Primate Cells Research Articles

E2F3-dependent activation of FAM111B restricts mouse cytomegalovirus replication in primate cells.

Viruses must counteract host cell defenses to facilitate viral replication. Viruses with a narrow host range, such as the cytomegaloviruses, are unable to counteract cellular defenses in cells of a foreign species. However, little is known about the cellular host range factors restricting cytomegalovirus replication. Here, we show that mouse cytomegalovirus (MCMV) induces the expression of the FAM111 proteases and that FAM111B, but not FAM111A that has previously been shown to restrict the replication of polyomavirus and orthopoxvirus host range mutants, acts as a cellular factor suppressing MCMV replication in human and rhesus monkey cells. The identification of FAM111B as a host range factor should provide new insight into the physiological functions of this poorly characterized protein.

Avian deltacoronaviruses encode fusion-associated small transmembrane proteins that can induce syncytia formation

Fusion-associated small transmembrane (FAST) proteins are nonstructural viral proteins that induce cell-cell fusion. FAST proteins, which previously were identified in the genomes of double-stranded RNA viruses, typically contain an acylated N-terminal ectodomain, central transmembrane domain, and C-terminal endodomain with a polybasic region. Using sequence homology and protein motif prediction, we identified accessory proteins in a subset of avian deltacoronaviruses as putative FAST proteins. Transient expression of thrush coronavirus NS7b or common moorhen coronavirus NS7a, but not night heron coronavirus NS7b, induced cell-cell fusion. Syncytia were detected in primate kidney epithelial cells or fibroblasts but not chicken embryo fibroblasts, and addition of an N-terminal FLAG peptide to the proteins ablated fusion activity. These findings suggest that multiple avian deltacoronaviruses, positive-sense RNA viruses, encode nonstructural proteins that can mediate cell-cell fusion and share features with known FAST proteins. Additional studies are needed to understand contributions of these proteins to deltacoronavirus biology.

A Novel Jeilongvirus from Florida, USA, Has a Broad Host Cell Tropism Including Human and Non-Human Primate Cells.

A novel jeilongvirus was identified through next-generation sequencing in cell cultures inoculated with spleen and kidney extracts. The spleen and kidney were obtained from a Peromyscus gossypinus rodent (cotton mouse) found dead in the city of Gainesville, in North-Central Florida, USA. Jeilongviruses are paramyxoviruses of the subfamily Orthoparamyxovirinae that have been found in bats, cats, and rodents. We designated the virus we discovered as Gainesville rodent jeilong virus 1 (GRJV1). Preliminary results indicate that GRJV1 can complete its life cycle in various human, non-human primate, and rodent cell lines, suggesting that the virus has a generalist nature with the potential for a spillover event. The early detection of endemic viruses circulating within hosts in North-Central Florida can significantly enhance surveillance efforts, thereby bolstering our ability to monitor and respond to potential outbreaks effectively.

Applying Super-Resolution and Tomography Concepts to Identify Receptive Field Subunits in the Retina.

Spatially nonlinear stimulus integration by retinal ganglion cells lies at the heart of various computations performed by the retina. It arises from the nonlinear transmission of signals that ganglion cells receive from bipolar cells, which thereby constitute functional subunits within a ganglion cell's receptive field. Inferring these subunits from recorded ganglion cell activity promises a new avenue for studying the functional architecture of the retina. This calls for efficient methods, which leave sufficient experimental time to leverage the acquired knowledge for further investigating identified subunits. Here, we combine concepts from super-resolution microscopy and computed tomography and introduce super-resolved tomographic reconstruction (STR) as a technique to efficiently stimulate and locate receptive field subunits. Simulations demonstrate that this approach can reliably identify subunits across a wide range of model variations, and application in recordings of primate parasol ganglion cells validates the experimental feasibility. STR can potentially reveal comprehensive subunit layouts within only a few tens of minutes of recording time, making it ideal for online analysis and closed-loop investigations of receptive field substructure in retina recordings.

Specific imaging of CD8 + T-Cell dynamics with a nanobody radiotracer against human CD8β.

While immunotherapy has revolutionized the oncology field, variations in therapy responsiveness limit the broad applicability of these therapies. Diagnostic imaging of immune cell, and specifically CD8+ T cell, dynamics could allow early patient stratification and result in improved therapy efficacy and safety. In this study, we report the development of a nanobody-based immunotracer for non-invasive SPECT and PET imaging of human CD8+ T-cell dynamics. Nanobodies targeting human CD8β were generated by llama immunizations and subsequent biopanning. The lead anti-human CD8β nanobody was characterized on binding, specificity, stability and toxicity. The lead nanobody was labeled with technetium-99m, gallium-68 and copper-64 for non-invasive imaging of human T-cell lymphomas and CD8+ T cells in human CD8 transgenic mice and non-human primates by SPECT/CT or PET/CT. Repeated imaging of CD8+ T cells in MC38 tumor-bearing mice allowed visualization of CD8+ T-cell dynamics. The nanobody-based immunotracer showed high affinity and specific binding to human CD8 without unwanted immune activation. CD8+ T cells were non-invasively visualized by SPECT and PET imaging in naïve and tumor-bearing mice and in naïve non-human primates with high sensitivity. The nanobody-based immunotracer showed enhanced specificity for CD8+ T cells and/or faster in vivo pharmacokinetics compared to previous human CD8-targeting immunotracers, allowing us to follow human CD8+ T-cell dynamics already at early timepoints. This study describes the development of a more specific human CD8+ T-cell-targeting immunotracer, allowing follow-up of immunotherapy responses by non-invasive imaging of human CD8+ T-cell dynamics.

A conserved opal termination codon optimizes a temperature-dependent tradeoff between protein production and processing in alphaviruses.

Alphaviruses are enveloped, single-stranded, positive-sense RNA viruses that often require transmission between arthropod and vertebrate hosts for their sustained propagation. Most alphaviruses encode an opal (UGA) termination codon in nonstructural protein 3 (nsP3) upstream of the viral polymerase, nsP4. The selective constraints underlying the conservation of the opal codon are poorly understood. Using primate and mosquito cells, we explored the role and selective pressure on the nsP3 opal codon through extensive mutational analysis in the prototype alphavirus, Sindbis virus (SINV). We found that the opal codon is highly favored over all other codons in primate cells under native 37°C growth conditions. However, this preference is diminished in mosquito and primate cells grown at a lower temperature. Thus, the primary determinant driving the selection of the opal stop codon is not host genetics but the passaging temperature. We show that the opal codon is preferred over amber and ochre termination codons because it results in the highest translational readthrough and polymerase production. However, substituting the opal codon with sense codons leads to excessive full-length polyprotein (P1234) production, which disrupts optimal nsP polyprotein processing, delays the switch from minus-strand to positive-strand RNA production, and significantly reduces SINV fitness at 37°C; this fitness defect is relieved at lower temperatures. A naturally occurring suppressor mutation unexpectedly compensates for a delayed transition from minus to genomic RNA production by also delaying the subsequent transition between genomic and sub-genomic RNA production. Our study reveals that the opal stop codon is the best solution for alphavirus replication at 37°C, producing enough nsP4 protein to maximize replication without disrupting nsP processing and RNA replication transitions needed for optimal fitness. Our study uncovers the intricate strategy dual-host alphaviruses use at a single codon to optimize fitness.

Temporally resolved single cell transcriptomics in a human model of amniogenesis.

Amniogenesis is triggered in a collection of pluripotent epiblast cells as the human embryo implants. To gain insights into the critical but poorly understood transcriptional machinery governing amnion fate determination, we examined the evolving transcriptome of a developing human pluripotent stem cell-derived amnion model at the single cell level. This analysis revealed several continuous amniotic fate progressing states with state-specific markers, which include a previously unrecognized CLDN10+ amnion progenitor state. Strikingly, we found that expression of CLDN10 is restricted to the amnion-epiblast boundary region in the human post-implantation amniotic sac model as well as in a peri-gastrula cynomolgus macaque embryo, bolstering the growing notion that, at this stage, the amnion-epiblast boundary is a site of active amniogenesis. Bioinformatic analysis of published primate peri-gastrula single cell sequencing data further confirmed that CLDN10 is expressed in cells progressing to amnion. Additionally, our loss of function analysis shows that CLDN10 promotes amniotic but suppresses primordial germ cell-like fate. Overall, this study presents a comprehensive amniogenic single cell transcriptomic resource and identifies a previously unrecognized CLDN10+ amnion progenitor population at the amnion-epiblast boundary of the primate peri-gastrula.

Dose-dependent regulation of immune memory responses against HIV by saponin monophosphoryl lipid A nanoparticle adjuvant.

The induction of durable protective immune responses is the main goal of prophylactic vaccines, and adjuvants play an important role as drivers of such responses. Despite advances in vaccine strategies, a safe and effective HIV vaccine remains a significant challenge. The use of an appropriate adjuvant is crucial to the success of HIV vaccines. Here we assessed the saponin/MPLA nanoparticle (SMNP) adjuvant with an HIV envelope (Env) trimer, evaluating the safety and impact of multiple variables including adjuvant dose (16-fold dose range), immunization route, and adjuvant composition on the establishment of Env-specific memory T and B cell responses (T Mem and B Mem ) and long-lived plasma cells in non-human primates. Robust B Mem were detected in all groups, but a 6-fold increase was observed in the highest SMNP dose group vs. the lowest dose group. Similarly, stronger vaccine responses were induced in the highest SMNP dose for CD40L + OX40 + CD4 T Mem (11-fold), IFNγ + CD4 T Mem (15-fold), IL21 + CD4 T Mem (9-fold), circulating T FH (3.6-fold), bone marrow plasma cells (7-fold), and binding IgG (1.3-fold). Substantial tier-2 neutralizing antibodies were only observed in the higher SMNP dose groups. These investigations highlight the dose-dependent potency of SMNP in non-human primates, which are relevant for human use and next-generation vaccines.

Single-cell analysis reveals transcriptional dynamics in healthy primary parathyroid tissue.

Studies on human parathyroids are generally limited to hyperfunctioning glands owing to the difficulty in obtaining normal human tissue. We therefore obtained non-human primate (NHP) parathyroids to provide a suitable alternative for sequencing that would bear a close semblance to human organs. Single-cell RNA expression analysis of parathyroids from four healthy adult M. mulatta reveals a continuous trajectory of epithelial cell states. Pseudotime analysis based on transcriptomic signatures suggests a progression from GCM2 hi progenitors to mature parathyroid hormone (PTH)-expressing epithelial cells with increasing core mitochondrial transcript abundance along pseudotime. We sequenced, as a comparator, four histologically characterized hyperfunctioning human parathyroids with varying oxyphil and chief cell abundance and leveraged advanced computational techniques to highlight similarities and differences from non-human primate parathyroid expression dynamics. Predicted cell-cell communication analysis reveals abundant endothelial cell interactions in the parathyroid cell microenvironment in both human and NHP parathyroid glands. We show abundant RARRES2 transcripts in both human adenoma and normal primate parathyroid cells and use coimmunostaining to reveal high levels of RARRES2 protein (also known as chemerin) in PTH-expressing cells, which could indicate that RARRES2 plays an unrecognized role in parathyroid endocrine function. The data obtained are the first single-cell RNA transcriptome to characterize nondiseased parathyroid cell signatures and to show a transcriptomic progression of cell states within normal parathyroid glands, which can be used to better understand parathyroid cell biology.

P-682 Malic enzyme 1 in granulosa cells promotes ovarian aging through inhibiting the AMPK-FoXO1-IDH2 signaling pathway

Abstract Study question Reduced levels of oxidative phosphorylation in granulosa cells are a key factor in regulating ovarian aging, but the upstream regulatory mechanisms are unknown. Summary answer Our study revealed that elevated malic enzyme 1 in granulosa cells promotes ovarian aging by inhibiting oxidative phosphorylation levels through the AMPK-FoXO1-IDH2 signaling pathway. What is known already With ovarian aging, the ovarian granulosa cells appear hallmarks of high apoptosis activity and low-proliferative activity, which lead to a reduction in ovarian reserve and a decrease in hormone synthesis, resulting in female infertility and reduced quality of life. And the decline of isocitrate dehydrogenase, IDH, in non-human primate ovarian granulosa cells is a key causative factor for the decline in the level of oxidative phosphorylation in granulosa cells. Recently, researchers reported that malic enzyme 1, which encodes a cytosolic, NADP-dependent Enzyme that generates NADPH for fatty acid biosynthesis inhibits IDH2 expression in tumor cells. Study design, size, duration Utilizing clinical samples, cells and animal models, with the help of research tools such as biochemical experiments, gene editing and single-cell transcriptome sequencing, at different levels, including molecular, cellular, animal and human samples, we systematically analyzed the characteristic changes and key molecules in granulosa cells along with ovarian aging and reveal the intracellular molecular pathways through which ME1 and IDH2 regulate the process of ovarian aging for 1 year. Participants/materials, setting, methods We first apply single-cell transcriptome sequencing analysis of young and old mouse ovaries to reveal cellular subpopulation changes and transcriptomic hallmarks of ovarian granulosa cell with aging. And then we validate the biological function and revealing the molecular mechanism to regulate ovarian aging by using physiological ovarian aging mouse model, clinical ovarian granulosa cells from young and old female and human ovarian granulosa cell line KGN utilizing technologies of protein immunology, qPCR and CRISPR/Cas9. Main results and the role of chance Ovarian single-cell transcriptome sequencing data from young and old mice showed that ovarian aging was accompanied by a dramatic decrease in the number of granulosa cells and drastic changes in the proportions of different subpopulations. The proportion of mitotically active granulosa cells was significantly lower in aging ovaries than in young mouse ovaries, and GO enrichment analysis showed that granulosa cell cycle, antioxidant and hormone synthesis-related signaling pathways were significantly down-regulated in aging ovaries. Using immunoblotting, immunofluorescence and qPCR, we demonstrated that the expression of ME1 in granulosa cells of aging mice and human ovaries was significantly elevated, while the expression of IDH2 was significantly decreased. Performing the overexpression and knockdown of ME1 in human ovarian granulosa cell line KGN, which further demonstrated that ME1 could inhibit the transcriptionally regulated IDH2 expression of downstream genes, such as FoXO1, by suppressing the phosphorylation of AMPK. The decrease of IDH2 in mitochondria reduces the level of oxidative phosphorylation in granulosa cells, resulting in the increase of intracellular reactive oxygen species, activation of the intracellular p53-p21 signaling pathway, inhibition of the cell cycle, and promotion of apoptosis, which in turn impairs the function of granulosa cells to synthesize hormones and promotes ovarian aging. Limitations, reasons for caution Malic enzyme 1, which links the glycolytic cycle to the citric acid cycle, expressing in other ovarian cells including oocytes, and the regulation of its expression is complex. Future studies will focus on its biological functions in other ovarian cells and its molecular mechanism in regulating ovarian aging. Wider implications of the findings Our study suggests that granulosa cell ME1 and IDH2 co-regulate oxidative phosphorylation levels to regulate ovarian aging, and that ME1 and IDH2 may serve as potential targets for the finding and treatment of premature ovarian aging and delaying ovarian aging in women. Trial registration number not applicable

Comparative Anatomy of the Dentate Mossy Cells in Nonhuman Primates: Their Spatial Distributions and Axonal Projections Compared With Mouse Mossy Cells.

Glutamatergic mossy cells (MCs) mediate associational and commissural connectivity, exhibiting significant heterogeneity along the septotemporal axis of the mouse dentate gyrus (DG). However, it remains unclear whether the neuronal features of MCs are conserved across mammals. This study compares the neuroanatomy of MCs in the DG of mice and monkeys. The MC marker, calretinin, distinguishes two subpopulations: septal and temporal. Dual-colored fluorescence labeling is utilized to compare the axonal projection patterns of these subpopulations. In both mice and monkeys, septal and temporal MCs project axons across the longitudinal axis of the ipsilateral DG, indicating conserved associational projections. However, unlike in mice, no MC subpopulations in monkeys make commissural projections to the contralateral DG. In monkeys, temporal MCs send associational fibers exclusively to the inner molecular layer, while septal MCs give rise to wide axonal projections spanning multiple molecular layers, akin to equivalent MC subpopulations in mice. Despite conserved septotemporal heterogeneity, interspecies differences are observed in the topological organization of septal MCs, particularly in the relative axonal density in each molecular layer along the septotemporal axis of the DG. In summary, this comparative analysis sheds light on both conserved and divergent features of MCs in the DG of mice and monkeys. These findings have implications for understanding functional differentiation along the septotemporal axis of the DG and contribute to our knowledge of the anatomical evolution of the DG circuit in mammals.

IL2 Targeted to CD8+ T Cells Promotes Robust Effector T-cell Responses and Potent Antitumor Immunity.

IL2 signals pleiotropically on diverse cell types, some of which contribute to therapeutic activity against tumors, whereas others drive undesired activity, such as immunosuppression or toxicity. We explored the theory that targeting of IL2 to CD8+ T cells, which are key antitumor effectors, could enhance its therapeutic index. To this aim, we developed AB248, a CD8 cis-targeted IL2 that demonstrates over 500-fold preference for CD8+ T cells over natural killer and regulatory T cells (Tregs), which may contribute to toxicity and immunosuppression, respectively. AB248 recapitulated IL2's effects on CD8+ T cells in vitro and induced selective expansion of CD8+T cells in primates. In mice, an AB248 surrogate demonstrated superior antitumor activity and enhanced tolerability as compared with an untargeted IL2Rβγ agonist. Efficacy was associated with the expansion and phenotypic enhancement of tumor-infiltrating CD8+ T cells, including the emergence of a "better effector" population. These data support the potential utility of AB248 in clinical settings. Significance: The full potential of IL2 therapy remains to be unlocked. We demonstrate that toxicity can be decoupled from antitumor activity in preclinical models by limiting IL2 signaling to CD8+ T cells, supporting the development of CD8+ T cell-selective IL2 for the treatment of cancer. See related article by Kaptein et al. p. 1226.

B-cell–directed CAR T-cell therapy activates CD8+ cytotoxic CARneg bystander T cells in patients and nonhuman primates

AbstractChimeric antigen receptor (CAR) T cells hold promise as a therapy for B-cell–derived malignancies, and despite their impressive initial response rates, a significant proportion of patients ultimately experience relapse. Although recent studies have explored the mechanisms of in vivo CAR T-cell function, little is understood about the activation of surrounding CARneg bystander T cells and their potential to enhance tumor responses. We performed single-cell RNA sequencing on nonhuman primate (NHP) and patient-derived T cells to identify the phenotypic and transcriptomic hallmarks of bystander activation of CARneg T cells following B-cell–targeted CAR T-cell therapy. Using a highly translatable CD20 CAR NHP model, we observed a distinct population of activated CD8+ CARneg T cells emerging during CAR T-cell expansion. These bystander CD8+ CARneg T cells exhibited a unique transcriptional signature with upregulation of natural killer-cell markers (KIR3DL2, CD160, and KLRD1), chemokines, and chemokine receptors (CCL5, XCL1, and CCR9), and downregulation of naïve T-cell-associated genes (SELL and CD28). A transcriptionally similar population was identified in patients after a tisagenlecleucel infusion. Mechanistic studies revealed that interleukin-2 (IL-2) and IL-15 exposure induced bystander-like CD8+ T cells in a dose-dependent manner. In vitro activated and patient-derived T cells with a bystander phenotype efficiently killed leukemic cells through a T-cell receptor–independent mechanism. Collectively, to our knowledge, these data provide the first comprehensive identification and profiling of CARneg bystander CD8+ T cells following B-cell–targeting CAR T-cell therapy and suggest a novel mechanism through which CAR T-cell infusion might trigger enhanced antileukemic responses. Patient samples were obtained from the trial #NCT03369353, registered at www.ClinicalTrials.gov.

Abstract 5811: Ultralong CDR3 engineered interleukin-15 antibody fusion for T and NK cell expansion

Abstract Interleukin (IL)-15 is a promising immunotherapeutic cytokine for cancer treatment because it can stimulate the proliferation and cytotoxicity of CD8+ T lymphocytes and nature killer (NK) cells, similar to the action of IL-2, which has been used to treat metastatic melanoma and renal cancer. IL-15 may have improved pharmacologic properties over IL-2 because it doesn’t cause vascular leak syndrome or stimulate regulatory T cells. However, IL-15 is difficult to express as a stable soluble protein and has a short half-life in vitro and in vivo. To solve the problem, we designed an IL-15 ultralong CDR3 humanized bovine antibody fusion. Bovine ultralong CDR3 antibodies have a unique “stalk and knob” structure in which two antiparallel β-strands support a disulfide bonded knob protruding out of the antibody surface and forms a mini antigen binding domain. By replacing the knob domain with IL-15, we have created a chimeric CDR3-IL-15 IgG which functions as IL-15 in vitro and in vivo but can be easily produced in mammalian cells with increased stability. Thus, the CDR3 can stabilize IL-15 in the absence of its high affinity receptor α (Rα) chain. Furthermore, as IL-15 utilizes Rα for increased trans signaling to the receptor β and γ subunits, one variant candidate (CDR3-IL-15_Rα) was produced by co-expressing Rα sushi domain with the chimeric IgG. Both constructs have been demonstrated to be highly active in stimulating proliferation of NK cells and CD8+ T cells in vitro as well as in stimulating proliferation of NK cells and CD8+ T cells in rats and non-human primates (NHP). Although the CDR3-IL-15_Rα exhibited higher in vitro signaling activity than CDR3-IL-15, both were equally potent in stimulating the proliferation of NK cells and CD4+ and CD8+ T cells in NHP without any immunogenicity detected. Moreover, in vivo studies in NHP indicated that CDR3-IL-15 acted faster than CDR3-IL-15_Rα in stimulating the proliferation of these lymphocytes. CDR3-IL-15 is the first thermodynamically stabilized variant of IL-15 produced in the absence of its Rα chain, is highly active in vivo, and has multiple potential cancer applications as an T and NK cell stimulating agent. Citation Format: Ruiqi Huang, Duncan McGregor, Vaughn Smider. Ultralong CDR3 engineered interleukin-15 antibody fusion for T and NK cell expansion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5811.

Abstract 6342: Anti-tumor in vivo efficacy of different classes of anti-CLEC-1 myeloid checkpoint antibodies in monotherapy and in combination with chemotherapeutic agents

Abstract The c-type lectin receptor CLEC-1 is a pattern recognition receptor [1] expressed by endothelial and myeloid cells in mice, non-human primates, and humans. While genetic deletion of CLEC-1 in mice does not lead to any developmental defect, CLEC-1 deletion or CLEC-1 targeting promotes anti-tumor response by increasing cross-presentation of necrotic or damaged cells by cDC1 dendritic cells and by enhancing T-cell activation [2]. To date, the identification of CLEC-1 endogenous ligand(s) and their biological activity into myeloid or endothelial cells remained to be fully investigated. We aimed to further characterize ligands that are involved into CLEC-1 activation and develop monoclonal antagonist anti CLEC-1 antibody anti-tumor therapy that are able fully counteract inhibitory signaling induced into myeloid cells. Using LC/MS campaign, we confirmed CLEC-1 specific binding to the E3 ubiquitin ligase TRIM21and to the secreted histidine rich glycoprotein (HRG) as previously described [2] and also identify several novel intra-cellular and cell surface proteins that bind to CLEC-1 receptor in protein specific-manner. To block interaction of CLEC-1 ligands, we developed different class of antagonist monoclonal antibodies and evaluated their anti-tumor efficacy in humanized CLEC-1 KI orthotopic and ectopic tumor mouse models fully resistant or partially resistant to ICI therapy. While blocking of CLEC-1 binding to its secreted ligand HRG moderately increases anti-tumor responses, inhibition of CLEC-1 binding to its cytoplasmic and membrane ligands significantly impairs MC38 tumor growth (n=12, p=0.04). Importantly, high anti-tumor efficacy was observed in combination therapy with cyclophosphamide in MC38 mouse model. Additionally, antagonist anti-CLEC-1 antibodies significantly increase overall survival of Hepa1.6-bearing mice (n=32 for each treatment, p<0.002), as compared to corresponding isotype control treatment in 3 independent experiments. Altogether, our results further dissect the mechanism of action of the myeloid checkpoint CLEC-1 in its ability to impair anti-tumor immunity and support its use as a novel and highly promising anti-CLEC antagonist antibody for cancer immunotherapy. Citation Format: Aurore Morello, Mylène Deramé, Marion Drouin, Emmanuelle Wilhelm, Stéphanie Neyton, Irène Baccelli, Caroline Mary, Vanessa Gauttier, Isabelle Girault, Kévin Biteau, Cécile Batty, Géraldine Teppaz, Ariane Desselle, Marion Colonello, Marine Malloci, Elise Chiffoleau, Nicolas Poirier. Anti-tumor in vivo efficacy of different classes of anti-CLEC-1 myeloid checkpoint antibodies in monotherapy and in combination with chemotherapeutic agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6342.

An equine iPSC-based phenotypic screening platform identifies pro- and anti-viral molecules against West Nile virus.

Outbreaks of West Nile virus (WNV) occur periodically, affecting both human and equine populations. There are no vaccines for humans, and those commercialised for horses do not have sufficient coverage. Specific antiviral treatments do not exist. Many drug discovery studies have been conducted, but since rodent or primate cell lines are normally used, results cannot always be transposed to horses. There is thus a need to develop relevant equine cellular models. Here, we used induced pluripotent stem cells to develop a new in vitro model of WNV-infected equine brain cells suitable for microplate assay, and assessed the cytotoxicity and antiviral activity of forty-one chemical compounds. We found that one nucleoside analog, 2'C-methylcytidine, blocked WNV infection in equine brain cells, whereas other compounds were either toxic or ineffective, despite some displaying anti-viral activity in human cell lines. We also revealed an unexpected proviral effect of statins in WNV-infected equine brain cells. Our results thus identify a potential lead for future drug development and underscore the importance of using a tissue- and species-relevant cellular model for assessing the activity of antiviral compounds.

Bronchioalveolar organoids: A preclinical tool to screen toxicity associated with antibody-drug conjugates

Despite extensive preclinical testing, cancer therapeutics can result in unanticipated toxicity to non-tumor tissue in patients. These toxicities may pass undetected in preclinical experiments due to modeling limitations involving poor biomimicry of 2-dimensional in vitro cell cultures and due to lack of interspecies translatability in in vivo studies. Instead, primary cells can be grown into miniature 3-dimensional structures that recapitulate morphological and functional aspects of native tissue, termed “organoids.” Here, human bronchioalveolar organoids grown from primary alveolar epithelial cells were employed to model lung epithelium and investigate off-target toxicities associated with antibody-drug conjugates (ADCs). ADCs with three different linker-payload combinations (mafodotin, vedotin, and deruxtecan) were tested in bronchioalveolar organoids generated from human, rat, and nonhuman primate lung cells. Organoids demonstrated antibody uptake and changes in viability in response to ADC exposure that model in vivo drug sensitivity. RNA sequencing identified inflammatory activation in bronchioalveolar cells in response to deruxtecan. Future studies will explore specific cell populations involved in interstitial lung disease and incorporate immune cells to the culture.

Genetic characterization and pathogenicity in a mouse model of newly isolated bat-originated mammalian orthoreovirus in South Korea.

Mammalian orthoreoviruses (MRVs) infect a wide range of hosts, including humans, livestock, and wildlife. In the present study, we isolated a novel Mammalian orthoreovirus from the intestine of a microbat (Myotis aurascens) and investigated its biological and pathological characteristics. Phylogenetic analysis indicated that the new isolate was serotype 2, sharing the segments with those from different hosts. Our results showed that it can infect a wide range of cell lines from different mammalian species, including human, swine, and non-human primate cell lines. Additionally, media containing trypsin, yeast extract, and tryptose phosphate broth promoted virus propagation in primate cell lines and most human cell lines, but not in A549 and porcine cell lines. Mice infected with this strain via the intranasal route, but not via the oral route, exhibited weight loss and respiratory distress. The virus is distributed in a broad range of organs and causes lung damage. In vitro and in vivo experiments also suggested that the new virus could be a neurotropic infectious strain that can infect a neuroblastoma cell line and replicate in the brains of infected mice. Additionally, it caused a delayed immune response, as indicated by the high expression levels of cytokines and chemokines only at 14 days post-infection (dpi). These data provide an important understanding of the genetics and pathogenicity of mammalian orthoreoviruses in bats at risk of spillover infections.IMPORTANCEMammalian orthoreoviruses (MRVs) have a broad range of hosts and can cause serious respiratory and gastroenteritis diseases in humans and livestock. Some strains infect the central nervous system, causing severe encephalitis. In this study, we identified BatMRV2/SNU1/Korea/2021, a reassortment of MRV serotype 2, isolated from bats with broad tissue tropism, including the neurological system. In addition, it has been shown to cause respiratory syndrome in mouse models. The given data will provide more evidence of the risk of mammalian orthoreovirus transmission from wildlife to various animal species and the sources of spillover infections.

Favorable culture conditions for spermatogonial propagation in human and non-human primate primary testicular cell cultures: a systematic review and meta-analysis.

Introduction: Autologous transplantation of spermatogonial stem cells (SSCs) isolated from cryopreserved testicular biopsies obtained before oncological treatment could restore fertility in male childhood cancer survivors. There is a clear necessity for in vitro propagation of the limited SSCs from the testicular biopsy prior to transplantation due to limited numbers of spermatogonia in a cryopreserved testicular biopsy. Still, there is no consensus regarding their optimal culture method. Methods: We performed a systematic review and meta-analysis of studies reporting primary testicular cell cultures of human and non-human primate origin through use of Pubmed, EMBASE, and Web of Science core collection databases. Of 760 records, we included 42 articles for qualitative and quantitative analysis. To quantify in vitro spermatogonial propagation, spermatogonial colony doubling time (CDT) was calculated, which measures the increase in the number of spermatogonial colonies over time. A generalized linear mixed model analysis was used to assess the statistical effect of various culture conditions on CDT. Results: Our analysis indicates decreased CDTs, indicating faster spermatogonial propagation in cultures with a low culture temperature (32°C); with use of non-cellular matrices; use of StemPro-34 medium instead of DMEM; use of Knockout Serum Replacement; and when omitting additional growth factors in the culture medium. Discussion: The use of various methods and markers to detect the presence of spermatogonia within the reported cultures could result in detection bias, thereby potentially influencing comparability between studies. However, through use of CDT in the quantitative analysis this bias was reduced. Our results provide insight into critical culture conditions to further optimize human spermatogonial propagation in vitro, and effectively propagate and utilize these cells in a future fertility restoration therapy and restore hope of biological fatherhood for childhood cancer survivors.

Generation of induced pluripotent stem cells from Bornean orangutans.

Introduction: Orangutans, classified under the Pongo genus, are an endangered non-human primate (NHP) species. Derivation of induced pluripotent stem cells (iPSCs) represents a promising avenue for conserving the genetic resources of these animals. Earlier studies focused on deriving orangutan iPSCs (o-iPSCs) from Sumatran orangutans (Pongo abelii). To date, no reports specifically target the other Critically Endangered species in the Pongo genus, the Bornean orangutans (Pongo pygmaeus). Methods: Using Sendai virus-mediated Yamanaka factor-based reprogramming of peripheral blood mononuclear cells to generate iPSCs (bo-iPSCs) from a female captive Bornean orangutan. In this study, we evaluate the colony morphology, pluripotent markers, X chromosome activation status, and transcriptomic profile of the bo-iPSCs to demonstrate the pluripotency of iPSCs from Bornean orangutans. Results: The bo-iPSCs were successfully derived from Bornean orangutans, using Sendai virus-mediated Yamanaka factor-based reprogramming of peripheral blood mononuclear cells. When a modified 4i/L/A (m4i/L/A) culture system was applied to activate the WNT signaling pathway in these bo-iPSCs, the derived cells (m-bo-iPSCs) manifested characteristics akin to human naive pluripotent stem cells, including high expression levels of KLF17, DNMT3L, and DPPA3/5, as well as the X chromosome reactivation. Comparative RNA-seq analysis positioned the m-bo-iPSCs between human naive and formative pluripotent states. Furthermore, the m-bo-iPSCs express differentiation capacity into all three germlines, evidenced by controlled in vitro embryoid body formation assay. Discussion: Our work establishes a novel approach to preserve the genetic diversity of endangered Bornean orangutans while offering insights into primate stem cell pluripotency. In the future, derivation of the primordial germ cell-like cells (PGCLCs) from m-bo-iPSCs is needed to demonstrate the further specific application in species preservation and broaden the knowledge of primordial germ cell specification across species.