Biology subjects

Banuelos, A.

Publications and source records attributed to Banuelos, A..

9 recordsLinked to original sources

bioRxiv2026

Neogenin-1 marks myeloid-primed fetal hematopoietic stem cells that undergo progressive lineage-restriction with age

During aging, hematopoietic stem cells (HSCs) increasingly shift from balanced to myeloid-biased differentiation, resulting in reduced lymphoid output and impaired adaptive immunity. The question of whether this lineage bias is established in a subset of HSCs during early development or primarily emerges with aging warrants further investigation. Here, we investigate whether myeloid-biased HSCs (my-HSCs) are established at the fetal liver stage by specifically examining Neogenin-1 (NEO1), a previously defined marker of my-HSCs. We identify two distinct populations of Hoxb5+ HSCs in the fetal liver: NEO1+ and NEO1-, with NEO1+ HSCs exhibiting transcriptional and functional characteristics consistent with my-HSCs. With age, my-HSC-associated transcriptional programs become increasingly reinforced across the Hoxb5+ pHSC compartment, with NEO1+ cells showing early enrichment of this program and both NEO1+ and NEO1- cells acquiring broader myeloid-biased features in aging. These findings suggest that lineage programming can begin early in development and is further shaped by age-related changes, potentially contributing to the functional decline observed in the aging hematopoietic system.

developmental biology↗

bioRxiv2026

Hoxb5+ fetal liver hematopoietic stem cells establish lifelong hematopoiesis and exhibit enhanced ITGA4-dependent engraftment

Adult long-term hematopoietic stem cells (LT-HSCs) are classically defined by self-renewal, multilineage regenerative capacity, and relative quiescence, but how and when lifelong LT-HSCs are established during development remains unclear. Here, we demonstrate that Hoxb5 fetal liver HSCs exhibit bona fide LT-HSC activity, including long-term multilineage reconstitution and serial transplantation capacity, whereas Hoxb5- fetal liver HSCs display limited regenerative potential. Embryonic lineage tracing further demonstrates that E14.5 Hoxb5-expressing hematopoietic cells contribute broadly to adult hematopoiesis, including the adult HSC compartment, and give rise to functional adult LT-HSCs. Across developmental stages, single-cell transcriptional profiling revealed that fetal Hoxb5 HSCs remain highly proliferative while maintaining canonical LT-HSC transcriptional programs and superior repopulating activity relative to predominantly quiescent adult Hoxb5 HSCs. Fetal Hoxb5 HSCs also exhibited elevated ITGA4-mediated adhesion programs, and disruption of the ITGA4-VCAM1 axis impaired engraftment following transplantation. Together, these findings establish a developmental continuum linking fetal and adult LT-HSCs and identify enhanced ITGA4-mediated adhesion as a defining feature of fetal LT-HSCs.

developmental biology↗

bioRxiv2026

Hoxb5 Enriches Long-Term Hematopoietic Stem Cell Activity within the Mouse Fetal Liver Phenotypic HSC Compartment

Hematopoietic stem cells (HSCs) in the adult mouse can be prospectively isolated to near-purity through phenotypic markers, enabling detailed analysis of stem cell function. Homeobox B5 (Hoxb5) was previously identified as a definitive marker of long-term (LT) HSCs in adult bone marrow1. In contrast, fetal HSCs have not been purified to the same extent. Here, we show that Hoxb5 is expressed in fetal liver (FL) HSCs at embryonic day (E) 12.5-16.5 using a single-color tri-mCherry reporter driven by endogenous Hoxb5 regulation. Prospective purification by stringent multiparameter flow cytometry revealed Hoxb5 FL-HSCs to exhibit robust, multilineage reconstitution upon serial transplantation. Quantitative assays reveal that Hoxb5 enriches FL-HSCs to near-single-cell purity, analogous to its role in the adult bone marrow, underscoring its reliability in distinguishing LT-HSCs throughout hematopoietic ontogeny. Notably, Hoxb5 expression is not exclusive to FL-HSCs, as it is also detected across the fetal liver hematopoietic hierarchy and in fetal liver endothelial cells, suggesting developmental stage-specific regulation of its expression. In addition, single-cell RNA sequencing of FL-HSCs identified distinct transcriptional states defined by Hoxb5 expression. These findings establish Hoxb5 as a robust marker for enhancing the purification of fetal liver phenotypic HSCs (pHSC) and provide a framework for dissecting the molecular regulation of HSC ontogeny.

developmental biology↗

bioRxiv2023

Engineered CD47 protects T cells for enhanced antitumor immunity

Adoptively transferred T cells and agents designed to block the CD47/SIRP axis are promising antitumor therapeutics, which activate distinct arms of the immune system. We administered anti-CD47 (CD47) with adoptively transferred T cells with the goal of enhancing antitumor efficacy but observed rapid macrophage-mediated clearance of T cells expressing chimeric antigen receptors (CARs) or engineered T cell receptors, which blunted therapeutic benefit. CD47 mediated CAR T clearance was potent and rapid enough to serve as an effective safety switch. To overcome this challenge, we engineered a CD47 variant (47E) that engaged SIRP and provided a "dont-eat-me" signal that was not blocked by CD47 antibodies. TCR or CAR T cells expressing 47E were resistant to clearance by macrophages following CD47, and mediated significant, sustained macrophage recruitment into the TME. Although many of the recruited macrophages manifested an M2-like profile, the combined therapy resulted in synergistic enhancement in antitumor efficacy. This work identifies macrophages as major regulators of T cell persistence and illustrates the fundamental challenge of combining T cell directed therapeutics with those designed to activate macrophages. It further delivers a therapeutic approach capable of simultaneously harnessing the antitumor effects of T cells and macrophages that manifests markedly enhanced potency against solid tumors.

bioengineering↗

bioRxiv2023

Pannexin-1 channels promote CD8+ T cell effector and memory responses through distinct intracellular pathways

Sensing of extracellular ATP (eATP) controls CD8+ T cell function. Their accumulation can occur through export by specialized molecules, such as the release channel Pannexin-1 (Panx1). Whether Panx1 controls CD8+ T cell immune responses in vivo, however, has not been previously addressed. Here, we report that T cell-specific Panx1 is needed for CD8+ T cell responses to viral infections and cancer. We found that CD8-specific Panx1 promotes both effector and memory CD8+ T cell responses. Panx1 favors initial effector CD8+ T cell activation through extracellular ATP (eATP) export and subsequent P2RX4 activation, which helps promote full effector differentiation through extracellular lactate accumulation and its subsequent recycling. In contrast, Panx1 promotes memory CD8+ T cell survival primarily through ATP export and subsequent P2RX7 engagement, leading to improved mitochondrial metabolism. In summary, Panx1-mediated eATP export regulates effector and memory CD8+ T cells through distinct purinergic receptors and different metabolic and signaling pathways.

immunology↗

bioRxiv2022

Androgen receptor condensates as drug targets

Transcription factors are among the most attractive therapeutic targets but are considered largely undruggable due to the intrinsically disordered nature of their activation domains. Here we show that the aromatic character of the activation domain of the androgen receptor, a therapeutic target for castration resistant prostate cancer, is key for its activity as a transcription factor by allowing it to partition into transcriptional condensates. Based on this knowledge we optimized the structure of a small molecule inhibitor, previously identified by phenotypic screening, that targets a specific transactivation unit within the domain that is partially folded and rich in aromatic residues. The optimized compounds had more affinity for their target, inhibited androgen receptor-dependent transcriptional programs, and had antitumorigenic effect in models of castration-resistant prostate cancer in cells and in vivo. These results establish a generalizable framework to target small molecules to the activation domains of oncogenic transcription factors and other disease-associated proteins with therapeutic intent.

biochemistry↗

bioRxiv2021

Anti-GD2 antibody disrupts GD2:Siglec-7 interactions and synergizes with CD47 blockade to mediate tumor eradication

The disialoganglioside GD2 is consistently overexpressed in neuroblastoma and osteosarcoma, and is variably expressed in other sarcomas, gliomas, neuroendocrine tumors, and epithelial cancers. Anti-GD2 antibodies have improved the survival rates of patients with neuroblastoma only when administered as part of intense chemotherapy-based cytotoxic regimens, which are associated with debilitating late effects including hearing loss, growth retardation, and secondary leukemias. Despite broad expression of GD2 on osteosarcoma, anti-GD2 antibody has not mediated significant antitumor activity in that disease or any other GD2+ cancers. CD47 is a checkpoint molecule overexpressed on tumor cells that inhibits macrophage activity, and CD47 blockade has demonstrated promising clinical activity in early human trials. We investigated whether anti-CD47 antibody could enhance the efficacy of anti-GD2 antibody in neuroblastoma and other GD2+ malignancies. We demonstrate substantial synergy of these two agents, resulting in the recruitment of tumor associated macrophages (TAMs) to mediate robust and durable anti-tumor responses. The responses are driven by GD2-specific factors that reorient the balance of macrophage activity towards phagocytosis of tumor cells, including disruption of a newly described GD2:Siglec-7 axis. These results demonstrate the unique synergy of combining anti-GD2 with anti-CD47, which has the potential to significantly enhance outcomes for children with neuroblastoma and osteosarcoma and will soon be investigated in a first-in-human clinical trial.

immunology↗

bioRxiv2019

Mass spectrometry analysis of mouse hematopoietic stem cells and their progenitors reveals differential expression within and between proteome and transcriptome throughout adult and aged hematopoiesis

SummaryHematopoietic stem cells (HSCs) are responsible for the generation of blood and immune cells throughout life. They have the unique ability to self-renew and generate more HSCs or differentiate into a progenitor cell in response to cell-intrinsic and -extrinsic stimuli. The balance of HSC fate commitment is critical for a healthy blood supply. Imbalances during hematopoiesis, which are frequent in aging, can result in hematological malignancies and pre-malignancies as well as increase risk of atherosclerosis. Given the importance of HSCs and their progenitors, they have been extensively characterized in genomic and transcriptomic studies. However, an understanding of protein expression within the HSC compartment and more broadly throughout hematopoiesis remains poorly understood, and it has been widely reported that the correlation between mRNA and proteins is more complicated than previously thought. Previous mouse mass spectrometry studies have focused either specifically on stem and the first early progenitor or broadly across mixed populations of stem and progenitor cells, which do not allow for cell-type specific protein resolution across stages of differentiation. Mass cytometry has been employed to characterize transcription factor expression in human HSCs and progenitors but does not apply an unbiased discovery approach. New mass spectrometry technology now allows for deep proteomic coverage with no more than 200 ng of sample input. We report here a proteomics resource characterizing protein expression in mouse adult and aged HSCs, multipotent progenitors and oligopotent progenitors, 12 cell types in total. We validated differential expression by flow cytometry analysis and immunofluorescence staining. Additionally, we investigated the relationship between mRNA and protein levels of individual genes in HSCs compared to progenitors through RNA sequencing studies and identified two proteins that appear to be uniquely regulated in the HSC compartment, Cpin1 and Adnp. In summary, this resource provides proteomic coverage of adult and aged hematopoietic stem cells and their progenitors and reveals changes in protein abundance between cell types, with potential future implications in understanding mechanisms for stem-cell maintenance, niche interactions and fate determination.

cell biology↗

bioRxiv2019

Overexpression of CD47 is associated with brain overgrowth in 16p11.2 deletion syndrome

One of the most common genetic linkages associated with neuropsychiatric disorders, such as autism spectrum disorder and schizophrenia, occurs at the 16p11.2 locus. Copy number variants (CNVs) of the 16p gene can manifest in opposing head sizes. 16p11.2 deletion carriers tend to have macrocephaly (or brain enlargement), while those with 16p11.2 duplication frequently have microcephaly. Increases in both gray and white matter volume have been observed in brain imaging studies in 16p11.2 deletion carriers with macrocephaly. Here, we use human induced pluripotent stem cells (hiPSCs) derived from controls and subjects with 16p11.2 deletion and 16p11.2 duplication to understand the underlying mechanisms regulating brain overgrowth. To model both gray and white matter, we differentiated patient-derived iPSCs into neural progenitor cells (NPCs) and oligodendrocyte progenitor cells (OPCs). In both NPCs and OPCs, we show that CD47 (a dont eat me signal) is overexpressed in the 16p11.2 deletion carriers contributing to reduced phagocytosis both in vitro and in vivo. Treatment of 16p11.2 deletion NPCs and OPCs with an anti-CD47 antibody to block CD47 restores phagocytosis to control levels. Furthermore, 16p11.2 deletion NPCs and OPCs upregulate cell surface expression of calreticulin (a pro-phagocytic eat me signal) and its binding sites, indicating that these cells should be phagocytosed but fail to be eliminated due to elevations in CD47. While the CD47 pathway is commonly implicated in cancer progression, we document a novel role for CD47 in regulating brain overgrowth in psychiatric disorders and identify new targets for therapeutic intervention.

neuroscience↗