Immature Population Research Articles

Anterior Cruciate Ligament Injury in Skeletally Immature Patients

The incidence of anterior cruciate ligament (ACL) injury continues to increase in the skeletally immature population. These injuries were historically treated with nonsurgical measures in this age group due to concerns for iatrogenic damage to the physis during ACL reconstruction. However, delayed surgery often led to recurrent instability, meniscal tears, and chondral damage. With the development of newer and safer surgical techniques with satisfactory outcomes, ACL reconstruction has become much more common in this age group. The patient's skeletal age is often used to determine remaining growth, which helps to decide the surgical technique chosen. These techniques include physeal sparing, partial transphyseal, and transphyseal surgical options, using soft-tissue autografts. Each technique has been shown to have favorable patient-reported outcomes, but no technique is without the risk of complications. This article will review the management of skeletally immature ACL injuries, including relevant anatomy, risk factors for injury, assessment of skeletal age, and different treatment options and outcomes.

Fatty acid metabolism in neutrophils promotes lung damage and bacterial replication during tuberculosis.

Mycobacterium tuberculosis (Mtb) infection induces a marked influx of neutrophils into the lungs, which intensifies the severity of tuberculosis (TB). The metabolic state of neutrophils significantly influences their functional response during inflammation and interaction with bacterial pathogens. However, the effect of Mtb infection on neutrophil metabolism and its consequent role in TB pathogenesis remain unclear. In this study, we examined the contribution of glycolysis and fatty acid metabolism on neutrophil responses to Mtb HN878 infection using ex-vivo assays and murine infection models. We discover that blocking glycolysis aggravates TB pathology, whereas inhibiting fatty acid oxidation (FAO) yields protective outcomes, including reduced weight loss, immunopathology, and bacterial burden in lung. Intriguingly, FAO inhibition preferentially disrupts the recruitment of a pathogen-permissive immature neutrophil population (Ly6Glo/dim), known to accumulate during TB. Targeting carnitine palmitoyl transferase 1a (Cpt1a)-a crucial enzyme in mitochondrial β-oxidation-either through chemical or genetic methods impairs neutrophils' ability to migrate to infection sites while also enhancing their antimicrobial function. Our findings illuminate the critical influence of neutrophil immunometabolism in TB pathogenesis, suggesting that manipulating fatty acid metabolism presents a novel avenue for host-directed TB therapies by modulating neutrophil functions.

Studies on the influence of water quality on community assemblage of immature mosquitoes in different ecosystems along the Vaigai river, Tamil Nadu, South India

BackgroundOver the last few decades, river ecosystem is highly modified through various anthropogenic activities which are resulted to alter ecosystem functions and services. This modified ecosystem rendering conducive environment to mosquitoes through various ecological links for the self-sustaining populations. However, deciphering the community assemblage of immature mosquitoes with reference to water quality at modified ecosystem is very essential to make suitable control measure to curtail mosquito populations. In order to understand how the water quality influences the larval density, habitat specificity and community assemblage of immature mosquito populations, a study was conducted at different ecosystems (urban, semi-urban and rural) along the Vaigai river. The physicochemical parameters such as pH, TDS, salinity, conductivity, turbidity, DO, were analyzed at each study site.ResultsOur results clearly revealed that Anopheline species were highly preferred to breed less polluted habitat than Culicine species. Community assemblage by Anopheline and Culicine mosquitoes were found to be higher at all the studies whilst community assemblage by Anopheline were maximum at rural and semi-urban sites. Among the Anopheline species, Anopheles subpictus able to breed at high polluted habitat, particularly higher turbid level (28.49 ± 2.18 NTU) than other Anopheles species. Cx. gelidus mostly breed at sewage disposal habitats with high salinity level (1.01 ± 0.08) whilst Cx. bitaeniorhynchus bred in only fresh water bodies particularly low turbid habitats (3.97 ± 0.40 NTU). Grouping of immature mosquitoes based on the habitat similarity, An. subpictus, Cx. vishnui, An. vagus, Cx. tritaeniorhynchus, Cx. gelidus and Cx. quinquefasciatus were able to breed in highly polluted habitats which are resulted fell in group A than group B mosquitoes. Cx. vishnui and An. subpictus have strong habitat similarity (0.96) and can able to share their habitats with more number of Anopheline and Culicine mosquitoes.ConclusionsFrom the study we concluded that, Cx. vishnui and An. subpictus were most prevalent species and strong habitats similarity along the Vaigai river basin. An. subpictus and An. vagus can adapt to breed in polluted habitats and this may be adequate to extend the vectorial capacity and disease outbreak along the Vaigai river basin.

Identification and characterization of early human photoreceptor states and cell-state-specific retinoblastoma-related features.

Human cone photoreceptors differ from rods and serve as the retinoblastoma cell-of-origin, yet the developmental basis for their distinct behaviors is poorly understood. Here, we used deep full-length single-cell RNA-sequencing to distinguish post-mitotic cone and rod developmental states and identify cone-specific features that contribute to retinoblastomagenesis. The analyses revealed early post-mitotic cone- and rod-directed populations characterized by higher THRB or NRL regulon activities, an immature photoreceptor precursor population with concurrent cone and rod gene and regulon expression, and distinct early and late cone and rod maturation states distinguished by maturation-associated declines in RAX regulon activity. Unexpectedly, both L/M cone and rod precursors co-expressed NRL and THRB RNAs, yet they differentially expressed functionally antagonistic NRL and THRB isoforms and prematurely terminated THRB transcripts. Early L/M cone precursors exhibited successive expression of several lncRNAs along with MYCN , which composed the seventh most L/M-cone-specific regulon, and SYK , which contributed to the early cone precursors' proliferative response to RB1 loss. These findings reveal previously unrecognized photoreceptor precursor states and a role for early cone-precursor-intrinsic SYK expression in retinoblastoma initiation.

Lenalidomide and pomalidomide modulate hematopoietic cell expansion and differentiation in the presence of MSC

Cytopenia is a well-documented complication in the treatment of hematological malignancies with lenalidomide and pomalidomide. Although prior studies have highlighted direct effects on hematopoietic cells to explain this adverse effect, the involvement of hematopoietic-supportive stroma remains less understood. This study examined the effects of lenalidomide/pomalidomide on the expansion and differentiation of human CD34+ hematopoietic stem/progenitor cells (HSPCs) in vitro, in co-culture with human bone-marrow mesenchymal stromal/stem cells (MSCs). Our findings indicate that lenalidomide/pomalidomide increases the population of immature CD34+CD38− cells while decreasing the number of mature CD34+CD38+ cells, suggesting a mechanism that inhibits early HSPC maturation. This effect persisted across myeloid, megakaryocytic, and erythroid lineages, with MSCs playing a key role in preserving immature progenitors and inhibiting their differentiation. Furthermore, in myeloid differentiation assays augmented by granulocyte-colony stimulating factor, lenalidomide/pomalidomide not only enhanced the presence of CD34+ cells with mature myeloid markers such as CD11b but also reduced the populations lacking CD34 yet positive for these markers, irrespective of MSC presence. Thus, while MSCs support the presence of these immature cell populations, they simultaneously inhibit their maturation. This finding provides novel mechanistic insights into lenalidomide- and pomalidomide-induced cytopenia, and could guide therapeutic strategies for its mitigation.

Impact of obesity‑associated myeloid‑derived suppressor cells on cancer risk and progression (Review).

Obesity is a chronic disease caused by the accumulation of excessive adipose tissue. This disorder is characterized by chronic low‑grade inflammation, which promotes the release of proinflammatory mediators, including cytokines, chemokines and leptin. Simultaneously, chronic inflammation can predispose to cancer development, progression and metastasis. Proinflammatory molecules are involved in the recruitment of specific cell populations in the tumor microenvironment. These cell populations include myeloid‑derived suppressor cells (MDSCs), a heterogeneous, immature myeloid population with immunosuppressive abilities. Obesity‑associated MDSCs have been linked with tumor dissemination, progression and poor clinical outcomes. A comprehensive literature review was conducted to assess the impact of obesity‑associated MDSCs on cancer in both preclinical models and oncological patients with obesity. A secondary objective was to examine the key role that leptin, the most important proinflammatory mediator released by adipocytes, plays in MDSC‑driven immunosuppression Finally, an overview is provided of the different therapeutic approaches available to target MDSCs in the context of obesity‑related cancer.

Quadriceps Tendon Anterior Cruciate Ligament Reconstruction in Skeletally Immature Patients: 3-Year Clinical and Patient-Reported Outcomes.

The rate of anterior cruciate ligament (ACL) rupture in active, skeletally immature patients is increasing. Although hamstring tendon autograft (HTA) was previously deemed the gold standard, recent studies have shown HTA to have a high failure rate in this high-risk population of young competitive athletes, and quadriceps tendon autograft (QTA) has yielded excellent preliminary outcomes in some studies examining this population. To evaluate 3-year clinical and patient-reported functional outcomes of primary ACL reconstruction (ACLR) with soft tissue QTA in skeletally immature patients. Case series; Level of evidence, 4. Skeletally immature patients who underwent ACLR with a full-thickness soft tissue QTA were included. Preoperative patient and surgical data were collected. The ACLR technique was selected predicated upon skeletal age and included all-epiphyseal and complete transphyseal techniques. Patients were followed for a minimum of 2 years with successive clinical visits or were contacted via telephone. Patients who did not have minimum 2-year follow-up after 3 contact attempts via telephone were excluded. Information regarding return to sports (RTS) and concomitant or subsequent surgical procedures was collected. Pediatric International Knee Documentation Committee (Pedi-IKDC), Hospital for Special Surgery Functional Activity Brief Scale (HSS Pedi-FABS), and Single Assessment Numeric Evaluation (SANE) scores were collected. Of 85 adolescent patients aged 11.1 to 17.6 years (mean age, 14.1 ± 1.2 years), 2 patients were determined to be lost to follow-up after 3 failed contact attempts. Of the patients included in this study (N = 83), 26 patients (31%) underwent all-epiphyseal and 57 patients (69%) underwent complete transphyseal ACLR. Additionally, 48 patients (58%) underwent concomitant lateral extra-articular tenodesis using the iliotibial band with a modified Lemaire technique. The mean follow-up time was 3.7 ± 1.2 years (range, 2-7 years). Twenty (24%) patients had subsequent surgical procedures, of which 3 (4%) were due to graft failures. At a mean 3-year follow-up, the mean Pedi-IKDC, HSS Pedi-FABS, and SANE scores were 90, 23, and 94 respectively; the RTS rate was 100%; and the rate of RTS at the previous level of performance was 93%. Use of a soft tissue QTA for ALCR in a high-risk skeletally immature population of athletes resulted in excellent postoperative outcomes with low rates of graft failure and high return to sport rates.

Exploring the Potential of Glycolytic Modulation in Myeloid-Derived Suppressor Cells for Immunotherapy and Disease Management.

Recent advancements in various technologies have shed light on the critical role of metabolism in immune cells, paving the way for innovative disease treatment strategies through immunometabolism modulation. This review emphasizes the glucose metabolism of myeloid-derived suppressor cells (MDSCs), an emerging pivotal immunosuppressive factor especially within the tumor microenvironment. MDSCs, an immature and heterogeneous myeloid cell population, act as a double-edged sword by exacerbating tumors or mitigating inflammatory diseases through their immune-suppressive functions. Numerous recent studies have centered on glycolysis of MDSC, investigating the regulation of altered glycolytic pathways to manage diseases. However, the specific changes in MDSC glycolysis and their exact functions continue to be areas of ongoing discussion yet. In this paper, we review a range of current findings, including the latest research on the alteration of glycolysis in MDSCs, the consequential functional alterations in these cells, and the outcomes of attempts to modulate MDSC functions by regulating glycolysis. Ultimately, we will provide insights into whether these research efforts could be translated into clinical applications.

Dynamics of a stage-structured predator-prey system with fear-induced group defense in autonomous and nonautonomous settings.

In this investigation, we construct a predator-prey model that distinguishes between immature and mature prey, highlighting group defense strategies within the mature prey. First, we embark on exploring the positivity and boundedness of the solution, unraveling sustainable equilibrium points, and deducing their stability conditions. Upon further investigation, we observe that the system exhibits diverse bifurcations, including Hopf, saddle-node, transcritical, generalized Hopf, cusp, and Bogdanov-Takens bifurcations. The results reveal that heightened fear decreases mature prey density, potentially causing prey extinction beyond a certain threshold. Increased maturation rates lead to the coexistence of immature and mature prey populations and higher predator density. Stronger group defense boosts mature prey density, while weaker defense results in weak persistence. Lower values of the maturation rate of prey and the decline rate of predators sustain only the predator population, reliant on resources other than focal prey. Furthermore, our model demonstrates intriguing and diverse dynamical phenomena, including various forms of bistability across distinct bi-parameter planes. We also explore the dynamics of a related nonautonomous system, where certain parameters are considered to vary with time. In the seasonally forced model, we set out to define criteria regarding the existence and stability of positive periodic solutions. Numerical investigations into the seasonally forced model uncover a spectrum of dynamics, ranging from simple periodic solutions to higher periodicities, bursting patterns, and chaotic behavior.

Metabolic dependencies of acute myeloid leukemia stem cells.

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy primarily driven by an immature population of AML cells termed leukemia stem cells (LSCs) that are implicated in AML development, chemoresistance, and relapse. An emerging area of research in AML focuses on identifying and targeting the aberrant metabolism in LSCs. Dysregulated metabolism is involved in sustaining functional properties of LSCs, impeding myeloid differentiation, and evading programmed cell death, both in the process of leukemogenesis and in response to chemotherapy. This review discusses recent discoveries regarding the aberrant metabolic processes of AML LSCs that have begun to change the therapeutic landscape of AML.

Flow cytometric immunophenotypic differentiation patterns of bone marrow eosinophilopoiesis.

Flow cytometry has been widely used to study immunophenotypic patterns of maturation of most hematopoietic lineages in normal human bone marrow aspirates, thus allowing identification of changes in patterns in many myeloid malignancies. Eosinophils play an important role in a wide variety of disorders, including some myeloid neoplasms. However, changes in flow cytometric immunophenotypic patterns during normal and abnormal bone marrow eosinophilopoiesis have not been well studied. Fresh bone marrow aspirates from 15 healthy donors, 19 patients with hypereosinophilic syndromes (HES), and 11 patients with systemic mastocytosis (SM) were analyzed for candidate markers that included EMR-1, Siglec-8, CCR3, CD9, CD11a, CD11b, CD11c, CD13, CD16, CD29, CD34, CD38, CD45, CD44, CD49d, CD49f, CD54, CD62L, CD69, CD117, CD125 (IL-5Rα), HLA-DR, using 10 parameter flow cytometry. Putative CD34-negative immature and mature normal eosinophil populations were first identified based on changes in expression of the above markers in healthy donors, then confirmed using fluorescence-based cell sorting and morphological evaluation of cytospin preparations. The normal immunophenotypic patterns were then compared to immunophenotypic patterns of eosinophilopoiesis in patients with HES and SM. The eosinophilic lineage was first verified using the human eosinophil-specific antibody EMR-1 in combination with anti-IL-5Rα antibody. Then, a combination of Siglec-8, CD9, CD11b, CCR3, CD49d, and CD49f antibodies was used to delineate normal eosinophilic maturational patterns. Early stages (eosinophilic promyelocytes/myelocytes) were identified as Siglec-8 dim/CD11b dim to moderate/CD9 dim/CCR3 dim/CD49d bright/CD49f dim, intermediate stages (eosinophilic myelocytes/metamyelocytes) as Siglec-8 moderate/CD11b moderate to bright/CD9 moderate/CCR3 moderate/CD49d moderate/CD49f moderate and mature bands/segmented eosinophils as Siglec-8 bright/CD11b bright/CD9 bright/CCR3 bright/CD49d dim/CD49f bright. Overall maturational patterns were also similar in patients with HES and SM; however, the expression levels of several surface markers were altered compared to normal eosinophils. A novel flow cytometric antibody panel was devised to detect alterations in immunophenotypic patterns of bone marrow eosinophil maturation and evaluated in normal, HES and SM samples. This approach will allow us to elucidate changes in immunophenotypic patterns of bone marrow eosinophilopoiesis in other hematological diseases.

New Pterosaur Tracks from the Hwasun Seoyuri Tracksite (Turonian) of South Korea: Implications for their Ecological Niche and Habitat

Pterosaurs are powered flight vertebrates that first emerged in the Late Triassic and persisted until the end of the Cretaceous. Recent studies on the ecological niches and habitats of pterosaurs, based on discoveries of Cretaceous bone beds in China, Brazil, Morocco, and other locations, suggest a decline in pterosaur diversity during the mid-Cretaceous. Various hypotheses have been proposed to interpret this phenomenon. This study focuses on newly discovered pterosaur tracks at the Upper Cretaceous (Truonian) Hwasun Seoyuri tracksite in Korea. The analysis is based on pterosaur tracks from in situ and ex situ specimens that originate from a variety of stratigraphic levels. The evidence found suggests that the Hwasun Seoyuri tracksite was occupied by small pterosaurs over an extended period, with indications of recurrent activity across at least six levels. Furthermore, the footprint sizes observed across these multiple layers follow a non-skewed normal distribution. Considering the known logarithmic growth characteristics of pterosaurs, this result suggests a predominantly immature population at this site. The prevalence of mid-Cretaceous non-marine pterosaur tracksites on the Korean Peninsula may reflect broader ecological shifts for pterosaurs during this period. This evidence is considered alongside the habitat shifts suggested by other non-marine pterosaur fossil sites in China and Brazil, as well as with indications of the decline in Late Cretaceous non-marine basins.

On Population Models with Delays and Dependence on Past Values

The current values of many populations depend on the past values of the population. In many cases, this dependence is caused by the time certain processes take. This dependence on the past can be introduced into mathematical models by adding delays. For example, the growth rate of a population depends on the population τ time units ago, where τ is the maturation time. For an epidemic, there is a time τ between the contact of an infected individual and a susceptible one, and the time the susceptible individual actually becomes infected. This time τ is also a delay. So, the number of infected individuals depends on the population at the time τ units ago. A second way of introducing this dependence on past values is to use non-local operators in the description of the model. Fractional derivatives have commonly been used to provide non-local effects. In population growth models, it can also be done by introducing a new compartment, the immature population, and in epidemic models, by introducing an additional exposed population. In this paper, we study and compare these methods of adding dependence on past values. For models of processes that involve delays, all three methods include dependence on past values, but fractional-order models do not justify the form of the dependence. Simulations show that for the models studied, the fractional differential equation method produces similar results to those obtained by explicitly incorporating the delay, but only for specific values of the fractional derivative order, which is an extra parameter. But in all three methods, the results are improved compared to using ordinary differential equations.

Early life vaccination reprograms the metabolism and function of myeloid cells in neonates.

Vaccination after birth provides protection against pathogen infection and immune related disorders in healthy children. The detailed effects of vaccination on neonatal immunity, however, remain largely unknown. Here, we reported that vaccination using Bacillus Calmette-Guérin (BCG) diminished the immunosuppressive function of myeloid-derived suppressor cells in neonatal mice, an immature myeloid population. A combination of single-cell transcriptome, metabolite profiling, and functional analysis demonstrated that upregulation of mTOR/HIF1a signalling and the enhanced glycolysis explained the effects of BCG on neonatal myeloid cells. Pharmalogical inhibition of glycolysis or mTOR signalling efficiently rescued the effects of BCG on neonatal myeloid cells. These observations suggest that BCG facilitates the maturation of myeloid cells in early life, which may contribute to its beneficial effects against immune disorders later in life.

MAGIC-DR: An interpretable machine-learning guided approach for acute myeloid leukemia measurable residual disease analysis.

Multiparameter flow cytometry is widely used for acute myeloid leukemia minimal residual disease testing (AML MRD) but is time consuming and demands substantial expertise. Machine learning offers potential advancements in accuracy and efficiency, but has yet to be widely adopted for this application. To explore this, we trained single cell XGBoost classifiers from 98 diagnostic AML cell populations and 30 MRD negative samples. Performance was assessed by cross-validation. Predictions were integrated with UMAP as a heatmap parameter for an augmented/interactive AML MRD analysis framework, which was benchmarked against traditional MRD analysis for 25 test cases. The results showed that XGBoost achieved a median AUC of 0.97, effectively distinguishing diverse AML cell populations from normal cells. When integrated with UMAP, the classifiers highlighted MRD populations against the background of normal events. Our pipeline, MAGIC-DR, incorporated classifier predictions and UMAP into flow cytometry standard (FCS) files. This enabled a human-in-the-loop machine learning guided MRD workflow. Validation against conventional analysis for 25 MRD samples showed 100% concordance in myeloid blast detection, with MAGIC-DR also identifying several immature monocytic populations not readily found by conventional analysis. In conclusion, Integrating a supervised classifier with unsupervised dimension reduction offers a robust method for AML MRD analysis that can be seamlessly integrated into conventional workflows. Our approach can support and augment human analysis by highlighting abnormal populations that can be gated on for quantification and further assessment. This has the potential to speed up MRD analysis, and potentially improve detection sensitivity for certain AML immunophenotypes.

Single-Cell RNA Sequencing Reveals Alterations in Patient Immune Cells with Pulmonary Long COVID-19 Complications.

Since the emergence of the COVID-19 pandemic, the effects of SARS-CoV-2 have been extensively researched. While much is already known about the acute phase of the infection, increasing attention has turned to the prolonged symptoms experienced by a subset of individuals, commonly referred to as long COVID-19 patients. This study aims to delve deeper into the immune landscape of patients with prolonged symptoms by implementing single-cell mRNA analysis. A 71-year-old COVID-19 patient presenting with persistent viral pneumonia was recruited, and peripheral blood samples were taken at 3 and 2 years post-acute infection onset. Patients and control peripheral blood mononuclear cells (PBMCs) were isolated and single-cell sequenced. Immune cell population identification was carried out using the ScType script. Three months post-COVID-19 patients' PBMCs contained a significantly larger immature neutrophil population compared to 2-year and control samples. However, the neutrophil balance shifted towards a more mature profile after 18 months. In addition, a notable increase in the CD8+ NKT-like cells could be observed in the 3-month patient sample as compared to the later one and control. The subsequent change in these cell populations over time may be an indicator of an ongoing failure to clear the SARS-CoV-2 infection and, thus, lead to chronic COVID-19 complications.

Abstract B010: Revealing the Influence of the Infantile Immune System on the Developmental Origins of Childhood Ependymoma: An Integrative Multi-Omic Investigation of Supratentorial Ependymoma Tumorigenesis Driven by ZFTA-RELA Fusion

Abstract Despite advances in treatment, the recurrence rate of childhood supratentorial ependymomas remains distressingly high, with approximately 60% of patients experiencing relapse. Moreover, the rigorous treatment regimen poses significant challenges to the quality of life of affected children. Among supratentorial ependymomas, those driven by the ZFTA-RELA fusion represent over 70% of cases. Here, we present the pioneering development of a spontaneous genetically engineered mouse model (GEMM) for ZFTA-RELA fusion-driven ependymoma. By targeting the Nestin-Flx-STOP-Flx-ZFTA-RELA open reading frame with luciferase to the Rosa-26 locus of C57B/L6 mice, and subsequently crossing them with the Nestin-CreERT2 line, we successfully induced recombination in neural progenitor cells. Consequently, resulting in the development of forebrain tumors that could be monitored through non-invasive bioluminescence imaging from postnatal day 20. Recognizing the neonatal origins of ZFTA-RELA ependymoma, our study employed an integrated multi-omic approach to investigate the complete developmental trajectory of this tumor, spanning from embryonic stages to postnatal tumor formation. Notably, we uncovered a reservoir of immature immune populations in the developing brain, which we implicate in the central tolerance of ependymoma tumorigenesis. The coordinated interaction between these immature immune populations in the developing brain and immune cell progenitors in the skull bone marrow niche establishes a unique environment that fosters tumor formation. In light of these findings, we propose that immunotherapeutic strategies aimed at augmenting these immature immune populations hold promise as potential therapeutic interventions. By exploiting the intricate interplay between these populations, we may unlock new avenues for targeted immunotherapies in ZFTA-RELA ependymoma. Our research sheds vital light on the critical role played by the pediatric immune system in the developmental origins of this devastating malignancy, opening up exciting possibilities for improved treatments and outcomes in the future. Citation Format: Elizabeth Cooper, Sigourney Bonner, Amir Jassim, Gunjan Katyal, Alisha Kardian, Stephen C. Mack, Katherine Masih, Jessica T. Taylor, Claire King, Katherine Wickham Rahrmann, Linda Hu, Richard J. Gilbertson. Revealing the Influence of the Infantile Immune System on the Developmental Origins of Childhood Ependymoma: An Integrative Multi-Omic Investigation of Supratentorial Ependymoma Tumorigenesis Driven by ZFTA-RELA Fusion [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B010.

Single Cell Analysis Identifies Expression of PD-L2 on Human Bone Marrow Hematopoietic Stem Cells

The identification of specific and reliable markers for human hematopoietic stem cells (HSCs) is crucial to enable their prospective isolation allowing the investigation of unique physiological HSC functions within the blood cell hierarchy. Despite significant advancements in identification of surface markers for human bone marrow HSC isolation, the enriched populations remain heterogeneous. Here we applied single cell CITE-Seq (cellular indexing of transcriptomes and epitopes) of FACS-enriched hematopoietic stem and progenitor cells (HSPCs) to decipher differentiation hierarchies and to identify markers of most immature populations. We FACS-isolated over 60.000 high-quality human bone marrow HSPCs by sorting CD34 + and CD34 +CD38 - cells from 15 different healthy donors. These were subjected to scCITE-Seq using the BD Rhapsody system with a targeted gene panel comprising 600 most relevant genes of early HSC differentiation and 50 antibodies. To validate differentially expressed surface marker genes, we performed extensive in vitro and in vivo studies of FACS-purified subpopulations of human bone marrow and mobilized peripheral blood samples (mPB) from healthy donors. Our multi-omics based single HSPC profiling indicated Programmed death ligand 2 (PD-L2 / CD273) as a surface marker expressed on the most immature HSCs within the hematopoietic compartment defined as CD34 +CD38 -CD45RA -CD90 +. Prospective FACS-based isolation allowed a deeper understanding of transcriptomic differences depending on PD-L2 positivity. A transcriptomic principal component analysis of CD273 hi and CD273 low expressing HSCs isolated pairwise from four healthy donors proved distinct clustering accompanied by discriminative molecular signatures. Stem cell genes as HOPX, MLLT3 and MPL were enriched in CD273 hi HSCs, and CDK6 was downregulated, further validating our single-cell analysis. The differential expression of stem cell genes was validated at protein level. Single cell tracking using video-microscopy confirmed a delayed entry into cell cycle in CD273 hi HSCs, suggesting deeper quiescence of these cells. Furthermore, their mitochondrial potential was reduced. In vivo transplantations of both populations in immunocompromised NSGs are ongoing and will reveal the long-term repopulation potential of CD273 hi HSCs. Multiple in vitro differentiation assays demonstrated CD273 positivity to be associated with an increased stem cell phenotype with higher expression of EPCR (CD201) and CD90 in liquid culture, a delayed differentiation and high capability of generating multipotent colonies. Interestingly, CD273 surface expression was strongly upregulated in HSCs upon in vitro culture. Since PD-L2 is known as a ligand for the Programmed death receptor 1 (PD1), we investigated the immunomodulating function of PD-L2 expressing HSPCs by establishing an allogeneic lymphocyte reaction assay. In addition to their stem cell-like characteristics, PD-L2 expressing HSPCs mediated diminished T cell proliferation in allogeneic co-culture settings. Furthermore, cytokine profiling of co-culture supernatants indicated increased secretion of anti-inflammatory cytokines (IL-10 and TGF-β1), accompanied by reduced secretion of pro-inflammatory cytokines (IL-17A) and a diminished expression of the pro-inflammatory receptor CD69 on CD8 + T cells. These findings showed that CD34 + cells isolated from mobilized peripheral blood of healthy donors possess immunomodulating capabilities, and that PD-L2 expression indicates an immunocompromising HSC phenotype. In conclusion, this study demonstrates differential surface expression of PD-L2 within the most immature human HSCs. PD-L2 expression is associated with HSC quiescence and delayed in vitro differentiation. Furthermore, these results provide valuable insights into the immunomodulating features of HSCs expressing PD-L2.

Jmjd3 Drives Immature T-Cell Lymphoblastic Leukemia

Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that accounts for 15% of pediatric and 25% of adult ALL cases. Despite an improved survival rate due to intensified chemotherapy and bone marrow transplantation, still 20% of T-ALL patients are primary refractory to the standard treatment or relapse, and have a poor clinical outcome. T-ALL patients can be classified into molecular genetic subgroups based on the expression or genomic alterations of specific oncogenic transcription factors. One such subgroup the early T-cell precursor ALL (ETP-ALL) is a heterogeneous subgroup of immature leukemias that often display high expression of LYL1 and LMO2. It's important to improve our knowledge of T-ALL biology and developing novel targeted anti-TALL therapies. We previously showed that the H3K27 demethylase Jmjd3 plays an important role in T-ALL and is a good candidate for the treatment of T-cell leukemia. Aim: Identify the role of Jmjd3 in the initiation of immature T-ALL. Methods: We generated a conditional Rosa26- Jmjd3 overexpression mouse model (Figure 1A), and crossed it with a hematopoietic-specific Vav-icre mice, to obtain Jmjd3 Vav mice. To investigate the role of Jmjd3 in tumor initiation, we monitored survival of a Jmjd3 Vav cohort. Arising tumors were characterized by immunohistochemistry, flow cytometry, and were transplanted into secondary immunodeficient recipients. To characterize the pre-leukemic compartment, we performed single cell feature barcoding and RNA sequencing. Also bulk RNAseq and proteomics were performed on primary and transplanted Jmjd3-driven tumors. To investigate the role of Jmjd3 in immature T-cell biology in human and mice, we analyzed thymi from 8-weeks-old control and Jmjd3 Vavmice, and performed OP9-DL4 stromal co-cultures with transduced CD34+ human cord blood cells that overexpressed JMJD3, respectively. We used ETP-ALL patient-derived xenografts (PDX) models to validate markers identified in single cell analysis. Results: We found that hematopoietic Jmjd3 overexpression resulted in hematological malignancies with 38% penetrance (n= 8/21) and a median survival of 63 weeks (Figure 1B). Immunohistochemistry confirmed overexpression of Jmjd3, decreased levels of H3K27me3, and increased levels of H3K27ac in Jmjd3 Vav tumors. Immunophenotypic analysis revealed immature Thy1.1 +CD4 -CD8 - cells in all obtained Jmjd3 Vav tumors (Figure 1C), indicative of an immature the early T-cell precursor (ETP)-like ALL. These immature T-ALLs gave rise to primary (7 weeks) and secondary (3 weeks) transplants upon their transplantation in immunocompromised mice. Bulk RNAseq confirmed that the transcriptional feature of the primary tumor were maintained upon transplantation. The role of Jmjd3 in stimulating immature thymocyte development was confirmed in thymi from 8-weeks-old Jmjd3 Vavmice and could be validated as well in human T-cell development using stromal OP9-DL4 co-cultures. Finally, we used single cell sequencing technologies to characterize both transcriptional features and cell-surface markers within the tumor fraction (Figure 1D). We confirmed that the tumor cluster was ETP-like with absence of CD1, CD5 and mature T-cell markers, and expression of CD44, Lyl1, and myeloid markers. Furthermore, we could identify cell-surface in immature Jmjd3-driven tumors that were also present in human T-cell stromal culture and ETP-ALL PDXs. Conclusion: We identified Jmjd3 as a driver of immature thymocyte populations that give rise to ETP-ALL.

A Multiomic Single-Cell Atlas of Human Myelopoiesis Reveals Cellular and Molecular Drivers of Immunomodulatory Drug-Induced Neutropenia

Neutrophils are crucial innate immune cells which protect the host by killing infectious pathogens. Single cell RNA-sequencing analyses have previously described transcriptional heterogeneity of neutrophils and precursors, however the genome regulatory events underlying the transcriptional changes are less well characterised. Understanding this is important as neutropenia can be caused by dysregulation of the genome regulatory events that orchestrate neutrophil maturation. A good example is the use of immunomodulatory drugs (IMiDs, lenalidomide [LEN] & pomalidomide [POM]) for the treatment of multiple myeloma (MM) which are now standard-of-care, but their use is frequently complicated by neutropenia because of neutrophil maturation impairment. This is due to Cereblon (CRBN)-driven substrate ubiquitination and Ikaros degradation, causing PU.1 downregulation. However, restoration of Ikaros levels cannot fully alleviate the IMiD-induced myeloid differentiation block suggesting other mechanisms might also contribute. Our aim was to build a multiomic atlas of human myelopoiesis and use this to interrogate the molecular drivers of IMiD-induced neutropenia. We developed an ex vivo myeloid cell differentiation assay using human mobilized peripheral blood CD34 + cells from healthy donors and optimised methods for single cell multiomic (combined RNA-seq & ATAC-seq) analysis. This platform was validated orthogonally (immunophenotype, morphology & functional) and employed to construct a multi-omic single-cell landscape of normal human neutrophil differentiation and it's perturbation by IMiDs. Single cell transcriptomes from >110,000 cells from untreated and IMiD-treated samples were analyzed, capturing the full spectrum of human myelopoiesis spanning from early myeloid progenitors through to neutrophil populations (band, segmented, mature) (Fig.1A-left). Ex vivo differentiated neutrophils retained transcriptional signatures reflective of effector functions (granule biogenesis, chemotactic activity, respiratory burst) and mapped to previously described human neutrophil populations in vivo. Ex vivo generated neutrophils showed expected functional properties of phagocytosis and neutrophil extracellular trap formation upon stimulation. Exposure to IMiDs caused a maturation arrest with a 40-50% (p<0.001) decrease in abundance of differentiating metamyelocytes, and neutrophil populations. Crucially, this maturation arrest was due to an accumulation of transcriptionally distinct myeloid precursors that differentiated though an aberrant pathway (population 6, Fig 1A). Furthermore, immature erythroid clusters representing 10% of live cells accumulated specifically in IMiD-treated samples (p<0.001) in absence of erythropoiesis-stimulating cytokines. Simultaneous profiling of gene expression and chromatin accessibility in individual cells confirmed the presence of distinct phenotypes for untreated and IMiD-treated myeloid cells (Fig.1A) enabling the characterisation of the genome landscape of myelopoiesis, which has previously proven challenging. As expected, we observed dysregulation of Ikaros targets including PU.1 upon IMiD treatment (Fig. 1B-frame). The ability to study distinct and aberrant cellular populations at transcriptional and genome regulation level in parallel allowed us to identify several novel regulators of normal and perturbed neutrophil development. For example, we observed perturbed activation patterns of the transcription factors (TFs) zinc finger protein (ZNF467) and interferon regulatory factor 5 (IRF5) and the expression of their downstream targets as illustrated in Fig.1B. ZNF467 orchestrates a transcriptional program supporting terminal neutrophil differentiation in untreated cells (Fig.1B-top right), whilst the regulatory repertoire of IRF5, a hallmark of neutrophil activation is confined primarily to treatment-specific immature populations (Fig.1B-bottom right). We present a multiomic atlas of normal myelopoiesis which we believe will provide an important resource for the study or normal and perturbed neutrophil development. We use this platform to study IMiD-induced neutropenia and identify two aberrant cellular progenitor populations (aberrant myeloid precursors & erythroid progenitors) with distinct molecular properties as the likely cause of myelosuppression.