutations, is associated with advanced age and hyperinflammation. Given the similar age range and hyperinflammatory phenotype between frequent CH and severe COVID-19, CH could impact the risk of severe COVID-19. Human cohort studies have attempted to prove this relationship, but conclusions are conflicting. Rhesus macaques (RMs) are being utilized to test vaccines and therapeutics for COVID-19. However, RMs, even other species, have not yet been reported to develop late inflammatory COVID-19 disease. Here, RMs with either spontaneous DNMT3A or engineered TET2 CH along with similarly transplanted and conditioned controls were infected with SARS-CoV-2 and monitored until 12 days post-inoculation (dpi). Although no significant differences in clinical symptoms and blood counts were noted, an aged animal with natural DNMT3A CH died on 10 dpi. CH macaques showed evidence of sustained local inflammatory responses compared to controls. Interestingly, viral loads in respiratory tracts were higher at every timepoint in the CH group. Lung sections from euthanasia showed evidence of mild inflammation in all animals, while viral antigen was more frequently detected in the lung tissues of CH macaques even at the time of autopsy. Despite the lack of striking inflammation and serious illness, our findings suggest potential pathophysiological differences in RMs with or without CH upon SARS-CoV-2 infection. HighlightsO_LINo evidence of association between CH and COVID-19 clinical severity in macaques. C_LIO_LIThe presence of CH is associated with prolonged local inflammatory responses in COVID-19. C_LIO_LISARS-CoV-2 persists longer in respiratory tracts of macaques with CH following infection. C_LI

genetics↗

bioRxiv2022

Ongoing Production of Tissue-Resident Macrophages from Hematopoietic Stem Cells in Healthy Adult Macaques

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

immunology↗

bioRxiv2020

barcodetrackR: an R package for the interrogation of clonal tracking data

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.

bioinformatics↗

bioRxiv2020

Validation and Long-Term Follow Up of CD33 Off-Targets Predicted In Vitro and In Silico Using Error-Corrected Sequencing in Rhesus Macaques

ABSTRACTThe programmable nuclease technology CRISPR/Cas9 has revolutionized gene editing in the last decade. Due to the risk of off-target editing, accurate and sensitive methods for off-target characterization are crucial prior to applying CRISPR/Cas9 therapeutically. Here, we utilized a rhesus macaque model to ask whether CIRCLE-Seq (CS), an in vitro off-target prediction method, more accurately identifies off-targets compared to in silico prediction (ISP) based solely on genomic sequence comparisons. We use AmpliSeq HD error-corrected sequencing to validate off-target sites predicted by CIRCLE-Seq and ISP for guide RNAs designed against TET2 and CD33 genes. A gRNA targeting TET2 designed using modern algorithms and predicted to have low off-target risk by both ISP and CIRCLE-Seq created no detectable mutations at off-target sites in hematopoietic cells following transplantation, even when applying highly sensitive error-corrected sequencing. In contrast, a CD33 gRNA designed using less robust algorithms with over 10-fold more off-targets sites predicted by both ISP and CIRCLE-Seq, however there was poor correlation between the sites predicted by the two methods. When almost 500 sites identified by each method were searched for in hematopoietic cells following transplantation, 19 detectable mutations in off-target sites were detected via error-corrected sequencing. Of these 19 sites, 8 sites were predicted in the top 500 sites by both methods, 8 by CIRCLE-Seq only, and 3 by ISP only. Cells with off-target editing exhibited no expansion or abnormal behavior in vivo in animals followed for up to 2 years. In conclusion, neither methodology predicted all sites, and a combination of careful gRNA design, followed by screening for predicted off-target sites in target cells by multiple methods may be required for optimizing safety of clinical development.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗

bioRxiv2019

Aberrant Clonal Hematopoiesis Following Lentiviral Transduction of HSPC in a Rhesus Macaque

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.

molecular biology↗